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M ETHODS IN MOLECULAR BIOLOGY™

Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes: http://www.springer.com/series/7651

Neuropeptides

Methods and Protocols

Edited by Adalberto Merighi

Dipartimento di Morfofisiologia Veterinaria, Università degli Studi di Torino, Grugliasco, TO, Italy; Istituto Nazionale di Neuroscienze (INN), Università degli Studi di Torino, Grugliasco, TO, Italy Editor Adalberto Merighi Dipartimento di Morfofisiologia Veterinaria Università degli Studi di Torino and Istituto Nazionale di Neuroscienze (INN) Università degli Studi di Torino Grugliasco, TO, Italy [email protected]

Please note that additional material for this book can be downloaded from http://extras.springer.com

ISSN 1064-3745 e-ISSN 1940-6029 ISBN 978-1-61779-309-7 e-ISBN 978-1-61779-310-3 DOI 10.1007/978-1-61779-310-3 Springer New York Dordrecht Heidelberg London

Library of Congress Control Number: 2011936011

© Springer Science+Business Media, LLC 2011 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights.

Printed on acid-free paper

Humana Press is part of Springer Science+Business Media (www.springer.com) Preface

The term was originally coined to indicate small molecules that are contained in neurons. In the late 1970s and the 1980s of the last century, several tens of were localized by immunocytochemistry to discrete cell populations of the central and peripheral nervous system, and the concept of chemical neuroanatomy, originally developed by Tomas Hökfelt and coworkers, entered the scene of neurobiology. Since then, the field of neuropeptide biology has dramatically widened, and today the ultimate frontiers in neuropeptide research lie in the development of pharmacologically active com- pounds that are capable of crossing the blood–brain barrier to exert their biological role(s) in vivo and in the construction of genetic vectors to be employed in gene therapy. This book represents a readily reproducible collection of established and emerging techniques for neuropeptide research. Such a collection is preceded by a general introductory chapter (Chapter 1) that discusses a series of new concepts leading to a broader neuropeptide definition in light of the huge amount of data accumulated after more than half a century of neuropeptide research. The methods presented include immunocytochemical localization, biochemical charac- terization, functional analysis, development and production of genetic probes, and the design of neuropeptide derivatives for cellular neurobiology as well as the potential therapeutic applications. As a general indication to the readers, Chapters 2–10 are focused on a series of tech- niques for localization studies. They cover a broad range of protocols, such as the immuno- cytochemical detection of neuropeptides in nonmammalian vertebrates together with a detailed description of procedures for anesthesia and tissue preparations in these species (Chapter 2); the combined neuropeptide/receptor localization at the light and transmission electron microscope for connectivity studies (Chapter 3); the analysis of neuropeptide genes’ transcription by localization of pre-mRNA (Chapter 6) or mRNA/microRNA with in situ hybridization (Chapter 4), in situ PCR (Chapter 5), and laser capture/microdissection (Chapter 7); the visualization in vivo of neuropeptide secretion (Chapter 8) and translocation across the plasma membrane (Chapter 9); and the functional analysis of neuropeptide inter- actions in vitro with cells of the immune system (Chapter 10). Chapter 11 describes a series of electrophysiological protocols for functional studies in vitro and in vivo. Chapters 12–19 are devoted to biochemical/molecular biology techniques, ranging from radioimmunoassay (Chapter 12) to neuropeptidomics employing reverse-phase HPLC (Chapter 13) or mass spectrometry (Chapter 14), RNA analysis by suppression subtractive hybridization (Chapter 15), determination of neuropeptide release in vivo by microdialysis (Chapter 16) or antibody microprobes (Chapter 17), and measurement of neuropeptidases (Chapter 18) and neuropeptide autoantibody levels (Chapter 19) in biological fluids. Chapters 20–24 deal with a number of techniques developed to optimize neuropeptide administration to central neurons or to interfere with biological effects in vivo. These pro- cedures include the intranasal delivery of neuropeptides (Chapter 20), the development of

v vi Preface neuropeptide pro-drugs (Chapter 21), the use of phosphorothioate oligodeoxynucleotides that are capable of crossing the blood–brain barrier to knock down neuropeptides in the CNS (Chapter 22), the development of liposome-encapsulated neuropeptides for assessing the chronic actions of physiologically short-lived molecules (Chapter 23), the construction of recombinant adeno-associated viral vectors that can be used to locally or systemically enhance or silence neuropeptide gene expression (Chapter 24). Finally, Chapter 25 describes a calcium mobilization assay in mammalian cells to identify novel G-protein-coupled receptor family members that transduce the neuropeptide signals. All scientists who have excellently contributed to this book have a direct experience in one or more fields of neuropeptide research. I am very much indebted to all of them for their successful effort in emphasizing the description of the more common pitfalls in the techniques that they have described and of the hints to reduce the possibility of failure for beginners. The collection of protocols that forms this book is surely not exhaustive of the wide range of approaches that today can be employed in top level neuropeptide research. Yet it is intended for a large audience of scientists, including histologists, biochemists, cellular and molecular biologists, and electrophysiologists that are currently active in the field or are willing to enter such an exciting and still expanding area of neurobiology.

Grugliasco, TO, Italy Adalberto Merighi Contents

Preface...... v Contributors...... ix

1 What Are Neuropeptides? ...... 1 J. Peter H. Burbach 2 Neuropeptide Localization in Nonmammalian Vertebrates ...... 37 Paolo de Girolamo and Carla Lucini 3 Combined Light and Electron Microscopic Visualization of Neuropeptides and Their Receptors in Central Neurons ...... 57 Chiara Salio, Laura Lossi, and Adalberto Merighi 4 Neuropeptide RNA Localization in Tissue Sections...... 73 Marc Landry, Shérine Abdel Salam, and Marie Moftah 5 Intron-Specific Neuropeptide Probes ...... 89 Harold Gainer, Todd A. Ponzio, Chunmei Yue, and Makoto Kawasaki 6 Direct In Situ RT-PCR ...... 111 Laura Lossi, Graziana Gambino, Chiara Salio, and Adalberto Merighi 7 Laser Capture Microdissection and Quantitative-PCR Analysis ...... 127 Sarah J. Paulsen and Leif K. Larsen 8 Visualization of Secretory Vesicles in Living Nerve Cells...... 137 Joshua J. Park and Y. Peng Loh 9 Fluorescence Imaging with Single-Molecule Sensitivity and Fluorescence Correlation Spectroscopy of Cell-Penetrating Neuropeptides ...... 147 Vladana Vukojevic´, Astrid Gräslund, and Georgy Bakalkin 10 Analysis of Neuroimmune Interactions by an In Vitro Coculture Approach...... 171 Tadahide Furuno and Mamoru Nakanishi 11 Electrophysiology ...... 181 Zhi-Qing David Xu 12 Localization of Neuropeptides by Radioimmunoassay ...... 191 Fred Nyberg and Mathias Hallberg 13 Reversed-Phase HPLC and Hyphenated Analytical Strategies for Peptidomics ...... 203 Anne-Marie Hesse, Sega Ndiaye, and Joelle Vinh 14 Neuropeptidomics: Mass Spectrometry-Based Qualitative and Quantitative Analysis ...... 223 Ping Yin, Xiaowen Hou, Elena V. Romanova, and Jonathan V. Sweedler 15 Suppression Subtractive Hybridization ...... 237 Mohamed T. Ghorbel and David Murphy

vii viii Contents

16 Neuropeptide Microdialysis in Free-Moving Animals ...... 261 Tetsuya Kushikata and Kazuyoshi Hirota 17 Antibody Microprobes for Detecting Neuropeptide Release ...... 271 Rebecca J. Steagall, Carole A. Williams, and Arthur W. Duggan 18 Neuropeptidases ...... 287 Manuel Ramírez, Isabel Prieto, Inmaculada Banegas, Ana B. Segarra, and Francisco Alba 19 Neuropeptide Autoantibodies Assay ...... 295 Sergueï O. Fetissov 20 Intranasal Delivery of Neuropeptides ...... 303 Michael C. Veronesi, Daniel J. Kubek, and Michael J. Kubek 21 Prodrug Design for Brain Delivery of Small- and Medium-Sized Neuropeptides ...... 313 Katalin Prokai-Tatrai and Laszlo Prokai 22 Measurement of Phosphorothioate Oligodeoxynucleotide Antisense Transport Across the Blood–Brain Barrier ...... 337 William A. Banks 23 Liposome-Encapsulated Neuropeptides for Site-Specific Microinjection ...... 343 Frédéric Frézard, Robson A.S. dos Santos, and Marco A.P. Fontes 24 Recombinant Adeno-Associated Viral Vectors ...... 357 Marijke W.A. de Backer, Keith M. Garner, Mieneke C.M. Luijendijk, and Roger A.H. Adan 25 Deorphanizing -Coupled Receptors by a Calcium Mobilization Assay ...... 377 Isabel Beets, Marleen Lindemans, Tom Janssen, and Peter Verleyen Index...... 393 Contributors

SHÉRINE ABDEL SALAM s Department of Zoology, University of Alexandria, Alexandria, Egypt ROGER A.H. ADAN s Department of Neuroscience and Pharmacology, Rudolf Magnus Institute of Neuroscience, Utrecht University Medical Centre Utrecht, Utrecht, The Netherlands FRANCISCO ALBA s Department of and Molecular Biology III and Immunology, University of Granada Medical School, Granada, Spain MARIJKE W.A. DE BACKER s Department of Neuroscience and Pharmacology, Rudolf Magnus Institute of Neuroscience, Utrecht University Medical Centre Utrecht, Utrecht, The Netherlands GEORGY BAKALKIN s Department of Pharmaceutical Biosciences, Uppsala University, Uppsala, Sweden INMACULADA BANEGAS s Unit of Physiology, Department of Health Sciences, University of Jaén, Jaén, Spain WILLIAM A. BANKS s Geriatrics Research, Education, and Clinical Center, Puget Sound Health Care System, Seattle, WA, USA; Division of Gerontology and Geriatric Medicine, Department of Internal Medicine, University of Washington, Seattle, WA, USA J. PETER H. BURBACH s Rudolf Magnus Institute of Neuroscience, Department of Neuroscience and Pharmacology, University Medical Center Utrecht, Utrecht, The Netherlands ISABEL BEETS s Research Group of Functional Genomics and Proteomics, K.U. Leuven, Leuven, Belgium ARTHUR W. DUGGAN s Department of Preclinical Sciences, Royal Dick School of Veterinary Medicine, Edinburgh University, Edinburgh, Scotland, UK SERGUEÏ O. FETISSOV s Digestive System and Nutrition Laboratory (ADEN EA4311), Rouen University, Rouen, France MARCO A.P. FONTES s Departamento de Fisiologia e Biofísica, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil FRÉDÉRIC FRÉZARD s Departamento de Fisiologia e Biofísica, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil TADAHIDE FURUNO s School of Pharmacy, Aichi Gakuin University, Nagoya, Japan HAROLD GAINER s Laboratory of Neurochemistry, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA GRAZIANA GAMBINO s Dipartimento di Morfofisiologia Veterinaria, Università degli Studi di Torino, Grugliasco, TO, Italy

ix x Contributors

KEITH M. GARNER s Department of Neuroscience and Pharmacology, Rudolf Magnus Institute of Neuroscience, Utrecht University Medical Centre Utrecht, Utrecht, The Netherlands MOHAMED T. GHORBEL s Bristol Institute, University of Bristol, Bristol, UK PAOLO DE GIROLAMO s Department of Biological Structures, Functions and Technology, University of Naples Federico II, Naples, Italy ASTRID GRÄSLUND s Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden MATHIAS HALLBERG s Division of Biological Research on Drug Dependence, Department of Pharmaceutical Biosciences, Uppsala University, Uppsala, Sweden ANNE-MARIE HESSE s Laboratory of Biological Mass Spectrometry and Proteomics (SMBP), CNRS USR3149, ESPCI ParisTech, Paris, France KAZUYOSHI HIROTA s Department of Anesthesiology, Hirosaki Graduate School of Medicine, Hirosaki, Japan XIAOWEN HOU s Center for Biophysics and Computational Biology, University of Illinois, Urbana, IL, USA; Beckman Institute, University of Illinois, Urbana, IL, USA TOM JANSSEN s Research Group of Functional Genomics and Proteomics, K.U. Leuven, Leuven, Belgium MAKOTO KAWASAKI s Department of Orthopaedics, School of Medicine, University of Occupational and Environmental Health, Kitakyushu-City, Fukuoka, Japan DANIEL J. KUBEK s Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, IN, USA MICHAEL J. KUBEK s Department of Anatomy and Cell Biology and Program in Medical Neuroscience, Indiana University School of Medicine, Indianapolis, IN, USA TETSUYA KUSHIKATA s Department of Anesthesiology, Hirosaki University Hospital, Hirosaki, Japan MARC LANDRY s INSERM U862, University of Bordeaux, Bordeaux, France LEIF K. LARSEN s Molecular Biology, Vipergen, Copenhagen, Denmark MARLEEN LINDEMANS s Research Group of Functional Genomics and Proteomics, K.U. Leuven, Leuven, Belgium LAURA LOSSI s Dipartimento di Morfofisiologia Veterinaria, Università degli Studi di Torino, Grugliasco, TO, Italy; Istituto Nazionale di Neuroscienze (INN), Università degli Studi di Torino, Grugliasco, TO, Italy CARLA LUCINI s Department of Biological Structures, Functions and Technology, University of Naples Federico II, Naples, Italy MIENEKE C.M. LUIJENDIJK s Department of Neuroscience and Pharmacology, Rudolf Magnus Institute of Neuroscience, Utrecht University Medical Centre Utrecht, Utrecht, The Netherlands ADALBERTO MERIGHI s Dipartimento di Morfofisiologia Veterinaria, Università degli Studi di Torino, Grugliasco, TO, Italy; Istituto Nazionale di Neuroscienze (INN), Università degli Studi di Torino, Grugliasco, TO, Italy MARIE MOFTAH s Department of Zoology, University of Alexandria, Alexandria, Egypt DAVID MURPHY s Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, University of Bristol, Bristol, UK MAMORU NAKANISHI s School of Pharmacy, Aichi Gakuin University, Nagoya, Japan Contributors xi

SEGA NDIAYE s Laboratory of Biological Mass Spectrometry and Proteomics (SMBP), CNRS USR3149, ESPCI ParisTech, Paris, France FRED NYBERG s Division of Biological Research on Drug Dependence, Department of Pharmaceutical Biosciences, Uppsala University, Uppsala, Sweden JOSHUA J. PARK s Neurosciences, University of Toledo College of Medicine, Toledo, OH, USA SARAH J. PAULSEN s Gubra, Hørsholm, Denmark Y. PENG LOH s Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA TODD A. PONZIO s Laboratory of Neurochemistry, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA ISABEL PRIETO s Unit of Physiology, Department of Health Sciences, University of Jaén, Jaén, Spain LASZLO PROKAI s Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, TX, USA KATALIN PROKAI-TATRAI s Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, TX, USA MANUEL RAMÍREZ s Unit of Physiology, Department of Health Sciences, University of Jaén, Jaén, Spain ELENA V. ROMANOVA s Department of Chemistry, University of Illinois, Urbana, IL, USA; Beckman Institute, University of Illinois, Urbana, IL, USA CHIARA SALIO s Dipartimento di Morfofisiologia Veterinaria, Università degli Studi di Torino, Grugliasco, TO, Italy ROBSON A.S. DOS SANTOS s Departamento de Fisiologia e Biofísica, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil ANA B. SEGARRA s Unit of Physiology, Department of Health Sciences, University of Jaén, Jaén, Spain JONATHAN V. SWEEDLER s Department of Chemistry, University of Illinois, Urbana, IL, USA; Beckman Institute, University of Illinois, Urbana, IL, USA REBECCA J. STEAGALL s Department of Physiology, Quillen College of Medicine, East Tennessee State University, Johnson City, TN PETER VERLEYEN s Research Group of Functional Genomics and Proteomics, K.U. Leuven, Leuven, Belgium MICHAEL C. VERONESI s Program in Medical Neuroscience, Indiana University School of Medicine, Indianapolis, IN, USA JOELLE VINH s Laboratory of Biological Mass Spectrometry and Proteomics (SMBP), CNRS USR3149, ESPCI ParisTech, Paris, France VLADANA VUKOJEVIC´ s Department of Clinical Neuroscience, Karolinska Institute, Stockholm, Sweden CAROLE A. WILLIAMS† s Department of Physiology, Quillen College of Medicine, East Tennessee State University, Johnson City, TN, USA ZHI-QING DAVID XU s Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden PING YIN s Department of Chemistry, University of Illinois, Urbana, IL, USA; Beckman Institute, University of Illinois, Urbana, IL, USA CHUNMEI YUE s Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

Chapter 1

What Are Neuropeptides?

J. Peter H. Burbach

Abstract

We know neuropeptides now for over 40 years as chemical signals in the brain. The discovery of neuropep- tides is founded on groundbreaking research in physiology, endocrinology, and biochemistry during the last century and has been built on three seminal notions: (1) peptide are chemical signals in the ; (2) neurosecretion of is a general principle in the nervous system; and (3) the nervous system is responsive to peptide signals. These historical lines have contributed to how neuropeptides can be defined today: “Neuropeptides are small proteinaceous substances produced and released by neurons through the regulated secretory route and acting on neural substrates.” Thus, neuropeptides are the most diverse class of signaling molecules in the brain engaged in many physiological functions. According to this definition almost 70 genes can be distinguished in the mammalian genome, encoding neuropeptide precursors and a multitude of bioactive neuropeptides. In addition, among cytokines, peptide hormones, and growth factors there are several subfamilies of peptides displaying most of the hallmarks of neuropeptides, for example neural chemokines, cerebellins, neurexophilins, and granins. All classical neuropeptides as well as putative neuropeptides from the latter families are presented as a resource.

Key words: Cerebellins, Chemokines, Granins, Adipose peptides, Neuropeptide synthesis

1. Introduction

Nerve cells communicate with each other by virtue of chemical signals that are released by one cell and received by another. All vertebrate and invertebrate species engage a multitude of signal molecules to meet the complexity of their nervous systems. The chemical nature of signal molecules ranges from simple gaseous

molecules, such as NO2, to aminergic and fatty molecules, amino acids, and . Each of them has its own biochemical path- ways of synthesis and degradation, and cell biological characteris- tics allowing it to participate in chemical communication. Neuropeptides are by far the largest and most diverse class of signaling molecules in the brain. They can act as neurotransmitters

Adalberto Merighi (ed.), Neuropeptides: Methods and Protocols, Methods in Molecular Biology, vol. 789, DOI 10.1007/978-1-61779-310-3_1, © Springer Science+Business Media, LLC 2011 1 2 J.P.H. Burbach

directly, as modulators of ongoing neurotransmission by other transmitters, as autocrine or paracrine regulators in a close cellular environment, and as hormones on long range. The modes of action can be recognized in many physiological processes and functions in which neuropeptides participate. But what are neuropeptides pre- cisely? When is a signal molecule a neuropeptide? There are no simple answers for all signal molecules. Here, an attempt is made toward criteria to define neuropeptides. These are based on chemi- cal nature, pathways of biosynthesis, cell biological characteristics, and modes of action. We know neuropeptides now for over 40 years as a term, but the historical lines that lead to their recognition have contributed to these criteria. These lines are outlined below as a brief history. A comprehensive historical account has been eloquently presented by Klavdieva (1–4).

2. How Were Neuropeptides Discovered? Neuropeptides were encountered before they were designated as such, even before chemical signaling was a precipitated concept in physiology. In ancient times, it was thought that organs of animals, including those of man, could affect the body and the mind when eaten. In the nineteenth century, when experimental life sciences developed, extracts of organs became systematically investigated for their effects on physiological systems. During these times the fundaments of endocrinology were laid. Substances such as secre- tin, , , and were encountered as biologi- cal activities in crude extracts, detected by bioassays in whole animals or organ systems (5–7). In 1905, such substances were named “hormones,” a term introduced by the English physiologist Ernest Starling (1866–1927). Starling coined the term, derived from the Greek verb “ormao” (to arouse or excite), during a Croonian Lecture at the Royal College of Physicians in London on the Chemical Control of the Functions of the Body (8–12). He defined hormones in terms of chemical messengers produced recurrently to answer the physiological needs of the organism, and carried from the organ where they are produced to the organ which they affect by means of the bloodstream. Among hormonally active substances were molecules that we now call neuropeptides, such as vasopressin, oxytocin, and (6, 7, 13). Only in the 1950s and beyond, the chemical identities of hormones were elu- cidated: almost all of them appeared to be short chains of amino acids, i.e., peptides (14). Large-scale purification of releasing fac- tors from hypothalamic extracts at immense scale was undertaken, resulting in the elucidation of the structures of releasing factors in the late 1960s, and award of the Nobel Prize to Guillemin and Schally in 1977 (15). From the late 1950s onward, Victor Mutt 1 What Are Neuropeptides? 3

(1923–1998) identified many new peptide hormones from gut organs, and later also from the brain based on chemical properties (16). Guided by biological activity, specifically activity, John Hughes and Hans Kosterlitz (1903–1996) identified the enkepha- lins in 1975 (17). It appeared a seminal moment, bringing neuro- peptides definitely from endocrinology into the neurosciences. While one historical line leading to neuropeptides is the con- ception of peptide hormones, the second is that of neurosecretion. Preceded by early studies of Carl Speidel in 1917 (18), Ernst Scharrer (1905–1965) proposed the concept of neurosecretion in 1934 (19). Early on, it was recognized by physiologists and his- tologists that nerve cells in the brain contained substances that were stored in the cell and could be secreted not into the blood stream, but internally in the nervous system. For example, by chemical staining of bridges, peptides could be detected in the and ventral brain with fibers to the brain sepa- rate from the gland, the canonical site of release for peripheral hormonal actions (20). These peptides were vaso- pressin, oxytocin, and their , later demonstrated by immunocytochemistry (21). Neurosecretion of peptidergic sub- stances is now a fundamental aspect of neuropeptides (22). A third line in the history of neuropeptides is the discovery of biological actions of peptides on the nervous system, and particu- larly of cognitive actions in the brain. In parallel to the discovery of hormones, bioassays were instrumental in the discovery of neuro- peptides. Bioassays for activity on the enteric nervous system existed already for a long time, but for the central nervous system this was new in the 1960s. David de Wied (1925–2004) pioneered on the activity of peptide hormones on rat behavior from the 1950s onward and discovered that ACTH, MSH, and vasopressin acted on the brain and affected learning and memory processes (23–25). In the 1970s he coined the term “neuropeptides” to designate neuroactive peptide hormones and fragments thereof. At that time receptors were still undiscovered mediators of the biological effects. With the development of radioligand binding assays and molecular technolo- gies, receptors for all neuropeptides were found, now explaining the central effects. Since then, the term “neuropeptides” has been adopted and has marked an exciting field in the neurosciences.

3. Criteria for Neuropeptides

The history of neuropeptides illuminates three scientific milestones (1) peptide hormones as regulators in the endocrine system, (2) neurosecretion of peptidergic substances, and (3) responsiveness of nerve cells to peptides. Along these lines, an answer to the question “What are neuropeptides?” is proposed here. The three historical 4 J.P.H. Burbach

lines leading to neuropeptides have not always been able to explain uncertainties like the following: “Are all peptide hormones neuro- peptides?”, “Are all proteineous substances secreted by neurons neuro- peptides?”, “Are all peptides with effects when injected in the brain, neuropeptides?” The basic question “What are neuropeptides?” requires a definition to pinpoint the essence of a neuropeptide. The hallmarks of neuropeptides are (1) gene expression and biosynthe- sis by neurons, (2) storage and regulated release upon demand, and (3) the ability to modulate or mediate neural functioning directly through neural receptors. These hallmarks are commented below.

3.1. Gene Expression Neuropeptides are used by neurons to signal to other cells. A priori and Biosynthesis gene expression and biosynthesis occur in neurons. For most by Neurons “­classical” neuropeptides, studies using in situ hybridization and immunocytochemistry­ have unambiguously shown that the ­transcript and peptide products are produced by neurons. In a few cases, bio- synthesis of peptides could actually be demonstrated by incorpora- tion of radioactive amino acids in pulse-chase experiments (26). An issue arises in cases where the peptide is produced in the nervous system by other cells than neurons. Formally, these are not neuropeptides, but are growth factors and cytokines produced by glial cells (see Subheading 4). Glial cells were believed to have only a constitutive secretory pathway, thus releasing intact or partly pro- cessed precursors in a nonregulated fashion. However, emerging data from astrocytes and glial cell lines show that these cells can also have a regulated secretory pathway (27, 28). Therefore, puta- tive neuropeptides may be recognized in peptide families expressed by glial cells.

3.2. Regulated Release Controlled secretion is a basis for regulation of chemical commu- nication. Neurons expressing classical neuropeptides use the regu- lated secretory pathway, allowing storage of biosynthesized peptides in large dense-cored vesicles and controlled release upon a stimulus (29–31). The constitutive and regulated secretory pathways are schematically compared in Fig. 1. A sequence is key for entry into secretion routes. Generally, this is a short 20–25 amino acid extension at the N-terminal of the precursor, the ­prepro-peptide. It is directly removed from the nascent precursor during protein synthesis and thus never found on the precursor or in the peptide pool. The regulated secretory route imposes specific structural characteristics to the neuropeptide precursor to be sorted away from the default constitutive route. These structural prereq- uisites for sorting are situated in the 3D structure of the precursor and have remained largely uncovered (32). Furthermore, the pro- teolytic processing of the precursor into active peptides is directed by short motifs of basic amino acids. Thus, neuropeptide precur- sors contain salient structural details, illustrated in Fig. 1. 1 What Are Neuropeptides? 5

Fig. 1. Secretory pathways and the biosynthesis of neuropeptides. The flow of newly biosynthesized secretory proteins through the cell is schematically depicted with emphasis on the subcompartments that are passed and the processing steps that occur. The default pathway is the constitutive pathway with small clear-cored secretory vesicles that immedi- ately fuse for secretion. Neuropeptides are generally released through the constitutive pathway. Neuropeptide precursors are sorted in the trans-Golgi network into vesicles of the secretory pathway. These vesicles acidify, condense the protein content, and activate proteolytic enzymes resulting in processing of the precursor protein into neuropeptides. Mature granules (dense-cored vesicles) are stored and await release upon a stimulus. Posttranslational modifications and their cellular sites are indicated. The figure was made available by Gerard Martens, Nijmegen.

1. The signal peptide. This 20–25 amino-acid long N-terminal portion is required for the entry of the newly synthesized gene product into the lumen of the endoplasmic reticulum (ER). It is cleaved off during passage of the ER membrane, leaving the proneuropeptide in the ER-Golgi for further sorting into the regulated secretory pathway. 2. The basic motifs. Pairs of the basic amino acids (Lys, K) and arginine (Arg, R), or, more rarely, a single Arg in the appropriate structural environment of the precursor serve as recognition sites and substrates of prohormone convertases (PCs). These proteolytic enzymes belong to a small family of subtilisin proteases with cell specific expression. Cleavage of the neuropeptide precursor leads to the generation of specific peptides, and cell-specific differences in PCs lead to different sets of peptides from the same precursor. 6 J.P.H. Burbach

3. C-terminal amidation. PC-generated peptides can be subject to further modification by peptidyl-aminotransferase (PAM). This enzyme uses a C-terminal (Gly, G) as amide donor for the preceding amino acid. This results in a peptide with an amidated C-terminus. This amide occurs in many neuropep- tides and is often essential for biological activity. This structural characteristic has been elegantly exploited by Mutt to isolated novel neuropeptides and peptide hormones on the bases of a chemical assay for C-terminal amides, such as , peptide YY, and peptide HI (all named after the C-terminal amidated amino acid) (16). 4. Post-translational modifications. Chemical modifications include N- and O-glycosylation, phosphorylation, sulfation, and acety- lation. These are found on stored and secreted peptides and can contribute to biological properties.

3.3. Modulation Neuropeptides have biological activities in neural systems that can of Neural Functions be observed at all levels of neural functioning: effects of neuropep- and Receptors tides have been established at the genetic, biochemical, cellular, behavioral, and organismal levels. In this respect, the biological activities of many neuropeptides have been indicated as “pleiotro- pic,” i.e., acting on a variety of cell types and tissues. Such effects are not always distinguishable from that of biologically active pep- tides indicated as growth factors or cytokines. Typical for the cel- lular action of neuropeptides are the relatively slow responses and actions as compared to “fast neurotransmitters” such as excitatory amino acids and amines. Neuropeptides often coexist with amino acid and amine neurotransmitters in nerve terminals, but are released only after intense or prolonged stimulation (22). This delayed response of neuropeptide secretion is due to storage in dense-cored vesicles that are not docked at the cellular release site, e.g., the synapse or synaptic button, requiring recruitment first, unlike the “fast neurotransmitters,” which are ready for release. This happens at elevated Ca2+ levels which require enhanced or repeated stimulation. The relatively slow response to released neu- ropeptides is due to the type of receptor that almost all neuropep- tides act on: G-protein coupled receptors (GPCRs). These receptors trigger an intracellular cascade of molecular enzymatic events that result in cellular responses. The time span over which such response is in effect (seconds or longer) is considerably longer than that of neurotransmitters that modulate ion fluxes directly through action on ion channels (milliseconds). Databases of neuropeptide receptors are available: http:// www.gpcr.org/7tm/ and http://www.iuphar-db.org. There are more neuropeptides known than there are receptors. Several pep- tides may still have unknown receptors. Moreover, receptors are often responsive to multiple related neuropeptides. 1 What Are Neuropeptides? 7

4. Neuropeptides Defined

Based on the above considerations, a strict definition of a neuro- peptide would be as follows: “A neuropeptide is a small proteina- ceous substance produced and released by neurons through the regulated secretory route and acting on neural substrates.” About 70 genes encoding for such neuropeptides have been recognized. These can be considered as “classical neuropeptides” and are listed in Table 1. The genes encoding these classical neuro- peptides can be grouped in 18 subfamilies according to precursor and peptide structure or according to function. Examples of the former are the CRH-related gene family and the /secre- tin gene family; the latter is illustrated by the opioid gene family. Apart from these, a group of miscellaneous peptide genes still remains (Table 1). Cerebellins are complicated with respect to fit- ting such a definition (Table 2). But how about peptides that are known as peptide hormones of the endocrine system, chemokines of the immune system, or growth factors, if they are also synthe- sized and active in the nervous system? To appreciate this issue, these classes of proteinaceous signaling molecules are briefly addressed below, in view of the characteristics of neuropeptides.

4.1. Peptide Hormones A is a substance secreted by one organ and acting on another after transport by the bloodstream, as already proposed by Starling in 1904 (8–12). In this way, cells at different locations in the organism communicate in an endocrine fashion. Many peptide hormones are also synthesized by neurons and thus belong no disputably to the classical neuropeptides (see Table 1). Reversely, many classical neuropeptides are also synthesized by endocrine glands and function peripherally as hormones. However, this defi- nition excludes hormones as being neuropeptides if they are not synthesized by neurons, even if they signal to the brain. and insulin are examples, and there is an emerging class of regulatory peptides originally found in adipose tissues (Table 3). Sometimes the relationships between the nervous system and peripheral endocrine glands are complicated by alternative process- ing and splicing. One example of alternative processing is the pep- tides derived from POMC. ACTH and b- are the prime peptide hormones derived from POMC in the periphery, while a-MSH and b-endorphin are the major POMC neuropeptides in the brain. This is due to differences in processing between adreno- corticotropes of the gland, secreting ACTH for peripheral action, and hypothalamic POMC neurons, secreting a-MSH and b-endorphin from neuronal processes (33). The dif- ferent sets of biologically active POMC products are accommo- dated by differences between peripheral and central receptors. Furthermore, differential expression of two paralogous 8 J.P.H. Burbach , , peptide B, BAM22P peptide E, F, ( a -MSH), g -melanocyte-stimulating hormone ( g -MSH), b -melanocyte- stimulating hormone ( b -MSH), adrenocorticotropic hormone (ACTH), b -endorphin, a g -endorphin, b -lipoprotein ( b -LPH), g -lipoprotein ( g -LPH), corticotropin- like intermediate peptide (CLIP) endorphin, b -neo-endorphin, ­ -32, leu-morphin Leu-, Met-enkephalin, a -Melanocyte-stimulating hormone , , a -neo- Active peptide(s) Prepro-enkephalin POMC Prepro-dynorphin Precursor Gene expression 2q12.2 20p13 8q12.1 Chromosomal localization gene (POMC) (PDYN) (PENK) Pro-opiomelanocortin Pro-dynorphin gene 1 Table neuropeptides classical Neuropeptide gene families: Gene (gene symbol) Opioid gene family Pro-enkephalin gene 1 What Are Neuropeptides? 9 (continued) neuropeptide 1, neuropeptide 2 II), C-terminal CPP (orphanin FQ), (VP), neurophysin II (NP Vasopressin Oxytocin (OT), (NP 1) -34, gastrin-17, gastrin-4 Active peptide(s) Prepro-orphanin neurophysin II neurophysin I Prepro-nociceptin, Prepro-vasopressin- Prepro-oxytocin- Prepro-gastrin Precursor Gene expression 20p13 20p13 17q21.2 8p21.1 Chromosomal localization prepro-nociceptin gene (PNOC) Vasopressin/oxytocin gene family Vasopressin/oxytocin gene (AVP) Vasopressin Oxytocin gene (OXT) CCK/gastrin gene family Gastrin gene (GAST) Gene (gene symbol) Orphanin gene, 10 J.P.H. Burbach CCK-8, CCK-33, CCK-58 SS-12, SS-14, SS-28, antrin cortistatin-29, cortistatin-17 Active peptide(s) Prepro-CCK Prepro- Prepro-cortistatin Precursor Gene expression 3q27.3 1p36.22 3p22.1 Chromosomal localization (CCK) Somastostatin gene family Somastostatin gene (SST) Cortistatin gene (CST) 1 Table (continued) Gene (gene symbol) gene 1 What Are Neuropeptides? 11 (continued) GnIH, p518, RF-related peptide-2), RF-related peptide-1, RF-related peptide-3, neuropeptide VF neuropeptide SF QRF-amide (neuropeptide RF-amide, neuropeptide AF, Neuropeptide FF, C-flanking peptide CPON NPY, PPY PYY-(3-36) PYY, Active peptide(s) Prepro-NPRF Prepro-NPFF Prepro-NPY Prepro-PPY Prepro-PYY Precursor Gene expression 12q13.13 7p15.3 17q21.31 17q21.31 7p15.2 Chromosomal localization (NPFF) (NPY) gene (PPY) hormone gene, RF-amide related peptide gene (RFRP) Neuropeptide FF gene Neuropeptide Y gene Peptide YY gene (PYY) Gene (gene symbol) gene family F- and Y-amide inhibitory 12 J.P.H. Burbach ( a -CGRP) ( b -CGRP) PrRP-31, PrRP-20 , katacalcin Calcitonin gene related peptide I Calcitonin gene related peptide II IAPP (, amyloid polypeptide) Active peptide(s) Prepro-PrRP Prepro-CALC Prepro-CGRP-alpha Prepro-CGRP-beta Prepro-IAPP Precursor Not available Gene expression 11p15.2 11p15.2 12p12.1 2q37.3 Chromosomal localization gene (CALCA) (CALCB) gene (IAPP), Amylin gene (PRLH) Calcitonin gene family Calcitonin I Calcitonin II gene Islet amyloid polypeptide 1 Table (continued) Gene (gene symbol) -releasing peptide 1 What Are Neuropeptides? 13 (continued) (IMDL), intermedin-short (IMDS) natriodilatine, ANP, peptide A, ANF, cardiodilatine-related peptide) BNP) peptide B, BNF, CNP-29, CNP-53 , AM, PAMP Adrenomedullin-2, intermedin-long Atrial natriuretic factor (natriuretic Brain natriuretic factor (natriuretic C-type (CNP-23), Active peptide(s) Prepro-adrenomedullin Prepro-adrenomedullin-2 Prepro-ANP Prepro-BNP Prepro-CNP Precursor Gene expression 22q13.33 1p36.22 1p36.22 2q37.1 11p15.4 Chromosomal localization - (ADM2) gene (NPPA) gene (NPPB) sor C gene (NPPC) (ADM) Adrenomedullin-2 gene factor gene family Natriuretic Atrial natriuretic factor Brain natriuretic factor Natriuretic peptide precur Gene (gene symbol) Adrenomedullin gene 14 J.P.H. Burbach (neuromedin C) (neuromedin C) (neuromedin C) RLP) RLP) GRP-27, GRP-14, GRP-10 GRP-27, GRP-14, GRP-10 GRP-27, GRP-14, GRP-10 (Ranatensin-like peptide, Neuromedin B (Ranatensin-like peptide, 1 (ET-1) Active peptide(s) (PPET1) Prero-GRP-1 Prepro-GRP-2 Prepro-GRP-3 Prepro-neuromedin B1 Prepro-neuromedin B2 Prepro- Precursor Gene expression 15q25.2-q25.3 6p24.1 18q21.32 Chromosomal localization (NMB) gene (GRP) 1 Table (continued) Neuromedin B gene Endothelin gene family Endothelin 1 gene (EDN1) Gene (gene symbol) -like peptide gene family Gastrin-releasing peptide 1 What Are Neuropeptides? 15 (continued) (GRPP); (OXY) (OXM); Glucagon; Glucagon-like peptide 1 (GLP-1); Glucagon-like peptide 1 (7-37) (GLP-1 (7-37)); Glucagon-like peptide 1 (7-36) (GLP-1 (7-36)); Glucagon-like peptide 2 (GLP-2) Active peptide(s) (ET-2) (ET-3) Glicentin; Glicentin-related polypeptide PHM-27/PHI-27, PHV-42 VIP, PHM-27/PHI-27, PHV-42 VIP, (PPET2)2 (PPET3) Prepro-endothelin 2 Prepro-endothelin 3 Prepro-glucagon Prepro-secretin Prepro-VIP-1 Prepro-VIP-2 Precursor Gene expression 1p34.2 20q13.32 2q24.2 11p15.5 6q25.2 Chromosomal localization gene (EDN2) gene (EDN3) peptide gene (VIP) Gene (gene symbol) Endothelin 2 Endothelin 3 gene family Glucagon/secretin Glucagon gene (CGC) Secretin gene (SCT) intestinal Vasoactive 16 J.P.H. Burbach nin, somatorelin, sermorelin) dependent insulinotropic polypeptide) PACAP-38, PACAP-27, PRP-48 PACAP-27, PACAP-38, somatocri - GHRH (somatoliberin,GRF, GIP (gastric inhibitory peptide, glucose- CRH Active peptide(s) Prepro-PACAP Prepro-GHRH Prepro-GIP Prepro-CRH Precursor Gene expression 20q11.23 17q21.32 8q13.1 18p11.32 Chromosomal localization hormone gene (GHRH) gene (GIP) hormone gene (CRH) activated peptide gene (ADCYAP1) releasing Gastric inhibitory peptide gene family CRH-related Corticotropin-releasing 1 Table (continued) Gene (gene symbol) Pituitary adenyl cyclase- 1 What Are Neuropeptides? 17 (continued) peptide,urotensin-2B UNC II, stresscopin-related peptide Urotensin-2 Urotensin-2 UNC I UNC III, stresscopin Urotensin-2-related Active peptide(s) isoform a isoform b Prepro-UNC II Prepro-urotensin-2, Prepro-urotensin-2, Prepro-UNC I Prepro-UNC III Prepro-urotensin-2B Precursor Not available Not available Gene expression 10p15.1 3q28 3p21.31 1p36.23 2p23.3 Chromosomal localization (UCN3) containing (UTS2D) III gene Urotensin-II domain Urocortin II gene (UCN2) Urotensin-II (UTS2) Gene (gene symbol) Urocortin gene (UCN) 18 J.P.H. Burbach substance K, neuromedin L), , neuropeptide gamma neuropeptide g neurokinin A Substance P, neurokinin A (NKA, Substance P, neuropeptide K, Substance P, neurokinin A, Substance P, neuropeptide K, Substance P, Neuromedin K, Neuromedin K, neurokinin B Active peptide(s) multiple isoforms Prepro-neuromedin S a -PPTA b -PPTA g -PPTA d -PPTA PPTB, isoform 1 PPTB, isoform 2 Prepro-neuromedin U, Precursor Not available Gene expression 4q12 12q13.3 2q11.2 7q21.3 Chromosomal localization (NMU) (TAC3) gene (NMS) (TAC1) Neuromedin U gene Preprotachykinin B gene Neuromedins Neuromedin S 1 Table (continued) Gene (gene symbol) and tensin gene family Preprotachykinin A gene 1 What Are Neuropeptides? 19 (continued) HMW-K-kinin HMW-K-kinin -(1–7) , motilin-associated peptide Motilin, motillin-associated peptide , kallidin, LMW-K-kinin, Bradykinin, kallidin, LMW-K-kinin, Bradykinin, kallidin, LMW-K-kinin, Angiotensin I, angiotensin II, (NT), , obestatin Ghrelin, obestatin Obestatin Obestatin Obestatin Active peptide(s) isoform 1 isoform 2 preprotein Pro-obestatin Pro-obestatin Prepro-motilin isoform 1 Prepro-motilin isofrom 2 -1 precursor, Kininogen-1 precursor, Kininogen-1 precursor, Angiotensinogen Prepro-neurotensin Prepro-ghrelin isoform 1 Prepro-ghrelin isoform 2 Prepro-ghrelin isoform 3, Prepro-ghrelin isoform 4, Prepro-ghrelin isoform 5 Precursor Not available Gene expression 3p25.3 1q42.2 12q21.31 6p21.31 3q27.3 Chromosomal localization Ghrelin gene (GHRL) Angiotensin gene (AGT) Neurotensin gene (NTS) Motilin family Motilin gene (MLN) Gene (gene symbol) and Tensins Kininogen-1 gene (KNG1) 20 J.P.H. Burbach peptide (GMAP) GnRH2 (LHRH II, gonadoliberin II) GnRH2 (LHRH II, gonadoliberin II) GnRH2 (LHRH II, gonadoliberin II) , galanin message associated Galanin-like peptide (GALP) GnRH (LHRH, gonadoliberin) Active peptide(s) Isoform-a Isoform-b Isoform-c precursor Prepro-GNRH2, Prepro-GNRH2, Prepro-GNRH2, Prepro-galanin Galanin-like peptide Prepro-GnRH1 Precursor Not in mouse Not in mouse Not in mouse Gene expression 19q13.42 8p21.2 20p13 11q13.2 Chromosomal localization precursor gene (GALP) hormone gene (GnRH1) hormone gene (GnRH2) Galanin-like peptide GnRH family Gonadotropin-releasing Gonadotropin-releasing 1 Table (continued) Gene (gene symbol) Galanin family Galanin gene (GAL) 1 What Are Neuropeptides? 21 (continued) neuropeptide B-29 neuropeptide W-30 -2 Relaxin-2 Neuropeptide B-23 (peptide L7), (peptide L8), Neuropeptide W-23 Relaxin-1 Relaxin-3 Active peptide(s) isoform 1 Isoform 2 PPL7 PPL8 Prepro-neuropeptide S Prepro-relaxin-2, Prepro-relaxin-2, Prepro-neuropeptide B, Prepro-neuropeptide W, Prepro-relaxin-1 Prepro-relaxin-3 Precursor Not available Not in mouse Gene expression 9p24.1 19p13.12 16p13.3 10q26.2 9p24.1 17q25.3 Chromosomal localization (NPW) (NPS) (NPB) Insulin/ Relaxin-1 gene (RLN1) Relaxin-3 gene (RLN3) Neuropeptide W gene Neuropeptides S gene Relaxin-2 gene (RLN2) Gene (gene symbol) Neuropeptide B/W family Neuropeptide B gene 22 J.P.H. Burbach PTHrP-(107-139) (osteostatin) neuropeptide Gly-Glu (NGE) ( B) TRH (thyroliberin) PTHrP-(1-36), PTHrP-(38-94), MCH, neuropeptide Glu-Ile (NEI), Hypocretin-1 (orexin A), hypocretin-2 Active peptide(s) hormone, isoform CRA_a hormone, isoform CRA_b Prepro-TRH Prepro-PTH-like Prepro-PTH-like Prepro-MCH Prepro-hypocretin Precursor Gene expression 12p11.22 12q23.2 17q21.2 3q21.3 Chromosomal localization hormone gene (PTHLH) hormone gene (PMCH) hormone gene (TRH) -like Melanin-concentrating Hypocretin gene (HCRT) Gene (gene symbol) No-family neuropeptides Thyrotropin-releasing 1 Table (continued) 1 What Are Neuropeptides? 23 (continued) ligand, AGTRL1 ligand) 1 homolog A,golt1a), -14, kisspeptin-13, kisspeptin-10 CART-(1-39), CART-(42-89) CART-(1-39), AGRP AGRP Prolactin Apelin-13, apelin-17, apelin-36 (APJ Metastin (kisspeptin-54), (golgi transport Active peptide(s) Prepro-CART AGRP precursor isoform 1 Prepro-AGRP isoform 2 Prolactin precursor Prepro-apelin Kiss-1 Precursor Gene expression 16q22.1 6p22.3 Xq25 1q32.1 5q13.2 Chromosomal localization homolog gene (AGRP) (KISS1) amphetamine-regulated transcript gene (CART) Agouti-related protein Prolactin (PRL) Apelin gene (APLN) Metastasis-suppressor KiSS Gene (gene symbol) Cocaine- and 24 J.P.H. Burbach derived VEGF (EGVEGF) 4, ECRG-4) -1, (PK1)endocrine gland- Augurin (esophageal cancer related gene Spexin Diazepam-binding inhibitory peptide Diazepam-binding inhibitory peptide Diazepam-binding inhibitory peptide Prokineticin-2 (PK2) Prokineticin-2 (PK2) Active peptide(s) isoform 1 isoform 2 Prokineticin-1 precursor Augurin precursor Spexin precursor DBI isoform 1 DBI isoform 2 DBI isoform 3 Prokineticin-2 precursor Prokineticin-2 precursor Precursor Gene expression 3p13 12p12.1 1p13.3 2q12.2 2q14.2 Chromosomal localization inhibitor (DBI) Prokineticin-2 (PROK2) Spexin (C12orf39) Prokineticin-1 (PROK1) Augurin (C2orf40) 1 Table (continued) Gene (gene symbol) Diazepam-binding 1 What Are Neuropeptides? 25

(OSF-2), fasciclin I-like) (OSF-2), fasciclin I-like) (OSF-2), fasciclin I-like) (OSF-2), fasciclin I-like) Osteocrin (musclin) Periostin (Osteoblast-specific factor 2 Periostin (Osteoblast-specific factor 2 Periostin (Osteoblast-specific factor 2 Periostin (Osteoblast-specific factor 2 Active peptide(s) isoform 1 isoform 2 isoform 3 isoform 4 Osteocrin precursor Periostin precursor, Periostin precursor, Periostin precursor Periostin precursor Periostin precursor Precursor europeptides are presented as the processed, biologically active products of the neuropeptide the of products active biologically processed, the as presented are n europeptides Gene expression 3q28 13q13.3 Chromosomal localization with permissionhttp://www.neuropeptides.nl and updated. Genes are abbreviated according to the official gene nomenclature ( http://www. Osteocrin (OSTN) Gene (gene symbol) Periostin (POSTN) precursors (prepro-peptide) and the encoding gene. for the color version of this figure. Brain ). See http://extras.springer.com/ ( http://www.brain-map.org expression data represent in situ hybridization data of the mouse brain obtained from the Allen Brain Atlas Data have been obtained from given. is genome human the on localization The gene.ucl.ac.uk/nomenclature/ ). 26 J.P.H. Burbach

Table 2 Putative neuropeptides: cerebellins

Gene (gene Chromosomal symbol) localization Precursor Active peptide(s) Mouse brain expression

Cerebellin-1 gene 16q12.1 Cerebellin-1 Cerebellin-1 Not available (CBLN1) precursor (Cbln1) Cerebellin-2 gene 18q22.3 Cerebellin-2- Cerebellin-2 (CBLN2) precursor (Cbln2)

Cerebellin-3 gene 14q12 Cerebellin-3 Cerebellin-3 (CBLN3) precursor (Cbln3)

Cerebellin-4 gene 20q13.2 Cerebellin-4 Cerebellin-4 (CBLN4) precursor (Cbln4, cerebellin-like glycoprotein-1)

Putative neuropeptides are presented as the processed, biologically active products of the neuropeptide precursors (prepro-peptide) and the encoding gene. The cerebellin family members do not all fulfill the criteria of the neuropeptide definition. Particularly, regulated release has not been established for all members. Genes are abbreviated according to the official gene nomenclature. (http://www.gene.ucl.ac.uk/nomenclature/), and the localization on the human genome is given. Brain expression data represent in situ hybridization data of the mouse brain obtained from the Allen Brain Atlas (http://www.brain-map.org). Data have been obtained from http://www.neuropeptides.nl with permission and updated for this chapter. See http://extras.springer.com/ for the color version of this figure.

genes, and differential splicing of one gene transcript can result in a nervous-system-specific population of neuropeptides which is distinct from the related peripheral peptide hormones. Examples are found in the calcitonin/CGRP and tachykinin families of genes and peptides (34, 35). A number of peptide hormones have not been found to be expressed in the brain or have not been specifi- cally investigated, such as the pituitary hormones, growth hor- mone, prolactin, LH, FSH, and others. Formally, they cannot be considered neuropeptides and have not been included in the tables. However, with ongoing examination of brain-expressed genes, several of these may still appear to be synthesized by certain neu- rons. Therefore, the list of classical neuropeptides may still grow.

4.2. Granins Granins form a family of peptides that do not fully fulfill all criteria of neuropeptides (Table 4). Members of this family, chromogranins and secretogranins, display the structural features of neuropeptide 1 What Are Neuropeptides? 27 (continued) Mouse brain expression related protein; adipocyte, C1Q and domain containing) (PBEF1), nicotinamide factor-1 phosphoribosyltransferase) FIZZ3, -specific secretory -rich secreted protein factor, Xcp4) A12-alpha-like 2, ADSF, Active peptide(s) -delta2 Leptin (obesin) (Acpr30, adipocyte complement Visfatin-1 (pre-B cell colony enhancing Resistin (Cysteine-rich secreted protein Precursor Resistin-delta2 precursor Prepro-leptin Adiponectin precursor Visfatin precursor Resistin precursor Chromosomal localization 7q32.1 3q27.3 7q22.2 19p13.2 (ADIPOQ) 3 Table Putative neuropeptides: regulatory in the brain peptides from adipose tissues and structural relatives expressed Gene (gene symbol) Adipose neuroepeptides Leptin/ob gene (LEP) Adiponectin gene Visfatin gene (PBEF1) Resistin gene (RETN) 28 J.P.H. Burbach Mouse brain expression not available inflammatory zone 1; FIZZ1, hypoxia- Xcp2) induced mitogenic factor, secreted protein FIZZ2, Colon and small intestine-specific cysteine-rich protein, Cysteine-rich secreted protein A12-alpha- like 1, Colon carcinoma-related gene protein, Xcp3) secreted protein FIZZ3, Xcp1) Active peptide(s) Resistin-like molecule alpha (found in Resistin-like molecule beta (Cysteine-rich Nesfatin-1 Resistin-like molecule gamma (Cysteine-rich precursor (RELMalpha) precursor (RELMbeta) gamma precursor (RELMgamma) Precursor Resistin-like molecule alpha Resistin-like molecule beta Nucleobindin-2 Resistin-like molecule Chromosomal localization Not in human 3q13.13 11p15.1 Not in human (RETLA) (RETLB) gene (NUCB2) (RETLG) 3 Table (continued) Gene (gene symbol) Resistin-like alpha gene Resistin-like beta gene Nucleobindin-2/NEFA Resistin-like gamma gene 1 What Are Neuropeptides? 29 http://extras.springer.com/ Mouse brain expression VEGF, EG-VEGF) VEGF, Active peptide(s) Prokineticin-2 isoform b Beacon Prokineticin-1 (endocrine gland-derived Prokineticin-2 isoform a precursor precursor Precursor Prokineticin-2 isoform b Beacon precursor Prokineticin-1 precursor Prokineticin-2 isoform a Chromosomal localization 19p13.2 1p36.12 3p13 with permission and updated. http://www.neuropeptides.nl Genes are abbreviated according to the official gene nomenclature ( http://www. Gene (gene symbol) -like 5 (UBL5) Prokineticin-1 (PROK1) Prokineticin-2 (PROK2) for the color version of this figure. Data have been obtained from gene.ucl.ac.uk/nomenclature/ ). The localization on the human genome is given. Putative neuropeptides are presented as the criteria. processed, all biologically for active established been products not of has neuropeptidethe as neuropeptide role A precursors brain. (prepro-peptide)the in and expressed relativesthe structural have encoding or gene. expression These brain have regulatorytissues adipose in found originally peptides See ). ( http://www.brain-map.org Atlas Brain Allen the from obtained brain mouse the of data hybridization situ in represent data expression Brain 30 J.P.H. Burbach Mouse brain expression peptide, GAWK peptide peptide, GAWK EM66, secretoneurin Active peptide(s) Chromogranin B (secretogranin I) Chromogranin A, beta-granin, vasostatin Chromogranin B (secretogranin I), CCB Secretogranin III Secretogranin II (chromogranin C), variant chromogranin C precursor Precursor Chromogranin B precursor Chromogranin A precursor Chromogranin B precursor Secretogranin III precursor Secretogranin II precursor, Secretogranin II precursor, Chromosomal localization 14q32.12 20p12.3 15q21.2 2q36.1 (CHGA) (CHGB) (SCG3) (SCG2) 4 Table neuropeptide-like proteins with ambiguous biological activities Putative neuropeptides: the granin family, Gene (gene symbol) Chromogranin A gene Chromogranin B gene Secretogranin III gene Secretogranin II gene 1 What Are Neuropeptides? 31 http://www. Mouse brain expression protein-1 (7B2, secretogranin 5) tretory protein), TLPQ-62, TLPQ-21, AQEE-30, LQEQ-19 Active peptide(s) Secretory granule neuroendocrine VGF (NGF-inducible protein, neurosec - crine precursor Precursor Secretory granule neuroendo - VGF-precursor Chromosomal localization 15q13.3 7q22.1 neuroendocrine protein 1, 7B2 gene (SGNE1) factor inducible protein (VGF) Gene (gene symbol) Secretory granule VGF nerve growth for the color version of this figure. neuropeptides.nl with permission and updated. See http://extras.springer.com/ Neuropeptidesareneuropeptidemembers products processed,the family presentedactive precursorsthe of granin biologically as (prepro-peptide)The gene. encoding the and been not have receptors and activities biological unequivocal display not do peptides derived and granins Particularly, definition. neuropeptide the of criteria the fulfill all not do defined. expres - Brain given. is genome human the on localization The ). ( http://www.gene.ucl.ac.uk/nomenclature/ nomenclature. gene official the to according abbreviated are Genes fromobtained been have Data ). representfrom( http://www.brain-map.org data obtained Atlas sion brain Brain mouse Allen the of data hybridization situ in 32 J.P.H. Burbach

precursors, and they are synthesized by neurons. Granins have been identified as peptides cosynthesized, costored, or coreleased with neuropeptides. Many reports on biological activities of some processed forms of granins exist, for instance for vasostatin, cat- estatin, pancreastatin, secretoneurin, EL35, EM66, and WE14 (36). However, a clear-cut biological function as signaling mole- cules as well as defined receptors lacks for these peptides. An alter- native view on these peptides is that they are chaperones of the regulated secretory pathway, assisting neuropeptides with sorting and transport (37).

4.3. Chemokines Chemokines, from “chemotactic cytokines,” are originally known as secreted factors that mediate leukocyte migration. They form a family of about 50 proteins of 8–14 kDa and are divided by struc- tural similarities that classify them in the C, CC, CXC, and CXXXC subfamilies, according to the cysteine configuration (38). The nomenclature of individual chemokines follows this configuration, for example XCL1, CCL1, CXCL1, and CX3CL1, respectively. An online database is available at http://cytokine.medic.kumamoto- u.ac.jp/. There is growing evidence for neuronal expression of chemok- ines, for chemokine receptor expression by neurons, and for elec- trophysiological effects of chemokines on neurons (38–40). Neural activities of chemokines have been implicated in neuronal migra- tion, neuroinflammation, and neuronal excitability. Chemokines, at least a number of them, may thus be considered as potential neuropeptides. The ones with proven neuronal origin have been listed in Table 5.

4.4. Growth Factors Growth factors are proteinaceous signaling molecules that are pro- duced and secreted by cells. They provide extracellular support to proliferation, differentiation and maintenance of other or the same cells in a paracrine or autocrine fashion, respectively. Many growth factors are expressed in the brain, where they act during embryonic development, and during neuronal adaptation and maintenance states of the adult nervous system. Growth factors come in many families, often with a large and diverse family tree. Examples are the neurotrophins, fibroblast growth factors (FGFs), Wnts, bone morphogenic factors (BMPs), (EGF), transforming growth factors alpha and beta (TGFs), and others. Although growth factors are expressed, synthesized, and released by neurons, they are generally not considered as neuropeptides, most growth factors are released by the constitutive secretory path- way and regulated at the level of gene expression. They act through receptor protein kinases. Still, in some cases growth factors may fulfill the criteria of neuropeptides. One example is BDNF, one of the five neurotrophins (41, 42). The primary gene product is prepro-BDNF, a precursor having a 1 What Are Neuropeptides? 33

Table 5 Putative neuropeptides: neurally expressed chemokine family members

Chromosomal Active Gene (gene symbol) localization Precursor peptide(s) Mouse brain expression

C–C motif 17q25.3 CCL2 CCL2 chemokine precursor ligand 2 (CCL2)

C–C motif chemokine 17q12 CCL3 CCL3 ligand 3 (CCL3) precursor

C–C motif 17q12 CCL4 CCL4 chemokine ligand 4 precursor (CCL4)

C–C motif 17q12 CCL5 CCL5 chemokine ligand 5 precursor (CCL5)

C–C motif 2q36.3 CCL20 CCL20 chemokine ligand precursor 20 (CCL20) isoform 1 CCL20 CCL 20 precursor isoform 2 C–C motif chemokine 9p13.3 CCL21 CCL21 ligand 21 (CCL21) precursor

CXC motif 4q21.1 CXCL10 CXCL10 chemokine 10 precursor (small (CXCL10) inducible cytokine B10)

(continued) 34 J.P.H. Burbach

Table 5 (continued)

Chromosomal Active Gene (gene symbol) localization Precursor peptide(s) Mouse brain expression

CXC motif 10q11.21 CXCL12 CXCL12 chemokine ligand precursor, (stromal 12 (CXCL12) SDF-1 cell-derived alpha factor 1, SDF-1a) SDF-1 beta Stromal cell-derived factor 1, SDF-1b SDF-1 Stromal gamma cell-derived factor 1, SDF-1c SDF-1 delta Stromal cell-derived factor 1, SDF-1d CX3C motif 16q21 Fractalkine CX3CL1 chemokine ligand 1 precursor, (fractalkine) (CX3CL1) CX3CL1 precursor

Putative neuropeptides are presented as the processed, biologically active products of the neuropeptide precursors (prepro-peptide) and the encoding gene. The chemokine family members do not all fulfill the criteria of the neuropep- tide definition. Particularly, expression by neurons and/or regulated release has not been established for all members. A database of all chemokines is available at dbCFC (http://cytokine.medic.kumamoto-u.ac.jp/) Genes are abbreviated according to the official gene nomenclature (http://www.gene.ucl.ac.uk/nomenclature/), and the localization on the human genome is given. Brain expression data represent in situ hybridization data of the mouse brain obtained from the Allen Brain Atlas (http://www.brain-map.org). Data have been obtained from http://www. neuropeptides.nl with permission and updated. See http://extras.springer.com/ for the color version of this figure.

signal peptide and cleavage sites for typical prohormone con- vertases. The BDNF gene is widely expressed in neurons, and BDNF is generated by processing and present in dense-cored vesi- cles, and is subject to stimulated release (43, 44). There is a sepa- rate role for pro-BDNF because it acts on a different receptor other than mature BDNF (45). Thus, BDNF fulfills all criteria to be a neuropeptide, albeit it is historically considered to be a growth ­factor. These properties may also appear for other neurotrophins, namely, NGF, NT-3, NT-4/5 (46), and even GDNF (28). 1 What Are Neuropeptides? 35

5. Conclusion

Neuropeptides have emerged as a distinct class of chemical signaling molecules in the 1960s and 1970s, mostly initiated by studies on the endocrine system and central nervous system effects. Over 60 genes exist in the mammalian genome, encoding classical neuro- peptides. Classical neuropeptides fulfill the criteria of synthesis and regulated release by neurons and action on brain receptors. In a broader perspective, peptides usually considered as peptide hor- mones, growth factors, and cytokines may also be considered ­neuropeptides. Together, these peptides help to fine-tune numerous functions of the nervous system.

References

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Neuropeptide Localization in Nonmammalian Vertebrates

Paolo de Girolamo and Carla Lucini

Abstract

Neuropeptides are particularly suited to comparative and evolutionary studies, since they have been highly conserved during evolution. Based on primary amino-acid structure, neuropeptides can be arranged into families and synthesized as multiple molecular variants. They may play different functional roles in differ- ent organs or tissues of the same species, but also among species and classes. Immunohistochemistry (IHC) is powerful technique for localizing the molecular expression of proteins in tissues and cells of dif- ferent classes of vertebrates and has been fully exploited in the study of the mammalian brain. The present chapter provides a detailed description of the protocols routinely used in our laboratory to analyze the presence and distribution of neuropeptides in nonmammalian vertebrate tissues. Single labeling protocols performed by both light and fluorescein IHC, and double labeling protocols using primary antisera raised in different species or in the same species are described. Antibody and method specificity are also discussed in detail.

Key words: Neuropeptides, Immunohistochemistry, PAP, ABC, HRP, Immunofluorescence, Fish, Amphibians, Reptiles, Birds

1. Introduction

Currently, the broader definition of a neuropeptide adopted by The International Neuropeptide Society includes those peptides, independently of whether they are secreted by neurons or nonneu- ral cells, which express the same genetic information and undergo identical processes of synthesis and transport, and bind to similar families of receptors (1). Neuropeptides are expressed in the cen- tral and peripheral nervous system and often coexist with other transmitters, either a classical transmitter or one or several other neuropeptides (2). Furthermore, neuropeptides or closely related molecules can be also expressed and released by endocrine cells to act as circulating of tissutal hormones (3).

Adalberto Merighi (ed.), Neuropeptides: Methods and Protocols, Methods in Molecular Biology, vol. 789, DOI 10.1007/978-1-61779-310-3_2, © Springer Science+Business Media, LLC 2011 37 38 P. de Girolamo and C. Lucini

Based on their primary amino acid structure, neuropeptides can be grouped into families, which are represented along the evo- lutionary scale, from the most simple of coelenterates (4–7). Nevertheless, they are often synthesized as multiple molecular vari- ants and may play different roles in different regions, species and classes. In some cases, these molecules even appear to be identical in structure: e.g. the head activator peptide of Hydra occurs in identical form in mammals (8), and the , originally extracted from mammals, were also shown to be present in the nervous system of the mollusks Mytilus edulis and Lymnaea ­stagnalis (9). On the contrary, as an example of neuropeptides that are not fully identical, but structurally closely related, the ­“–” of the vertebrate classes (10) can be men- tioned. These phenomena can be explained by considering the possible mutagenesis (gene duplication, gene conversion, point mutations) of genes coding for neuropeptides precursors (11). During this process, the neuropeptides diverge structurally, but they may be expected to still show similarities. With regard to the diver- sity, tissue-specific expression of related neuropeptide genes and dif- ferences in neuropeptide precursor processing can be also considered. For this reason, the study of the distribution of neuropeptides in vertebrates and their phenotypic plasticity leads to an understand- ing of the basic cytochemical organization, gives an appreciation of the wide diversity among animal classes, and represents a field par- ticularly suited to comparative and evolutionary studies (12–14). In numerous investigations where antibodies to vertebrate or invertebrates neuropeptides were used, immunohistochemical rela- tions were reported in animals unrelated to the donor species of the neuropeptides that served as immunogens (15–18). So, immu- nohistochemistry (IHC) has proven to be a valuable technique for the accurate mapping of the cellular distribution of neuropeptides in nonmammalian vertebrate tissues. However, when IHC is applied to lower vertebrates’ tissues some additional difficulties with respect to mammalian studies are encountered. The major limitations of the immunohistochemical approach are associated with (1) that neuropeptides often exist in closely related isoforms in a given species and antisera raised against specific synthetic holo- peptides tend to recognize all of the isoforms, (2) cross-reactivity with undefined tissue antigens, and (3) the “detection limit” of the assay, namely, the possibility to discriminate between signal and background in the tissue. For these reasons, in most cases without adequate positive controls (sites within tissue of mammalian spe- cies where known proteins are stored), there is a fat chance to arrive at a determination of the lower detection limit for an immu- nohistochemical assay of nonmammalian tissues. Furthermore, because a neuropeptide may be present at a specific location, but in amounts below the detection limit, it is not appropriate to draw definitive comments on where a neuropeptide is not located in a tissue of a given species. 2 Neuropeptides in Lower Vertebrates 39

In order to provide a starting point for peptide IHC, and try out changeable protocol points, it is fundamental to remember that virtually any laboratory that employs IHC for histological studies has modified the protocols, since every antiserum or tissue requires specific modifications according to specific experimental conditions. Thus, it is important to consult the data sheet describ- ing initial characterization of the antiserum and its application to specific tissues and animal species. For commercially available anti- sera, standard protocols are commonly supplied. Nonetheless, information on nonmammalian species is often missing. The ­present chapter provides a detailed description of the immunohis- tochemical protocols, including double labeling techniques, rou- tinely used in our laboratory to analyze the presence and distribution of neuropeptides in nonmammalian vertebrate tissues.

2. Materials

2.1. Anesthesia/ 1. MS-222: Commercially available as Tricaine Methane Sulfonate Euthanasia (TMS; 3-amino benzoic acid ethyl ester) for use in fishes (19) and amphibians (20) (see Note 1). 2. : For use in reptiles (21) (see Note 2). 3. Isoflurane: Inhalant anesthetic of choice for lizards (see Note 3) and birds (22).

2.2. Fixation 1. Bouin’s Fixative: Mix 75 mL saturated aqueous solution of and Tissue Processing picric acid (see Note 4), 25 mL formalin (~40% aqueous solu- tion of formaldehyde – see Note 5), 5 mL glacial acetic acid (see Note 6). 2. Buffered formalin 10%: Add 100 mL formalin (~40% aqueous solution of formaldehyde) (see Note 5) and 900 mL distilled water. Buffer the mixture, for precise buffering to neutrality

(pH 7.0), with 4 g NaH2PO4⋅H2O and 6.5 g Na2HPO4. 3. Paraformaldehyde 4% in 0.1 M phosphate buffer: Heat 400 mL

distilled H2O to 50–60°C. Add 40°g paraformaldehyde (see Note 5) and ten drops of 5°M NaOH until the solution is clear (to completely dissolve the aldehyde). Cool and add 500°mL

0.2°M phosphate buffer stock solution and enough H2O to 1°L. This fixative can be aliquoted and stored frozen in vials. After thawing, mix the solution well and check the pH. 4. 100, 95, 85, 70, and 50% ethanol. 5. Benzene (see Note 7). 6. Methyl benzoate (see Note 8). 7. Celloidin (see Note 9). 8. Paraffin with a melting point of 58–60°C (see Note 10). 40 P. de Girolamo and C. Lucini

2.3. Immuno- 1. 0.01°M sodium citrate buffer, pH 6: Prepare the solution histochemistry immediately before use. Mix 9°mL of solution A (2.1°g citric acid in 100°mL distilled water) and 41°mL of solution B (2.941°g sodium citrate in 100°mL distilled water) and bring to 500°mL with distilled water. Solutions A and B can be stored at 4°C for many weeks. 2. 0.01°M phosphate-buffered saline (PBS), pH 7.2–7.4: Prepare the solution immediately before use. Mix 31.5°mL solution A

(8.89°g Na2HPO4 × 2H2O in 100°mL distilled water) and

8.5°mL of solution B (6.9°g NaH2PO4 × 2H2O in 100 mL dis- tilled water) and add 16 g NaCl. Bring the volume to 2 L with distilled water. Adjust pH with NaOH or HCl. Solutions A and B can be stored at 4°C for many weeks. Stir and heat solu- tion A before use to dissolve crystals. 3. Hydrogen peroxide 3%: Can be easily purchased in prediluted solutions. Hydrogen peroxide should be stored at 4°C and, since it breaks down quickly when exposed to light, in an opaque container. 4. Damp chamber: It can easily obtained using a Petri dish with a wet paper and five slides at bottom where put the slides with sections. 5. Antibodies: Lyophilized antibodies are stable for two or even more years when stored from −20 to −70°C. Reconstitute with distilled water and store in concentrated form (up to 1/20) in small aliquots at −20°C in a manual defrost freezer for 1 year and more. Avoid freeze–thawing. Store small volumes of work- ing dilutions of the antibody at 4°C, for a maximum of 1 week. Liquid antibodies containing sodium azide as preservative are stable at 4°C for 6 months. Dilute antibodies according to the suppliers’ information. However, it is often useful to deter- mine the optimal concentration by carrying out a series of dilutions. 6. DAB solution: Before use, dissolve 10 mg 3,3¢-diaminobenzidine

(DAB) in 15 mL Tris–HCl, filter and add 1.5 mL H2O2 (see Note 11). DAB is commercially available in prediluted forms, powder, or 10-mg tablets. The latter are the most suitable because often prediluted DAB is at a higher concentration than needed, and can increase background staining, whereas DAB powder is more harmful. 7. 0.05 M Tris–HCl, pH 7.6: Dissolve 6.1 g tris-(hydroxymethyl) aminomethane in 50 mL distilled water, add 37 mL HCl 1 N, and bring to 1 L. Store the solution at 4°C for at maximum 1 week. 8. Fluorochrome-conjugated secondary antibodies: It can be stored in the dark at 4°C. If they are supplied in lyophilized form, they can be rehydrated with distilled water and centrifuged 2 Neuropeptides in Lower Vertebrates 41

if the solution is not clear. Then, add an equal volume of glycerol for a final concentration of 50% and store at −20°C. After the addition of glycerol, the concentration of protein and buffer salts is one half of the original. 9. Neuropeptides (antigens): Lyophilized neuropeptides are stable for 2 years or else more when stored from −20 to −70°C. Reconstitute with distilled water and store at 4°C for at maximum 1 week.

3. Methods

3.1. Anesthesia/ 1. Fish: Place animals in MS-222 anesthetic solution, which is Euthanasia then absorbed through the gills. Animals should be left in the (see Note 12) solution for at least 10 min following cessation of respiratory movement. MS-222, like all fish anesthetic agents, has a dose- dependent effect, which varies with species as well as individuals. A 0.03% solution of MS-222 is effective in zebrafish, although stronger solutions may be required in other species. MS-222 is acidic, and the solution should be kept at pH ­7.0–7.5 by adding sodium bicarbonate (typically 2 g per g of MS-222). 2. Amphibians: Place animals in a dish containing 2–3 mm of MS-222 anesthetic solution at the bottom. The anesthetic is thus absorbed through the ventral skin. A 0.5–2.0 mg/L solu- tion is the MS-222 effective dose in frog. For other amphibian species, inject 50–200 mg/kg MS-222 solution intramuscu- larly or subcutaneously (20, 21). 3. Reptiles: Injectable and inhalant anesthesia are commonly employed for treatment procedures in reptiles (21). For inject- able anesthesia, in lizards administrate intramuscularly or sub- cutaneously 50–150 mg/kg ketamine. For inhalant anesthesia, in lizards administrate, by means of a cone constructed of an appropriate size syringe casing covered by a rubber glove, a 3–4% isoflurane solution for induction and a 1–2% isoflurane solution for maintenance when exposed to the annoying odor. Adequate induction can take anywhere from several minutes to hours. 4. Birds: Induce inhalant anesthesia in chickens by a 0.5–4% ­isoflurane solution and maintain by 1–3% isoflurane solution. The bird’s head is placed into the glass anesthetic mask with a latex rubber forming a firm seal around the feathers. The latex is cut from a surgical glove and secured to the mask using a rubber band. The mask should be placed on an electric blanket heated to 40°C in a draft-free environment (not a fume hood) to minimize loss of body heat from conduction and convection. Feather removal should be kept to a minimum to maintain 42 P. de Girolamo and C. Lucini

thermal insulation. Control of heat loss is particularly critical during anesthesia of very young chicks (12, 13).

3.2. Fixation 1. Fixation: Since all fixatives can influence tissue antigenicity, a and Tissue Processing selection of the fixatives listed in Subheading 2.2 may need to be determined by trial and error. Adequate fixation is obtained by simple immersion of small tissue pieces into the fixative solution. This is the only mode of fixation possible for many tissues of lower vertebrates and when it is necessary to process samples in multiple fixatives (see Note 13). 2. Place tissue samples into an appropriate volume of fixative immediately upon removal or as soon as possible after death. The optimal size for tissues to be fixed for light microscopy is about 2 cm2 and about 3–4 mm in thickness, but these figures vary with tissue density. For example, compared with a com- pact tissue such as a , much larger pieces of spongy tissues such as fish heart can be adequately fixed (see Note 14). 3. Immerse samples in fixative for 2–4 up to 48 h at room tem- perature, depending on sample size, tissue, and subsequent treatment (see Note 15). 4. Wash specimens fixed in Bouin’s solution in 70% alcohol to precipitate soluble picrates and until all the yellow color is removed from the tissue (see Note 16). Rinse specimens fixed in buffered formalin 10% and paraformaldehyde must be in PBS (several changes) at room temperature for 2 h overnight at 4°C.

5. Rinse the tissue for 2 × 20 min in 0.137 M NaPO4 buffer, keeping the vials at 4°C during rinses. 6. Dehydrate in a graded ethanol–water series: 50, 70, 85, and 95% for 10–20 min each. Then, place in 100% ethanol for two changes from 20 min to 1 h each. All solutions should be at 4°C. 7. Clear samples in methyl benzoate–celloidin solution (celloidin 1% in methyl benzoate) for 24 h (see Notes 17 and 18). 8. Immerse in pure benzene for 1–2 h. 9. Transfer to a mixture containing of equal parts of paraffin wax and pure benzene in the vacuum for 1 h. 10. Immerse in two changes of pure paraffin wax from 15 min to 3 h each, depending upon the thickness and the nature of the tissue (see Note 19). Orientate and embed tissue in fresh wax using warm molds and embedding cassettes. 11. Cut thin sections (3–10 mm, 5 mm is commonly used) by using a sliding microtome. Trim the block until the cutting surface is optimal, transfer paraffin ribbons with a wet paintbrush to egg-albumen-coated slides. 2 Neuropeptides in Lower Vertebrates 43

12. Place the slides on a warming tray and add distilled water to float the paraffin sections and allow then to expand and straighten out. Excess water is thus removed and sections will adhere to the slides. 13. Allow the slides to dry in an oven at 35–37°C overnight.

3.3. Immuno- Single labeling (Fig. 1) can be performed by both light and fluo- histochemistry rescence IHC. In nonmammalian vertebrates single neuropeptide labeling is preferably detected by light IHC, by using peroxidase– 3.3.1. PAP, ABC, and HRP anti-peroxidase (PAP), avidin-biotin (ABC), and polymer horse- Protocols: Single Labeling radish-peroxidase (HRP) methods (see Note 20) that offer higher sensitivity than immunofluorescence. 1. Remove wax in xylene and bring the sections to water through graded alcohols. 2. Retrieve antigens by microwave oven treatment (23) on the sec- tions immersed in 0.01 M sodium citrate buffer (see Note 21). 3. Rinse in PBS for 5 min at room temperature. 4. Block endogenous peroxidase activity by immersion of the sec- tions at room temperature for 30 min in a solution of 3% hydrogen peroxide (for PAP and polymer-HRP methods). Block endogenous biotin by immersion of the sections in 0.05% avidin solution for 15 min followed by brief rinse in PBS and then incubate the sections in 0.005% biotin solution for 15 min (for ABC method). All these procedure are carried out at room temperature. 5. Rinse in PBS for 15 min. Dry the slides except for the area of section and place in a damp chamber. 6. Incubate for 30 min with 1:5 normal serum belonging to the species in which the secondary antibody was raised to block aspecific background staining. 7. Do not rinse. Draw off serum with a tissue, except for the area of the section and place in a damp chamber. 8. Dilute primary antibody in PBS to the appropriate concentra- tion (see Note 22). Apply the primary antibody in a volume of 50–100 mL (depending on the area of sections). 9. Incubate the sections overnight at 4°C or for shorter periods at room temperature or at 37°C (see Notes 23 and 24). 10. Rinse three times in PBS, 5 min each. Dry the slides except for the area of section and place in a damp chamber. 11. Dilute the secondary antibody in PBS to appropriate concen- tration (1:50–1:200). This antibody is unconjugated for PAP, conjugated to biotin for ABC and to a polymer for HRP. 12. Incubate the sections for 30 min up to 2 h at room temperature or for a shorter time at 37°C (see Note 25). 44 P. de Girolamo and C. Lucini

Fig. 1. Light microscopic ICC of peptides in nonmammalian vertebrates. (a–c) Beacon immunoreactivity within the Gallus domesticus hypothalamus – PAP method: (a) Immunoreactive neurons (arrows) in the dorsal (d), medial (m) and ventral (v) part of nucleus magnocellularis preopticus (MPO); (b) Innervation of the internal zone (i.z.) and external zone (e.z.) of the median eminence; (c) Dense immunoreactive fiber arrows( ) arrangement in the median eminence (3V, third ventricle); 2 Neuropeptides in Lower Vertebrates 45

13. Rinse three times in PBS, 5 min each. Dry the slides except for the area of section. 14. Dilute the PAP or ABC complex in PBS to appropriate con- centration. Incubate the sections for 30 min up to 2 h at room temperature or for a shorter time at 37°C. For the sections treated with the method HRP skip this step and proceed directly to step 16. 15. Rinse three times in PBS, 5 min each. Dry the slides except for the area of section. 16. Put a drop of DAB solution on the section in a dark chamber at room temperature. Monitor the reaction at low magnifica- tion under a microscope. The reaction should be complete in 10–20 min, but it can be prolonged up to 1 h (see Note 26). 17. Dehydrate the slides into progressive alcohols and mount with a commercially available medium (see Note 27). The sections stained with DAB are very stable and can be stored for many years.

3.3.2. Immunofluorescence To perform this method (Fig. 2), both primary antisera against the Protocols: Double Labeling two target neuropeptides, the related secondary antisera and nor- Using Primary Antisera mal sera can be mixed (see Note 28). All steps are carried out at Raised in Different Species room temperature unless otherwise stated. 1. Remove wax in xylene and bring the sections to water through graded alcohols. 2. Retrieve antigen by microwave oven treatment on the sections immersed in 0.01 M sodium citrate buffer (see Note 21). 3. Rinse in PBS for 5 min. Block background by incubation for 30 min with 1:5 normal sera belonging to the species in which the secondary antibodies were raised (see Note 29). 4. Do not rinse. Draw off the sera with a tissue, except for the area of the section and place in a damp chamber. 5. Apply a mixture of primary antibodies diluted in PBS to the appropriate concentration in a volume of 50–100 mL (depend- ing on the section area). Incubate the sections for 24–48 h.

Fig. 1. (continued) (d) Neurotensin immunoreactivity in endocrine cells (arrows) of the domestic duck antrum, PAP method; (e) Ghrelin-immunostained cells (arrows) in the mucosal layer at the base of glandular lobuli in the Gallus domesticus proventriculus, PAP method; (f–g) Vasoactive Intestinal Peptide in neurons (arrows) of the muscular of a 7-day-old duck embryo at low (f ) and high (g) magnification, PAP method; h( ) Vasoactive Intestinal Peptide positive neurons and fibers in the myenteric plexus ganglion of the muscular stomach of the domestic duck at hatching, PAP method; i( ) Gastrin/ CCK immunoreactive cells (arrows) in the antrum of the domestic duck at hatching, PAP method; (l) Cells displaying apical Orexin A immunoreactivity (arrows) in the stomach of Xenopus laevis, ABC method; (m) Orexin A immunoreactive cell (arrow ) in the proximal region of the Lacerta viridis small intestine, ABC method; (n) Orexin B immunoreactive cells (arrows) in the proximal region of the Lacerta viridis small intestine, HRP method; (o) Immunohistochemical detection of Vasoactive Intestinal Peptide in the taste buds of Carassius auratus gill arch, PAP method. Scale bars: (c, f ) = 50 mm; (e, h, i, l) = 25 mm; (a, b, d, g, m, n) = 10 mm; (o) = 5 mm. 46 P. de Girolamo and C. Lucini

Fig. 2. Single fluorescence immunohistochemistry.a ( , b) Somatostatin immunoreactivity in mixed (a) and large (b) ­pancreatic islet cells of the domestic duck, at low (a) and high (b) magnification. In b( ), somatostatin cells are lining large islet capillaries; (c) An individual PACAP immunoreactive nerve cell body with a prominent process is present at the base of the raker cushion in the oral side of the gill arch of Carassius auratus; (d) Vasoactive Intestinal Peptide immunore- active nerve cell bodies with long processes in the connective tissue of the oral side of the gill arch of Carassius auratus. Scale bars: (a) = 20 mm; (b, c, d) = 10 mm.

6. Rinse three times in PBS, 5 min each. Dry the slides except for the area of section and place in a damp chamber. 7. Apply a mixture of secondary antibodies (against the IgGs of the species in which the primary antibodies were raised) diluted in PBS to appropriate concentration (1:25–1:50) (see Note 30). These antibodies are conjugated to fluorescein isothiocyanate (FITC) and to tetramethyl rhodamine isothiocyanate (TRITC) (24) (see Notes 31 and 32). Incubate the sections for 2 h in the dark. Incubation can be carried out also at 37°C. 8. Rinse three times in PBS, 5 min each. Dry the slides except for the area of section. 9. Mount the slides with 1:1 glycerin and PBS or a commercially available aqueous medium, fixing coverslips with four glue drops at angles. 10. Observe and photograph slides as soon as possible or store in the dark at 4°C for few weeks or at −20°C for few months (see Note 33). 2 Neuropeptides in Lower Vertebrates 47

3.3.3. Immunofluorescence The simultaneous visualization of two antigens is also difficult for Protocols: Double problems related to the availability of commercial antibodies raised Immunofluorescence in different species. This problem is particularly true in nonmam- Labeling Using Primary malian vertebrates because, usually, there are not mouse mono- Antisera Raised in the clonal antibodies available against nonmammalian antigens, thus Same Species eliminating the mouse from the panels of antibody donor species. Double immunofluorescence labeling using primary antisera raised in the same species consists of two sequential labeling techniques, using monovalent F(ab)-fluorochrome conjugates as secondary reagents (see Note 34) to avoid cross talk of subsequent antibody probes. 1. Repeat steps 1–4 of Subheading 3.3.2. 2. Apply the first primary antibody diluted in PBS to the appro- priate concentration in a volume of 50–100 mL (depending on the section area). Incubate the sections for 24–48 h (see Notes 30 and 35). 3. Rinse three times in PBS, 5 min each. Dry the slides except for the area of section and place in a damp chamber. 4. Apply the TRITC-conjugated F(ab) secondary antibody (against the species in which the primary antibodies of the first labeling was raised) diluted in PBS to appropriate concentra- tion (1:25–1:50) (see Notes 36 and 37). Incubate the sections for 2 h in the dark. Incubation can also be carried out at 37°C. 5. Rinse three times in PBS, 5 min each. Dry slides except for area of section. 6. Block background by incubation for 30 min with 1:5 normal serum belonging to the species in which the secondary anti- body of the second labeling was raised. 7. Do not rinse. Draw off the mixture with a tissue, except for the area of the section and place in a damp chamber. 8. Apply the second primary antibody diluted in PBS to the appropriate concentration in a volume of 50–100 mL (depend- ing on the section area). Incubate sections for 24–48 h ­ (see Note 35). 9. Rinse three times in PBS, 5 min each. Dry slides except for area of section and place in a damp chamber. 10. Apply FITC-conjugated IgG secondary antibody (against the species in which was raised the primary antibody of the ­second labeling) diluted in PBS to appropriate concentration ­(1:25–1:50) (see Note 32). Incubate the sections for 2 h in the dark. Incubation can also be carried out at 37°C. 11. Repeat steps 9–10 of Subheading 3.3.2 (see Note 38). 48 P. de Girolamo and C. Lucini

3.3.4. Control Experiments Each single experiment should be run positive as well as negative for Single Immunolabeling controls to test that the detection system works and that the primary antibody used is responsible for generation of the immunolabeling ((25, 26) – see Note 39). 1. Positive controls using known tissues: Carry on parallel experi- ments by using tissue(s) in which the molecule(s) of interest is(are) known to be expressed. These controls test a protocol or procedure and make sure that it works. If positive control tissue(s) display(s) negative staining, the procedure needs to be checked until a good positive staining is obtained (see Note 40). 2. Negative controls – Antibody replacement: Substitute the ­primary antibody, the secondary antibody or the PAP/ABC complex in repeated trials with buffer (PBS) or normal serum to check for specificity of the immunoreactivity. During these controls, all the other steps must be kept the same. If any stain- ing results, then the antibodies are binding nonspecifically to different sites on the tissue. These controls are easy to achieve and can be used routinely. 3. Negative controls – Absorption of the primary antibody: Incubate (prior to its use) the primary antibody with an excess of its corresponding or a related purified antigen at the same temperature and for the same time of the tested primary anti- body reaction on experimental tissue (see Notes 41 and 42). In order to minimize methodological variations, process at least two adjacent sections, using absorbed and unabsorbed anti- bodies respectively.

3.3.5. Control Experiments Double immunofluorescence labeling needs, besides the controls for Double Immunolabeling described in Subheading 3.3.4, additional specific controls to exclude cross-reactions of the secondary antibodies. 1. Controls for double labeling experiments using primary anti- sera raised in different species: Incubate the sections with one of the two primary antisera and with the mismatched second- ary antibody. This control should give a negative reaction, if the secondary antibody of one line is specific and does not recognize the primary antiserum of the other line. 2. Controls for double labeling experiments using primary anti- sera raised in the same species: Test the specificity of the immu- noreactivity in double labeling with F(ab) fragments by omitting the primary antibody in the second staining, and by the two assays described below (27). 3. Assay 1: After the first staining, incubate the sections for 48 h at room temperature with a PAP rabbit complex, which serves as the second-staining primary antibody to prove the monovalence of the F(ab) fragment. After DAB treatment 2 Neuropeptides in Lower Vertebrates 49

(see Subheading 3.3.1, step 16), no immunolabeling should be observed at the light microscope. 4. Assay 2: After the first training, incubate the sections for 24 h at 4°C with a peroxidase-coupled donkey anti-rabbit F(ab) fragment, which serves as the second-staining secondary anti- body, to prove the complete F(ab) saturation of rabbit IgG epitopes. After DAB treatment (see Subheading 3.3.1, step 16), no light immunolabeling should be observed.

4. Notes

1. Tricaine is a benzoic acid derivative. Therefore, a solution of MS-222 that is not buffered to neutral pH is acidic and poorly absorbed, resulting in a prolonged induction time. The stock solution should be stored in a dark brown bottle, and refriger- ated or frozen (if possible). The solution should be replaced monthly and any time a brown color is observed. All bottles should be labeled and be provided the expiration date. Warning: This compound is a potent carcinogen, so precautions must be taken when handling. 2. Perhaps it is the most popular reptile anesthetic agent. As a dis- sociative psychotropic agent, this drug is effective and has a high level of safety in the reptile patient. 3. It is widely used in reptiles and birds. Isoflurane tends to have a high margin of safety and displays extremely rapid induction and recovery times. Since it is very lipid soluble, increasing the temperature of the animal may release more isoflurane and result in a very prolonged recovery. This anesthetic can be used for box induction if system has calibrated vaporizer, and has been used in bell jars following brief exposure. Of all inhalants, this is the least metabolized and is preferable for birds and high-risk patients. Isoflurane should only be handled in fume hood. If used in bell jar, use as small of diameter jar as possible and ensure a tight-fitting lid. Keep exposure times extremely brief, as death can occur rapidly. 4. Picric acid crystals are highly explosive when dry. Therefore, picric acid is best handled as a commercially prepared saturated aqueous solution. 5. Formaldehyde is a colorless gas with a strong, pungent odor. It is commonly used in liquid form as a 40% aqueous solution known as formalin and in solid form as a white powder called paraformaldehyde. Because of its volatility, both formalin and paraformaldehyde will readily give off irritating formaldehyde vapor with a strong odor. Warning: Formaldehyde is a sensitizer 50 P. de Girolamo and C. Lucini

by skin and respiratory contact, and toxic by ingestion and inhalation. Its main targets are organs of the respiratory system. Formaldehyde is a corrosive and carcinogenic agent and must be handled in appropriate fume hoods. 6. This dry salt mixture can be prepared in advance and carried into the field in plastic containers. Warning: Glacial acetic acid is corrosive and must be handled with appropriate care. It causes skin burns, permanent eye damage, and irritation to mucous membranes. 7. Warning: Benzene is a very flammable cancer-causing agent. The fluid, either pure or in solution, constitutes a fire risk. If benzene must be used in an experiment, it should be han- dled at all stages in a fume cupboard. Wear safety glasses and use protective gloves. 8. Methyl benzoate is an aromatic substance with good dehydrat- ing and alcohol detracting capacities. It is a colorless flammable solvent miscible with most organic solvents and with paraffin wax. 9. Warning: This product is handled safely when wet with water and is classified as an explosive when dry. If ignited after drying, it will burn very rapidly producing great amounts of heat and toxic smoke. Store under wet conditions until ready for use. Store in a cool, ventilated area, away from heat sources. 10. Warning: Keep paraffin away from heat and from sources of ignition. Empty containers pose a fire risk, evaporate the resi- due under a fume hood. Ground all equipment containing material. Keep away from incompatibles such as oxidizing agents. Keep container tightly closed, in a cool, well-ventilated area. 11. Warning: DAB is suspected carcinogen. Appropriate care should be exercised when using this reagent including gloves, eye protection, lab coats, and good laboratory procedures. Care for tips on handling and disposing of DAB solution and plasticware that has been in contact with DAB. 12. This is a reference of suggested and possible doses of anesthet- ics for a variety of lower vertebrate species. However, there is no claim made that this information is complete, original, or unique. When using agents, the concept of preemp- tive analgesia should be followed. That is, relieving the poten- tial pain before the pain is felt. To do so will result in a quicker, less stressful recovery of the patient. For other potential sources of information on anesthetic/analgesic or euthanasic doses, agents, and techniques, consult the veterinary and clinical animal care staff at facility, for advice during protocol preparation and during the conduct of the study. 2 Neuropeptides in Lower Vertebrates 51

13. Fixation is one of the most critical steps in immunostaining of lower vertebrates’ tissues. An ideal fixative should yield the fol- lowing: a good preservation of the cells and tissues in a life-like manner and, at the same time, preserve antigenicity, allow anti- body access to the antigen, harden naturally soft tissue which allow for easy manipulation during subsequent processing and sectioning. Fixation is also necessary to protect the sample from the deleterious effects of the immunostaining process. Chemical fixatives preserve tissue by artifactual diffusion of cell compo- nents, denaturing proteins through coagulation, cross-linking (e.g., formaldehyde), or both (e.g., mercuric formalin). 14. It is possible to evaluate several histology protocols for whole small fish specimens (e.g. Danio rerio, Nothobranchius furzeri, and Oryzias latipes). High-quality sections can be difficult to produce because small fish are composed of a variety of tissues that range in consistency from soft visceral organs to hard bones and scales. In this case, the best fixative is the Bouin’s solution that balances the hardening and minor shrinkage properties of formaldehyde by the softening and shrinking action of picric acid. 15. Storage in fixative for too long period generally causes loss of antigenicity, whereas overly short fixation times lead to inade- quate retention of antigen and suboptimal tissue preservation. If the tissues are fixed longer than 24 h, they tend to become brittle and difficult to section. If long fixation times are required, it is advisable to replace with fresh fixative each day. 16. Failure to remove the Bouin’s solution can result in inadequate staining. 17. Since paraffin is immiscible with water, the main constituent of tissue, samples need to be dehydrated by progressively more concentrated ethanol baths. This is followed by a clearing agent to remove the ethanol. Finally, molten paraffin wax infiltrates the sample and replaces the clearing agent. 18. IHC can be performed on cryostatic sections of differently embedded tissues and also on whole organs or embryos. However, in our experience, sections of routinely paraffin embedded tissues give no problems for neuropeptide detec- tion in lower vertebrates and, on the contrary, give an excellent definition of tissue morphology. 19. Tissues should be kept in the vacuum evaporator just long enough for the paraffin wax to fully infiltrate them. Prolonged heating in the oven causes shrinking and hardening of the tissue, rendering sections difficult to cut. Furthermore, many anti- gens are easily destroyed by high temperature and, therefore, prolonged exposure to molten wax. Infiltrate tissue with wax at the lowest possible temperature to keep it molten. Specimens 52 P. de Girolamo and C. Lucini

of brain and spinal cord about 5–10 mm thick, skin such as embryos need at least three changes of pure paraffin wax for a total time of about 2–6 h, whereas organs such as spleen, con- taining a large amount of blood, muscle fibrous tissue, require no longer than a total of 2–3 h in the paraffin baths. 20. PAP method, the more ancient of the three methods, is some- what less sensible of ABC, but it does not recognize biotin which is widely diffused and can give false positive or back- ground staining. The polymer-HRP method has highest sensi- tivity, but is more expensive. In nonmammalian vertebrate tissues, the distribution of endogenous peroxidases, biotin and the concentration of investigated antigen are generally not known; thus, it is always preferable to compare all the three methods. 21. Fixation can influence tissue antigenicity, but several studies have suggested that increased heating may reverse the effects of longer fixation (23). Because studies on nonmammalian vertebrates often involve many different specimens, it may be difficult to standardize the optimal time of fixation. Therefore, different heating times and pHs of sodium citrate buffer could be tested. However, using the solution at pH 6.0 and the microwave oven treatment for 10 min at 750 W is a good stan- dard to start with for most specimens. 22. The primary antibody concentration must be carefully deter- mined because it is an essential requisite to achieve a good immunoreaction. The concentrations suggested by suppliers are often too much higher and have to be gradually tested at progressive dilutions. Their final dilution depends on neuro- peptide concentrations in tissues and the sensitivity of the method. 23. Temperature and time of incubation are, after the dilution of the antibody, two other essential requisites for a good immu- noreaction. Temperature and time are in inverse relationship to each other. Usually, the best reactions are achieved by incu- bating slides overnight at 4°C because low temperatures decrease background. 24. Commercially available antibodies are generally directed against human or, at most, mouse antigens. Theoretically, these anti- bodies in nonmammalian vertebrate tissues could not recog- nize their target neuropeptides and might cross-react with other unknown peptides. This contingency is quite infrequent because neuropeptides are usually well conserved through ver- tebrates in their aminoacidic sequence. However, to obtain a good reaction and minimal cross-reaction staining, it is best practice to choose those primary antibodies which are directed against epitopes showing the most conservative sequence. 2 Neuropeptides in Lower Vertebrates 53

Many free programs are suitable for comparing peptides of different species. To this purpose we often employ Mega program (http://www.megasoftware.net/). Another control to check for the actual presence of a ­neuropeptide in nonmammalian tissue highly similar to a mam- malian neuropeptide is to treat the experimental nonmam- malian sections with several different antibodies that are directed against the same neuropeptide to show that the same structure(s) is(are) labeled. 25. Secondary antibody concentration, time, and temperature of incubation have to be tested for each primary antibody and tissue employed. For PAP method, the secondary antibody has to be clearly in excess to leave an antigenic site free to bind to PAP complex. 26. Time incubation in DAB solution and concentration of primary antibody are in inverse proportion. To avoid background and then misunderstanding of positive stainings, it is better diluting the primary antibody to obtain a slow reaction of DAB solu- tion. Warning: DAB is suspected carcinogen (see Note 11). 27. Warning: Histological mounting medium is flammable and hazardous to health when inhaled. Thus, mounting the slides must be conducted in a fume hood. 28. The visualization of two antigens is quite difficult for problems related to antibody penetration. Thus, the fluorescence tech- nique, even if less sensitive of light techniques, is more suitable because the secondary fluorescent dye-liked antibody is a com- plex sterically smaller than secondary and tertiary layer mole- cules of light techniques. 29. To obtain the final optimal dilution for the antibody mixture, remember that each antibody solution represents a dilution buffer for the other antibody. 30. Fluorescence needs more concentrated primary antibodies than light immunohistochemistry (the ratio is about one to ten). To improve the sensitivity of immunofluorescence, it might be better to increase the time and the temperature of incubation with primary antibody. 31. FITC is the form of fluorescein used for conjugation to anti- bodies. Fluorescein conjugates absorb light maximally at 492 nm and fluoresce maximally at 520 nm. FITC is a widely used fluorochrome due to its long history. The major disad- vantage of fluorescein is its rapid photobleaching (fading). TRITC conjugates absorb light maximally at 550 nm and fluo- resce at 570 nm. Better color separation could be achieved by using Texas Red, but it has been reported that use of Texas Red may lead to higher background staining (24). 54 P. de Girolamo and C. Lucini

32. Secondary antibodies for multiple labeling must be affinity-purified and it is mandatory that they do not recognize one another, or other primary antibodies used in the assay system or the endog- enous immunoglobulins that are present in tissues under investigation. 33. Attention: Steps 7–10 in Subheading 3.3.2 must be performed in the dark to avoid any bleaching of fluorochromes. 34. Monovalent F(ab) fragments of affinity-purified, secondary antibodies cover the surface of immunoglobulins for double labeling primary antibodies from the same host species. Thus, it is necessary to employ a high concentration of conjugated F(ab) to achieve effective blocking of the first primary antibody. 35. Best results are obtained when the primary antibody with the highest affinity is used in the second stage. When both primary antibodies have approximately the same affinity, better results are obtained when the primary antibody raised against the less abundant antigen is applied first. 36. Because monovalent F(ab) fragment must be applied in excess (see Note 34), the right concentrations have to be deducted by a series of controls (see Subheading 3.3.5). 37. In the first labeling, it is better to avoid using a FITC-conjugated F(ab) secondary antibody because this fluorochrome could fade during the second labeling reaction. 38. Attention: Steps 2–11 in Subheading 3.3.3 must be performed in the dark to avoid any bleaching of fluorochromes. 39. Specificity is the most important and difficult criterion of valid- ity to define in immunocytochemical stainings of nonmamma- lian vertebrate tissues and requires two independent sets of validation: specificity of the method and specificity of the anti- body. Thus, the requirements for a valid immunohistochemical method are as follows: (1) it should include a careful design of experiments with preliminary standardization of all steps of the control tests (e.g., incubation time, antibody dilution, the sec- ond and following antibodies used), which should be identical to those used for the immunohistochemical localization proce- dure; (2) handling of the antigens in the test system should be identical to those in the tissue section (e.g., specificity is often dependent on the fixative used); (3) it should include the use of other significant and appropriate information to support the validity of the localization (e.g., reliable positive and negative controls) and the correct interpretation of the results. 40. If the positive control tissue gives positive staining and the nonmammalian tissue gives negative reaction, it does not nec- essarily mean that in the latter the searched neuropeptide is lacking, since it may be only present in lower concentration. 2 Neuropeptides in Lower Vertebrates 55

Thus, in these occurrences further reactions employing more concentrated antibodies or more sensitive detection systems (see Note 20) are needed. 41. The absorption of primary antibody with its corresponding antigen should give a negative staining if the tested primary antiserum is specific and its antigenic sites were saturated by a sufficient amount of antigen. The absorption of primary anti- serum with related antigens should not influence the positive reaction if the tested primary antiserum is specific and does not cross-react with related antigens. 42. The amounts of antibody and antigen that have to be mixed will vary depending upon the titer and avidity of the antibody and upon the stability of the antigen. However, it is better to absorb the primary antibody at its maximum dilution that gives a good staining. The amount of related antigens (generally up to 100 mg/mL) is much more than the specific antigen (gener- ally up to 50 mg/mL).

References

1. Kastin, A. J. (2000) What is a neuropeptide? 8. Bodenmüller, H., and Schaller, H. C. (1981) Trends Neurosci. 23, 113–114. Conserved amino acids sequence of a neuro- 2. Salio, C., Lossi, L., Ferrini, F., and Merighi, A. peptide, the head activator, from the coelenter- (2006) Neuropeptides as synaptic transmitters. ates to humans. Nature 293, 579–580. Cell Tissue Res. 326, 583–598. 9. Leung, M. K., Boer, H. H., van Minnen, J., 3. Solcia, E., Usellini, L., Buffa, R., et al (1998) lundy, J. and Stefano, G. B. (1990) Evidence Endocrine cells producing regulatory peptides. for an enkephalinergic system in the nervous In: Polak JM, (ed.) Regulatory peptides. system of the pond snail Lymnea stagnalis. Experientia supplementum, vol. 56. Basel/ Brain Res. 531, 66–71. Boston/Berlin: Birkha user Verlag; 220–246. 10. Hadley, M. E. (1988) Endocrinology, Prentice- 4. Holmgren, S. and Jensen, J. (1994) Comparative Hall, Inglewood Cliffs, NJ, USA. aspects on the biochemical identity of neu- 11. Wilson, A. C. (1985) The molecular basis of rotransmitters of autonomic neurons. In: evolution. Sci. Am. 253, 164–173. Burnstock G, series ed. The autonomic nervous 12. Fasolo, A., and Vaudry, H. (1992) system. Nilsson S, Holmgren S, volume eds. Neuropeptides in evolution. Ann. Sci. Nat. 12, Comparative physiology and evolution of the 124–136. autonomic nervous system. London: Gordon and Breach Science Publishers; 69–95. 13. Holmgren, S. and Jensen, J. (2001) Evolution 5. Thorndyke, M. C. (1986) Immunocytochemistry of vertebrate neuropeptides. Brain Res. Bull. and evolutionary studies with particular refer- 55, 723–735. ence to peptides. In: Polak JM and Noorden S 14. Conlon, J.M. and Larhammar, D. (2005) The Van (eds.) Immunocytochemistry, J. Wright and evolution of neuroendocrine peptides. Gen. Sons, Bristol; 300–327. Comp. Endocrinol. 142, 53–59. 6. Thorndyke, M. C. and Goldsworthy, G.J. 15. Bjenning, C. and Holmgren, S. (1988) (1988) in invertebrates. Neuropeptides in the fish gut. Histochemistry Cambridge: Cambridge University Press. 88, 155–163. 7. Joosse, J. What is special about peptides as neu- 16. Conway, K. M. and Gainer, H. (1987) ronal messengers? (1988) In: Thorndyke MC; Immunocytochemical studies of , Goldsworthy GJ, eds. Neurohormones in inver­ mesotocin, and the neurophysins in the tebrates. Cambridge: Cambridge University Xenopus hypothalamo-neurohypophysial sys- Press; 1–3. tem. J. Comp. Neurol. 264, 494–508. 56 P. de Girolamo and C. Lucini

17. Schot, L. P. C., Boer, H. H. and Montagne- 23. Taylor, C. R., Shi, S. R., Chen, C., Young, L., Wajer, C. (1984) Characterization of multiple Yang, C., and Cote, R, J,. (1996) Comparative immunoreactive neurons in the central nervous study of antigen retrieval heating methods: system of the pond snail Lymnea stagnalis. microwave, microwave and pressure cooker, Neuroscience 81, 373–378. autoclave, and steamer. Biotech. Histochem. 71, 18. Veenstra, J. A., Romberg-Privee, H. M., 263–270. Schooneveld, H. and Polak, J. M. (1985) 24. Wessendorf, M. W., and Brelje, T. C. (1992) Immunocytochemical localization of peptider- Which fluorophore is brightest? A comparison gic neurons and neurosecretory cells in the of the staining obtained using fluorescein, neuroendocrine system of the Colorado potato tetramethylrhodamine, lissamine rhodamine, beetle with antisera to vertebrate regulatory Texas red, and cyanine 3.18. Histochemistry. peptides. Histochemistry 82, 9–18. 98, 81–85. 19. Nusslein-Volhard, C. and Dahm, R. (2002) 25. Burrt, R. W. (2000) Specificity controls for Zebrafish: a practical approach. Oxford immunocytochemical methods. J. Histochem. University Press. Cytochem. 48, 163–165. 20. Wright, K. M. and Whitaker, B. R. (2001) 26. Petrusz, P. (1983) Essential requirements for the Amphibian medicine and captive husbandry. validity of immunocytochemical staining proce- Krieger Publishing Company, Malabar, dures J. Histochem. Cytochem. 31, 177–179. USA. 27. Negoescu, A., Labat-Moleur, F., Lorimer, P., 21. Fish, R. E., Danneman, P.J., Brown, M., and Lamarcq, L., Guillermet, C., Chambaz, E., and Karas, A. (2008) Anesthesia and analgesia in Brambilla, E. (1994) F(ab) secondary antibod- laboratory animals. Academic Press. ies: a general method for double immunolabel- 22. Abou-Madi, N. (2001) Avian anesthesia. Vet. ing with primary antisera from the same species. Clinics of North America: Exotic Animal Efficiency control by chemiluminescence. Practice 4, 147–167. J. Histochem. Cytochem. 42, 433–437. Chapter 3

Combined Light and Electron Microscopic Visualization of Neuropeptides and Their Receptors in Central Neurons

Chiara Salio, Laura Lossi, and Adalberto Merighi

Abstract

The study of neuronal connections and neuron to neuron (or neuron to glia) communication is of fundamental importance in understanding brain structure and function. Therefore, ultrastructural investigation by the use of immunocytochemical techniques is a really precious tool to obtain an exact map of the localization of neurotransmitters (neuropeptides) and their receptors at different types of synapses. However, in immu- nocytochemical procedures one has always to search for the optimal compromise between structural pres- ervation and retention of antigenicity. This is often made difficult by the need to localize not only small transmitter molecules, as in the case of transmitter amino acids and neuropeptides, but also their specific receptors that are usually large proteins very sensitive to fixation procedures. We describe here a preembedding procedure employing the Fluoronanogold™ reagent, a probe con- sisting of fluorescein-tagged antibodies conjugated with ultrasmall gold particles that can be made visible under the electron microscope by a gold intensification procedure. This technique permits correlative fluo- rescence and electron microscopy observations, providing a very useful tool for the study of neuronal connectivity. Moreover, the Fluoronanogold™ procedure can be combined with conventional postem- bedding immunogold techniques in multiple labeling studies.

Key words: Neurons, Electron microscopy, Fluoronanogold™, Neuropeptides, Somatostatin, SSTR2a receptors, GDNF, GFRa1 receptors, BDNF, fl-trkB receptors

1. Introduction

The ultrastructural localization of neuropeptides and their receptors at synapses (and nonsynaptic sites) is of great importance to under- stand the mechanism(s) of action of this family of neurotransmitter molecules and the organization of neuronal networks. Nevertheless, our knowledge of the role of neuropeptides in different areas of the brain and spinal cord is still largely incomplete, since a thorough

Adalberto Merighi (ed.), Neuropeptides: Methods and Protocols, Methods in Molecular Biology, vol. 789, DOI 10.1007/978-1-61779-310-3_3, © Springer Science+Business Media, LLC 2011 57 58 C. Salio et al.

anatomical and functional description of neuronal circuitry is often lacking, such is the precise localization of messenger molecules and their receptors at synapses. Different immunocytochemistry (ICC) labeling methods have been developed starting from the seventies of the last century to detect neuropeptides (and other neurotransmitters) at the trans- mission electron microscope (TEM) level (see for example (1–4)). Initial work in the field was mainly carried out by a preembedding approach with adaptation of the widely employed peroxidase– anti-peroxidase (PAP) and/or avidin-biotin complex (ABC) meth- ods for light microscopy for use in ultrastructural studies. In parallel, the development of colloidal gold probes opened the way to the use of postembedding procedures that allowed a better pres- ervation of ultrastructure in labeled samples but were associated with an often too drastic reduction of antigenicity that seriously limited their suitability for detection of large receptor molecules (5). More recently, the preembedding Fluoronanogold™ technique (Fig. 1) that we describe here has emerged as a new approach for the detection of a wide number of neurotransmitters and receptors resulting as an excellent method that combines several advantages of the pre and postembedding procedures and allows for a precise correlation of anatomical distributional observations at the light microscope with subcellular localization TEM studies (6–10). As an example of the possibilities that have been opened to combined histological/ultrastructural investigations of central neurons by the use of the Fluoronanogold™ technique, we describe here the localization of the somatostatin (SST) receptor 2 (SSTR2) and the glial-derived neurotrophic factor (GDNF)-family receptor a1 (GFRa1) at central synapses. Moreover, the Fluoronanogold™ procedure can be combined with conventional postembedding immunogold (4) in multiple labeling studies, as shown here by the concurrent visualization of the high-affinity brain-derived neurotrophic factor (BDNF) tyrosine kinase receptor (trkB) with neuropeptides such as calcitonin-gene related peptide (CGRP) or substance P (SP). All these peptide molecules have multiple roles in sensory pathways, acting via specific high-affinity receptors (11–18). Namely, SSTR2, one of the five SST G-protein coupled receptors (SSTR1-SSTR5) (19, 20), is expressed in spinal cord (21) in two different isoforms, SSTR2a and SSTR2b (22, 23), and seems to mediate the effect of SST in the control of nociceptive informa- tion. GFRa1 and RET are the two receptors forming the multi- receptor complex that mediates the biological effects of GDNF. GFRa1 is the high-affinity ligand-binding component of the complex, anchored to the outer plasma membrane by a glycosyl phosphatidylinositol link (24–27). RET is the transmembrane tyrosine kinase receptor, acting as the signal transducing compo- nent (24–30). 3 Fluoronanogold™ Labeling of Neuropeptides and Their Receptors 59

Fig. 1. Fluoronanogold™ immunostaining technique. (A) Structure of Alexa Fluor 488, Streptavidin and the 1.4 nm Nanogold™ particle, covalently conjugated to form the complex Alexa Fluor 488-Fluoronanogold™-Streptavidin. (B) Enlargement of 1.4 nm gold particle with the Gold Enhancement kit. The time of incubation in the Gold Enhancement kit is from 5 to 15 min, during which gold ions are catalytically deposited all around the 1.4 nm gold particle to give rise to an enlarged electron- dense particle of 25–30 nm in size. (C) Fluorescent immunostaining of GFRa1 receptors in the mouse spinal cord dorsal horn obtained using a goat anti-GRFa1 primary antibody (Neuromics, Minneapolis, USA), a secondary biotinylated anti-goat antibody (Vector Laboratories, Burlingame, CA, USA) and the Alexa Fluor-488-Fluoronanogold-Streptavidin complex (Nanoprobes, Yaphank, NY). GFRa1-immunoreactive fibers are mainly distributed in laminae I and II of the dorsal horn. In the inset, a dense mesh of varicose processes is shown. (D) Electron micrograph of the same area of the dorsal horn shown in (C) after the gold-intensification procedure. A GFRa1-immunoreactive axon terminal is labeled by irregular electron-dense gold particles of 25–30 nm in size distributed along the plasma membrane. Scale bar: (C): 250 mm; inset in (C): 20 mm; (D): 200 nm. See http://extras.springer.com/ for the color version of this figure.

TrkB is the high-affinity tyrosine kinase receptor mediating BDNF cellular actions (31, 32). Three different trkB receptor isoforms are generated by alternative splicing of trkB mRNA (33, 34): the full-length trkB (fl-trkB) receptor and two truncated trkB receptors (tr-trkB; (33, 35)). All trkB isoforms share a ­common extracellular domain, whereas the fl-trkB receptor is the only one with the intracellular tyrosine kinase domain and is thus able to trigger the signal transduction pathways utilized by BDNF to exert its biological functions. 60 C. Salio et al.

2. Materials

2.1. Tissue Preparation 1. Sodium pentobarbital (30 mg/kg). for Preembedding 2. Phosphate buffer (PB): Prepare a stock solution “A” with Electron Microscopy 0.2 M NaH2PO4∙H2O and a stock solution “B” with 0.2 M with Fluoronanogold™ Na2HPO4. Prepare working solution by adding 19 mL “A” and 81 mL “B” solutions to 100 mL of distilled water. Adjust pH to 7.4 if necessary. Store at room temperature. 3. Phosphate buffered saline (PBS): Prepare a 9‰ NaCl solution in 900 mL distilled water and 100 mL 0.2 M PB. Adjust pH to 7.4 if necessary. Store at 4°C. 4. Ringer solution: Dissolve 2 g NaCl, 0.0625 g KCl, and 0.125 g

NaHCO3 in 237.5 mL distilled water. Add 14 mL PB 0.2 M. Adjust pH to 7.4. Store at 4°C. 5. Paraformaldehyde/glutaraldehyde fixative: prepare a 4% (w/v) paraformaldehyde solution (available from Sigma, St. Louis, MO), in PB. Dissolve the solution by carefully heating it with a stirring hot plate in a fume hood, then cool at room tempera- ture and finally store at 4°C. Immediately before use, add 0.01–0.5% glutaraldehyde (from a 25% glutaraldehyde stock solution, available from Sigma). 6. Surgical instrumentation and peristaltic pump for perfusion. 7. Vibrating microtome (Leica Microsystems, Weitzlar, Germany).

2.2. Fluorescence 1. 50 mM glycine solution in PBS. Microscopy Labeling 2. 0.1%(v/v) Triton X-100 (TX) in PBS (PBS-0.1%TX). Store with Fluoronanogold™ at 4°C. 3. 5% (v/v) Normal Serum (NS, available from Vector Laboratories, Burlingame, CA), made in the same species of the secondary antibody, diluted in PBS (PBS-5%NS) or PBS- 0.1%TX (PBS-5%NS-0.1%TX). As an alternative, use 6% (w/v) Bovine Serum Albumin, Fraction V (BSA, Sigma) in PBS (PBS-6%BSA) or PBS-0.1%TX (PBS-6%BSA-0.1%TX). 4. Primary antibodies: goat anti-SSTR2a (Santa Cruz, Biotech­ nology, Santa Cruz, CA); goat anti-GRFa1 (Neuromics, Minneapolis, MN), chicken anti-fl-trkB (Promega Corpora­ tion, Madison, WI), chicken anti-human BDNF (Promega Corporation, Madison, WI), rabbit anti-CGRP (obtained from JM Polak, Imperial College, UK), and rat anti-SP (BD Pharmingen, Franklin Lakes, NJ). 5. Secondary biotinylated anti-species antibody (available from Vector Laboratories) plus Alexa Fluor 488 (or 594)- FluoroNanogold™-Streptavidin (Nanoprobes, Yaphank, NY) (see Note 1) or directly secondary FluoroNanogold™-anti-species Fab’-Alexa Fluor 488 (or 594) (Nanoprobes) (see Note 2). 3 Fluoronanogold™ Labeling of Neuropeptides and Their Receptors 61

2.3. Gold 1. Gold Enhancement kit (Nanoprobes): Prepare the mixture Enhancement, Tissue immediately before use. Store at 4°C. Processing 2. Osmium tetroxide (available from Electron Microscopy and Ultrathin Sciences, Hatfield, PA): Prepare an aqueous stock solution Sectioning for TEM of 2% osmium tetroxide at least 24 h before use. Store at 4°C (see Note 3). 3. Osmium ferrocyanide: mix equal volumes of 2% aqueous osmium tetroxide and 3% potassium ferrocyanide in distilled water (see Note 4). 4. Maleate buffer stock solution: Dissolve 23.2 g. maleic acid in 200 mL distilled water and 8 g NaOH in 200 mL distilled water to obtain a 1 N solution. Mix the two solutions and dilute to 1000 mL with distilled water. 5. Maleate buffer pH 5.2: To 100 mL of stock solution, add approximately 14.4 mL 0.2 N NaOH and then dilute to 400 mL with distilled water. 6. Uranyl acetate (available from Electron Microscopy Sciences) in maleate buffer: Prepare a 1% (w/v) solution of uranyl acetate (available from Electron Microscopy Sciences) in maleate buffer pH 6. 100 mL of maleate buffer stock solution plus approximately 53.8 mL 0.2 N NaOH. Dilute to 400 mL with distilled water (see Note 5). 7. Ethanol 70%, Ethanol 90%, Absolute Ethanol. 8. Propylene oxide (available from Sigma Chemicals – see Note 6). 9. Base resin: Araldite M, Araldite M Hardener 964, and dibu- tylphthalate (all these reagents are available from Sigma Chemicals). Mix together 10 mL of Araldite M, 10 mL of Araldite M Hardener 964 and 0.25 mL of dibutylphthalate to obtain base resin. Prepare just before using. 10. Base resin plus accelerator: Prepare a solution of 2% Accelerator (available from Sigma Chemicals) in base resin. Put resin plus Accelerator at 37°C for 30 min before use. 11. Acetate foils (blank EM photographic negatives) or polyethylene capsules for section embedding (see Note 7). 12. Glass knife maker (Leica, Germany) for making glass knives starting from glass knife strips. 13. Glass knife strips (available from Electron Microscopy Sciences). 14. Diatome Diamond knife (Electron Microscopy Sciences) for ultrathin sections (optional). 15. Grid Coating Pen (Electron Microscopy Sciences). 16. Chloroform. 17. 200 mesh nickel grids (Electron Microscopy Sciences). 62 C. Salio et al.

18. Syringe filters – pore size 0.22 mm (available from Whatman, Maidstone, England). 19. Aqueous uranyl acetate solution: Prepare a saturated uranyl acetate (available from Electron Microscopy Sciences) solution in double-distilled water. Filter the solution with syringe filters – pore size 0.22 mm (see Note 5).

20. Lead citrate (Reynold’s): Dissolve 1.33 g Pb(NO3)2 in 30 mL

double-distilled water, then add 1.76 g C6H5Na3O7∙2H2O. Mix for 30 min, and then add 8 mL NaOH 4 N to clarify the solution. Add double-distilled water to 50 mL final volume. Store the solution at 4°C, in the dark. Filter the solution with syringe filters – pore size before use. 21. Parafilm M. 22. Glass Petri dishes.

3. Methods

The choice of the immunolabeling procedure for the ultrastructural localization of neuropeptides and their receptors is critical and should be carefully related to the structure, size, and molecular weight of the molecules that one wants to detect under the TEM. The preembedding immunoperoxidase method (PAP or ABC) that has been often used for the subcellular visualization of recep- tors, presents several disadvantages, primarily because the product of the reaction is often larger than the organelle(s) of interest, commonly masks the underlying cellular membranes, and thus does not lead to reliable identification of subcellular storage sites. An alternative technique is the preembedding Nanogold™ immu- nostaining procedure with ultrasmall gold clusters. In this case colloidal gold particles or gold clusters are silver-intensified before observation with TEM (5). Although this technique allows a good subcellular antigen localization, its main disadvantage is the diffi- culty in correlating light and electron microscopic observations. The Fluoronanogold™ preembedding technique is a rather new approach that, by using fluorescein-tagged antibodies conjugated with intensified small gold particles, allows a precise correlation between the fluorescence immunostaining observed at the light level and the gold-enhanced signal at the electron microscope, providing a useful tool for high-resolution correlative microscopy. The Fluoronanogold™ probes (Fig. 1) have different advanta- geous properties: (1) penetration into aldehyde-fixed tissues greater than colloidal gold particles (36), (2) penetration into tissues as readily as con- ventional immunofluorescent probes (7), (3) a covalent link between the fluorochrome, the antibody, and 1.4-nm gold particles 3 Fluoronanogold™ Labeling of Neuropeptides and Their Receptors 63

that avoids the dissociation of antibodies from gold particles, as reported for conventional colloidal gold immunoprobes (37), (4) good fluorescent labeling that is not reduced from the proxim- ity to the gold cluster (36), and (5) visualization of the labeling at both the fluorescence and electron microscope. By the use of these probes, receptor molecules are clearly local- ized over the plasma membrane of different neuronal processes that can be easily identified as being axonal or dendritic, pre or postsynaptic, etc. The content of labeled profiles (i.e., synaptic vesicles, mitochondria, etc.) is not obscured by immunostaining that takes the form of very electron-dense particles of relatively large sizes (25–30 nm) and irregular shapes. Gold-intensified par- ticles are easily spotted under the TEM (Fig. 2) and immediately distinguishable from colloidal gold in multiple labeling procedures (Fig. 3).

3.1. Tissue Preparation 1. Under deep pentobarbital anesthesia (30 mg/kg), perfuse for Preembedding ­animals through the descending aorta with Ringer solution Electron Microscopy followed by cold fixative solution (see Note 8). with Fluoronanogold™ 2. After perfusion, carefully dissect out areas of interest, cut them into small blocks (not exceeding 5 mm in thickness), and postfix for 2–4 additional hours in the same aldehyde mixture at 4°C. 3. Thoroughly rinse tissue blocks in PBS and cut sections with a vibratome at a thickness of 50–100 mm. 4. Store the sections in PBS and further process them as free- floating.

3.2. Florescence 1. Incubate the sections for 5 min in 50 mM glycine in PBS Microscopy Labeling (see Note 9). with Fluoronanogold™ 2. Transfer the sections in PBS-5%NS or PBS-5%NS-0.1%TX (see Note 10) or alternatively in PBS-6%BSA or PBS-6%BSA- 0.1%TX for 30 min at room temperature in continuous agita- tion (see Note 11). 3. Incubate the sections with the primary antibody of interest (goat anti-SSTR2a diluted 1:100; goat anti-GRFa1, diluted 1:300; chicken anti-fl-trkB diluted 1:500) in the same buffer of step 2 overnight at 4°C under continuous agitation (see Note 12). 4. Wash in PBS three times for 10 min each. 5. Incubate for 1 h with an anti-species biotinylated secondary antibody at the right concentration in PBS at room tempera- ture and then for 1 h with Alexa Fluor 488 (or 594)- Fluoronanogold™-Streptavidin at the right dilution, or directly with a secondary Fluoronanogold™-anti-species Fab¢-Alexa Fluor 488 (or 594) antibody (see Note 13). 64 C. Salio et al.

Fig. 2. Ultrastructural localization of GFRa1 and SSTR2a receptors in mouse spinal dorsal horn using the Fluoronanogold™ labeling technique. (A) A GFRa1-immunoreactive type I glomerular terminal (GIa) is surrounded by several unlabeled dendrites (d). (B) A GFRa1-immunoreactive central terminal, in a characteristic type II glomerular arrangement (GII), is surrounded by unlabeled dendrites (d). Both in GIa and GII glomerular terminals, GFRa1 gold-intensified labeling is associ- ated with the plasma membrane. Note in the insets that GFRa1-labeling is characterized by irregular electron-dense gold particles. The gold particle intensification was made directly on nickel uncoated grids, after the electron microscopy embedding procedure. The grids were rinsed in double-distilled water and then gold particles were intensified by the Gold Enhancement kit for 15 min, reaching a diameter of 25–30 nm. (C) An SSTR2a-immunoreactive nonglomerular axon terminal (At) contacts an unlabeled dendrite (d). (D) Two SSTR2a-immunolabeled dendrites (d1 and d2) are postsynaptic to an unlabeled axon terminal. SSTR2a immunostaining appears in the form of electron-dense particles of relatively large size and irregular shape, distributed along the plasma membrane. The gold particle intensification was made on free-floating sections before the electron microscopy embedding procedure. Sections were rinsed in PBS and then incubated in the Gold Enhancement kit for 15 min to intensify gold particles until a size of 25–30 nm. Scale bars: 300 nm; insets: 30 nm. 3 Fluoronanogold™ Labeling of Neuropeptides and Their Receptors 65

Fig. 3. Ultrastructural localization of fl-trkB receptors, BDNF, CGRP and/or SP in mouse spinal dorsal horn using the Fluoronanogold™ labeling technique combined with conventional postembedding immunogold procedure. (A) A BDNF + CGRP- immunoreactive type Ib terminal (GIb) is surrounded by several dendrites (d), one of which is fl-trkB immunolabeled. BDNF (10-nm gold particles; see inset) and CGRP (20-nm gold particles; see inset) are costored in large granular vesicles (LGVs) of the type Ib glomerulus, while trkB is distributed along the dendrite plasma membrane. (B) A BDNF + CGRP- immunoreactive nonglomerular axon terminal (At) makes a synapse (arrowheads) with a fl-trkB-labeled dendrite (d). BDNF (10-nm gold particles) and CGRP (20-nm gold particles) are costored in LGVs of the axon terminal, while trkB is distributed along the intracellular aspect of the dendrite plasma membrane. Note in the inset that trkB-labeling is char- acterized by irregular electron-dense gold particles. (C) A fl-trkB + BDNF + SP-immunopositive GIb central terminal is surrounded by several unlabeled dendrites (d). BDNF (10-nm gold particles) and SP (20-nm gold particles) show a selective localization in LGVs, while trkB displays intracellular plasma membrane labeling. The two labels are easily distinguishable. BDNF and SP postembedding immunolabeling is associated with the presence of gold particles of very regular round shapes and constant size (10 and 20 nm respectively; arrows in the inset), while trkB preembedding immunostaining is in the form of very electron-dense particles of relatively large sizes (25–30 nm) and irregular shapes (arrowhead in the inset). Scale bars: 300 nm; 20 nm (inset in A); 30 nm (inset in B, C). 66 C. Salio et al.

6. Thoroughly wash the sections in PBS. 7. Check the specificity of immunostaining with a fluorescence microscope (see Note 14). An example of the fluorescence immunolabeling for GFRa1 receptors in mouse spinal cord lamina II is shown in Fig. 1C.

3.3. Gold Two alternative protocols are given since the gold enhancement Enhancement, Tissue step can be carried out either before embedding in resin, or directly Preparation, on ultrathin sections on grids. In the preembedding protocol skip and Ultrathin step 11. In the on-grid protocol skip steps 1 and 2. Both are equally Sectioning for TEM suitable for analysis of CNS connectivity in term of morphological preservation and retention of antigenicity. Examples of the gold- intensified probes before resin embedding are shown in Fig. 2C, D. Examples of the gold-intensified probes directly on grid after elec- tron microscopy embedding are shown in Figs. 2A, B and 3. 1. Rinse free-floating sections in PBS and incubate in the Gold Enhancement kit for 5–15 min to intensify gold particles (Figs. 1B, D and 2C, D). The kit consists of four solutions that should be prepared immediately before use. Wait for 5 min after mixing solutions A (enhancer) and B (activator) and then add solutions C (initiator) and D (buffer) (see Note 15). The development is not highly light sensitive, so it may be con- ducted under normal room lighting. 2. Thoroughly wash gold-enhanced sections in PBS. 3. Postfix in osmium ferrocyanide for 1 h at 4°C. Remember to prepare the solution immediately before use. 4. Wash four times for 5 min each in maleate buffer pH 5. 5. Stain with 1% uranyl acetate in maleate buffer pH 6 for 1 h at 4°C. 6. Wash four times for 5 min each in maleate buffer pH 5. 7. Dehydrate in increasing concentrations of ethanol, starting from 70% ethanol for 5 min, followed by 95% ethanol for 5 min, and then 100% ethanol, three times for 10 min each. 8. Transfer sections in propylene oxide 2× 10 min each and then in propylene oxide-base resin 1:1 overnight at 37°C. 9. Flat-embed in base resin plus accelerator for 24 h at 60°C (see Note 6). 10. Cut ultrathin sections (70–80 nm) and collect them on pre- cleaned uncoated nickel grids (see Note 16). 11. Rinse the grids in double-distilled water and intensify the gold particles with the Gold Enhancement kit for 5–15 min (Fig. 1B, D). The kit consists of four solutions that should be prepared immediately before use. Wait for 5 min after mixing solutions A (enhancer) and B (activator) and then add solutions 3 Fluoronanogold™ Labeling of Neuropeptides and Their Receptors 67

C (initiator) and D (buffer). After mixing, a drop of the solution is placed on a sheet of parafilm in a glass Petri dish and the grids are floated on individual drops for the required time (see Note 15). The development is not highly light sensitive, so it may be conducted under normal room lighting. Once intensi- fication is terminated thoroughly wash in double-distilled water. 12. Counterstain sections on grids with aqueous uranyl acetate for 1 min, wash in double-distilled water, counterstain with lead citrate for an additional minute, and wash again in double- distilled water. 13. Allow grids to dry on filter paper and observe at TEM.

4. Notes

1. Alexa Fluor-FluoroNanogold™-Streptavidin contains a fluoro- chrome molecule, a 1.4-nm diameter gold particle and strepta- vidin, covalently bound together (see Fig. 1A). 2. Alexa Fluor-FluoroNanogold™-anti-species Fab¢ consists of a 1.4-nm diameter gold particle conjugated with a specific Fab¢ fragment and a fluorochrome. 3. Osmium tetroxide is highly poisonous, even at low exposure levels, in particular upon inhalation, and must be handled with precautions. Wear appropriate protective clothing, always work under a fume hood and discharge it in an apposite disposal container. 4. The osmium ferrocyanide solution must have a brown colour. If solution becomes black, discharge it. It is advisable to prepare fresh for each experiment. 5. Uranyl acetate is a nuclear fuel derivative, and thus its use and possession are sanctioned by international law. It is very toxic by ingestion and if inhaled as dust or by skin contact. Keep it in a special metal container, wear appropriate protective cloth- ing when using, and carefully transfer it to an apposite disposal container and arrange its removal by a disposal company. 6. Propylene oxide is a probable human carcinogen; it is harmful by ingestion, inhalation and through skin contact. Wear appro- priate protective clothing when using, always work under a fume hood, and discharge it in an apposite disposal container. 7. Different methods of section embedding are available accord- ing to specific needs. Acetate foils (blank EM photographic negatives) are used to flat embed large sections from which it is possible to select the area(s) of interest to be cut at the ultramicrotome. This method is really useful to preserve the 68 C. Salio et al.

right spatial orientation of the section for further TEM observations. Polyethylene capsules are used for embedding small pieces of tissue and when the orientation is not of primarily impor- tance. In this case, tissue specimens tend to shift inside the capsule during polymerization, changing the initial orienta- tion. It is convenient to place the surface of the specimen from which sections are desired next to the tip of the capsule. Different types of polyethylene capsules are available, depend- ing on the diameter size, length of the capsule, shape of the tip (conical or pyramid) or the bottom (flat). 8. Fixation is carried out with minimal concentrations of glutaral- dehyde to minimize the reduction of tissue antigenicity. The percentage of glutaraldehyde also depends on the primary antibody used (some antibodies need high concentrations of glutaraldehyde, whereas others do not work in presence of this aldehyde molecule). 9. Glycine solution in PBS is a useful step to inactivate residual aldehyde groups, which may still be present after aldehyde fixation without detrimental effects on tissue morphology. 10. To prevent nonspecific background staining (due to interac- tions between the primary antibody and the cell surface or intracellular structures of the specimen), it is important to use a nonimmune serum from the same animal species that produces the secondary antibody or a high concentration high-molecular- weight protein solution, such as BSA. This avoids nonspecific positive staining due to binding of excess secondary antibody to components in the protein solution. 11. Triton X-100 has some detrimental effects on tissue ultrastruc- ture, so use only if necessary and at very low concentration (do not exceed 0.1%). 12. The length and temperature of incubation should change depending on the primary antibody(ies) employed. Some anti- bodies work well only at room temperature; others need to stay at least for 24–48 h at 4°C. 13. Owing to some quenching of fluorescence by the gold particle, slightly higher concentrations (1:50–1:100) of Streptavidin/ secondary Fluoronanogold™-anti-species Fab’-antibody are recommended for incubations. Optimal antibody dilution should be determined by titration. 14. Alexa Fluor-488 has the maximum absorption near to 494 nm, and the maximum emission near to 519 nm. These values are very similar to those of fluorescein (green), so a fluorescein filter set is recommended for fluorescence observation. Alexa Fluor-594 has the maximum absorption near to 590 nm, and the maximum emission near to 619 nm. These values are very 3 Fluoronanogold™ Labeling of Neuropeptides and Their Receptors 69

similar to those of rhodamine (red) or Texas Red, and so one of these filter sets is recommended for observation. 15. The enlargement of particles with the GoldEnhance kit depends on the length of application: 5–15 min will give rise to particles from 5 to 30 nm in size, so the intensification time should be adjusted time to time according to specific needs. 16. In order to enhance specimen adhesion to the grid, it is recom- mended to clean grids before using. Different cleaning ­protocols are available depending on the type of grids (nickel, copper, gold). In particular, for copper and nickel grids, such a treat- ment is effective for long periods in preventing the oxidation of the metal surface that tend to interfere with specimen adhesion to the grid. For nickel grids, the following cleaning procedure is recommended: wash grids with chloroform for 5 min in a sonicator, then in 1% glacial acetic acid in double-distilled water in a sonicator, followed by ethanol 95% for 1–2 min, and finally wash three times in double-distilled water. The use of nickel grids is recommended because they are chemically inert and avoid the risk of unwanted chemical reac- tions during the labeling procedures.

Acknowledgments

This work was supported by grants of the Compagnia di San Paolo (Torino – Italy) and the Italian MiUR (Fondi PRIN 2008).

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Chapter 4

Neuropeptide RNA Localization in Tissue Sections

Marc Landry, Shérine Abdel Salam, and Marie Moftah

Abstract

In situ hybridization has become a routine technique to provide insights into RNA localization. However, different protocols exist for multiple purposes, and it is, therefore, important to clearly define specific needs to choose the most suitable one(s). For instance, in situ hybridization can target different types of RNA, including mRNA or small noncoding RNA such as micro RNA (miRNA). Detection protocols are developed for light or electron microscopy and can be combined with immunocytochemistry to study RNA coexpression with proteins or peptides. In this chapter, we present some protocols to illustrate the diversity of in situ hybridization methods. We focus on the detection of mRNA or miRNA and show that the protocols are quite similar but use dedicated probe types, namely, oligo- or riboprobes and locked nucleic-acid probes.

Key words: mRNA, miRNA, In situ hybridization, Oligoprobes, Riboprobes, Locked nucleic-acid probes

1. Introduction

The birth of new concepts in chemical neurotransmission such as cotransmission, volume transmission, or neuronal versatility fre- quently occurred thanks to the advances in neurocytochemical technologies. First, the identification and in situ localization of neurotransmitters, related enzymes, and receptors greatly benefited from the development of more and more specific and sensitive cytochemical techniques: after histochemistry of acetylcholinesterase, the chemical neuroanatomy of monoamine neurons originated from histofluorescence methods. But it is the development of immuno- cytochemistry (ICC) at optic and electron microscope levels that has given a universal and versatile tool for such cartogra- phies, first of neuropeptides, then of enzymes of neurotransmitter

Adalberto Merighi (ed.), Neuropeptides: Methods and Protocols, Methods in Molecular Biology, vol. 789, DOI 10.1007/978-1-61779-310-3_4, © Springer Science+Business Media, LLC 2011 73 74 M. Landry et al.

metabolism, and finally of the neurotransmitters themselves after the pioneering work of Steinbusch for [1). However, these mappings, completed by that of receptors through ligand binding or ICC, do not allow, for example, to assume that the protein molecules identified are truly synthesized within the sites where they are detected. The in situ hybridization (ISH) of mRNAs leads to the detection and cellular localization of another step of the protein and peptide synthesis. However, ISH is not restricted to the detection of coding RNAs and can also be applied to the visualization of small noncoding RNAs that have essential and diverse cellular functions. They may function as catalysts, adapters, and guides in processes such as pre-mRNA splicing, translation, and amino-acid transfer. Other categories of noncoding RNAs act as regulators of gene expression, such as micro RNAs (miRNA). MiRNA are implicated in the pathogenesis of many human diseases, including cancers and neurological affections (2). It is thus of the utmost importance to detail the tissue and cellular distribution of miRNA to provide new insights into their regulatory roles in pathological conditions. As for coding RNAs, ISH is the most suitable tool for the localization of noncoding RNAs, including miRNA. We describe and comment here some protocols for the detection of mRNA and miRNA with light and electron microscopy. We also give some clues for the combination of ISH with other histochemical approaches, i.e., ICC.

2. Materials

2.1. Preparation 1. Cryostat or paraffin embedding station and microtome (see of Biological Samples Note 1). 2. Super Frost Plus Gold slides (Menzel-Glaser, Braunschweig, Germany). 3. Water bath (only if using paraffin sections). 4. Slide oven (only if using paraffin sections).

2.2. Probes (see Note 2) 1. DNA oligoprobes (25–30 nucleotides). 2. RNA riboprobes. 3. Locked nucleic acid (LNA™) probes prelabeled with digoxi- genin or fluorochromes (Exiqon, Vedbaek, Denmark).

2.2.1. Probe Labeling and 1. dNTP (Roche Diagnostics, Mannheim, Germany). Purification (see Note 3) 2. Terminal transferase or RNA polymerase (available from Roche Diagnostics). 4 RNA Localization 75

3. Labeling buffer (see below). 4. Ethanol (molecular biology grade). 5. LiCl. 6. EDTA or DNase I (available from Roche Diagnostics). 7. SP6/T7 RNA polymerase labeling kit (Roche Diagnostics) for digoxigenin labeling of riboprobes. 8. Digoxigenin-11-dUTP (Roche Diagnostic) for digoxigenin labeling of oligoprobes. 9. Brightstar Psoralen Kit (BioRad, Hercules, CA) for biotin labeling of oligo- or riboprobes.

2.3. Reagents 1. 0.1 M and 0.01 M phosphate-buffered saline (PBS – available and Buffers from Sigma Chemicals, St. Louis, MO).

2.3.1. Tissue 2. Phosphate buffer (PB): Prepare a stock solution “A” with Pretreatments 0.2 M NaH2PO4∙H2O and a stock solution “B” with 0.2 M

Na2HPO4. Prepare working solution by adding 19 mL “A” and 81 mL “B” solutions to 100 mL of distilled water. Adjust pH to 7.4 if necessary. Store at room temperature. 3. Proteinase K stock solution: 1 mg/mL in water. 4. Acetylation solution: Add 1.165 mL triethanolamine to 100 mL distilled water and then add 250 mL acetic anhydride. 5. Ethanol. 6. Chloroform. 7. 4% paraformaldehyde in 0.1 M phosphate buffer.

2.3.2. Hybridization 1. Standard saline citrate (SSC 1×): 0.15 M NaCl, 0.015 M Na citrate, pH 7.2 (available from Sigma Chemicals). 2. Denhardt’s solution: 0.02% bovine serum albumin (BSA – available from Sigma Chemicals), 0.02% Ficoll, 0.02% polyvinylpyrrolidone. 3. Hybridization buffer for oligoprobes and LNA probes: 50% deionized formamide, 4× SSC, 1× Denhardt’s solution, 0.02 M

NaPO4, pH 7.0, 1% N-lauroylsarcosine, 10% dextran sulfate (to be omitted when using free-floating sections), 500 mg/L denatured salmon testis DNA. 4. Hybridization buffer for riboprobes: 50% deionized forma- mide, 5× SSC, 50 mg/mL yeast RNA, 1% sodium dodecyl sulfate (SDS). 5. RNAse A (available from Roche Diagnostics): 100 mg/mL in 1× SSC.

2.3.3. Posthybridization 1. Posthybridization rinsing solution for oligo- and riboprobes: Rinsing 1× SSC. 76 M. Landry et al.

2. Posthybridization rinsing solution 1 for LNA probes: SSC 1×, 50% formamide, 0.1% Tween. 3. Posthybridization rinsing solution 2 for LNA probes: SSC 0.2×.

2.3.4. Detection 1. TNT: 0.1 M Tris–HCl, 0.15 M NaCl, 0.5% blocking reagent (provided in Perkin Elmer Tyramide system amplification kit). 2. TNB: 0.1 M Tris–HCl, 0.15 M NaCl, 0.05% Tween. 3. BSA.

4. Buffer A: 1 M NaCl, 200 mM MgCl2, 0.1 M Tris–HCl, pH 7.5.

5. Buffer B: 1 M NaCl, 5 mM MgCl2, 0.1 M Tris–HCl, pH 9.5. 6. Alkaline phosphatase substrate: 5-bromo-4-chloro-3-­ indolylphosphate (0.45%, BCIP, Roche Diagnostics) and Nitro-blue tetrazolium chloride (0.35%, NBT, Roche Diagnostics) in buffer B. 7. 0.05 M Tris–HCl, pH 7.6. 8. 0.025% 3,3¢ diaminobenzidine in 0.05 M Tris–HCl, pH 7.6.

2.3.5. Mounting 1. Antifading mounting medium (Dako, Glostrup, Denmark). or Embedding 2. Osmium tetroxide. 3. Epon resin. 4. Uranyl acetate.

2.4. Antibodies 1. HRP- or AP-conjugated streptavidin (Roche Diagnostics). and Detection 2. HRP-conjugated anti-DIG (Roche Diagnostics). Systems 3. Avidin/biotin blocking kit (Vector laboratories, Burlingame, CA). 4. Tyramide system amplification kit (TSA): FITC- or biotin con- jugated tyramide (TSA-Plus Fluorescein (or Biotin) system-tyramide signal amplification Kit – Perkin Elmer, Waltham, MA). 5. Ultrasmall gold particles-conjugated anti-DIG antibody (Nanoprobes, Yaphank, NY, USA). 6. Silver enhancement kit for electron microscopy (Nanoprobes). 7. Anti-synaptoporin antibody (Synaptic systems, Goettingen, Germany). 8. AlexaFluor 568-anti-mouse secondary antibody (Invitrogen, Carlsbad, CA).

2.5. Disposables 1. Eppendorf tubes. 2. Plastic cuvettes. 3. 12-well tissue culture plates. 4. 24-well tissue culture plates. 4 RNA Localization 77

3. Methods

Various protocols are available to perform ISH with different probe types, as well as with different detection systems. Important criteria drive the choice of the method. In particular, the nature (mRNA or miRNA) and relative abundance of target RNA will help defining the type of probe (oligoprobe, riboprobe, or LNA probe) as well as the detection system to be applied. Oligoprobes are most convenient, as they do not need so much precaution such as RNAse-free medium for riboprobes (Fig. 1). However, riboprobes remain known to be more sensitive because of their RNA nature, and the high Tm of the hybrids made with riboprobes. As described above, LNA probes are most suitable for short targets such as miRNA (Fig. 2). Another element that guides the choice of the method is the possible requirement for multiple labeling. A double labeling experiment needs that the two reactions are compatible in terms of fixation, pretreatments, temperature of reaction, and detec- tion. There is no major hindrance to the detection of two mRNAs if the probes are of the same type. By contrast, a good preserva- tion of antigenicity during the ISH reaction is a major condition for coupling ICC and ISH. In addition, the signals given by each technique should be readily distinguishable. Moreover, the detectable molecule, i.e., the reporter molecule, introduced chemically or enzymatically should not interfere with the hybrid- ization reaction or the stability of the resulting hybrid. Also, it should remain accessible to the detection system used later on. If two enzymatic labelings have to be performed, the reaction products must be of sharp different colors (e.g., purple and brown) (3). New protocols have arisen that allow multiple fluo- rescent detections (4, 5). To improve the sensitivity of detection systems, it is often required to amplify the signal. One of the most effective proce- dures uses tyramide signal amplification (TSA). TSA may actually provide up to a 100-fold increase in signal as compared with conventional detection systems. This detection system is based on the reaction of peroxidase with hydrogen peroxide and the phe- nolic part of tyramide, which produces a quinone-like structure bearing a radical on the C2 group. This activated tyramide then covalently binds to tyrosine in close vicinity to the peroxidase (6). Such a reaction can be applied either to ICC or ISH detection with a similar protocol after peroxidase is bound to the target via an antibody. The tyramide can be conjugated to a fluorochrome or a hapten. Hence, direct or indirect detection of enzymatically deposited tyramides is possible and can be coupled to another labeling (3). Fig. 1. (a, b) Nonradioactive ISH with oligoprobes was applied to the detection of mRNA coding for the GABAB receptor subunit B1. TSA detection provides a high signal-to-noise ratio that allows identifying GABAB-containing neurons (arrow- heads) throughout the dorsal horn of the rat spinal cord (a). At a higher magnification b( ), subcellular distribution of B1 mRNA can be detailed and ISH shows the presence of transcripts in neuronal processes (double arrows). (c) ISH with oligoprobes was used to detect mRNA coding for a neuropeptide, galanin, with electron microscopy. The digoxigenin reporter molecule was visualized with silver-enhanced ultrasmall gold particles. High magnification shows the location of galanin mRNA (arrows) on the cytoplasmic side of the endoplasmic reticulum membrane in neurons of the rat locus coeruleus. B1: GABAB1 subunit of the GABAB receptor; AP-Dig: alkaline phosphatase detection of the digoxigenin reporter molecule; USgold-Dig: silver enhancement of ultrasmall gold particles coupled to anti-digoxigenin antibody. Bar: 50 mm (a, b); 200 nm (c). 4 RNA Localization 79

Fig. 2. (a, b): ISH with LNA probes was applied to the detection of miRNA miR-134 in the rat spinal cord. Hybrids were visualized with a fluorescent TSA method. Signal was intense on the ventral horn of the spinal cord, and especially in presumed motoneurons (arrowheads). At higher magnification, labeling appears punctuate both in cell bodies and neuronal processes (double arrows). (c) ISH with LNA probes was coupled to ICVC for synaptoporin, a marker of nociceptive sensory fibers, in the dorsal horn of the spinal cord. No colocalization can be seen while some apposition between immunopositive nerve endings and miR-134 containing cell bodies are detected (arrows). (d) ISH with LNA probes was detected with silver enhanced ultrasmall gold particles in the rat spinal cord. Hybrids are observed in lysosomes, corresponding to degrading compartments where miRNA/mRNA hybrids are likely to be addressed. LNA Locked nucleic acid, TSA Detection with Tyramide System Amplification of digoxigenin-labeled LNA probe,USgold ultrasmall gold detection of digoxigenin-labeled LNA probe, L Lysosome. Bar: (a) 50 mm; (b) 25 mm; (c) 10 mm. See http://extras.springer.com/ for the color version of this figure.

At the electron microscope level, an important feature is the resolution of the reporter molecules. Autoradiographic studies require the use of 3H instead of 35S to limit the diffusion of the signal. Nevertheless, beside the difficulties of such a technique, the resolution remains poor. Also, enzymatic products are known to diffuse throughout the cytoplasm. To date, the use of silver-enhanced gold particles represents the marker ensuring the best resolution for preembedding ICC and ISH (7) studies, provided that the duration and temperature of the silver-enhancement procedure are well optimized. 80 M. Landry et al.

3.1. Direct 1. Air-dry sections at room temperature (RT) for 2 h. Chromogenic 2. Fix with 4% paraformaldehyde for 20 min (see Note 4). Detection 3. Incubate with proteinase K diluted 1:2,000 for 15 min in PB of Oligoprobes (see Note 5). (mRNA) 4. Stop reaction with 1 M Tris–HCl, 0.5 M EDTA (v/v). 3.1.1. Prehybridization 5. Quickly wash the sections in RNase-free water. 6. Dehydrate through graded ethanols, once in 50, 70, 95% ethanol, and twice in 100% ethanol, 1 min each. 7. Incubate in chloroform for 5 min (for lipid removing). 8. Wash twice in 100% ethanol and once in ethanol 95%. 9. Air-dry the slides for at least 2 h.

3.1.2. Hybridization 1. Before hybridization, incubate the hybridization buffer at 42°C for 1 h. 2. Incubate the sections at 42°C overnight in hybridization buffer containing the oligoprobe at 2 pmol/mL (labeled probe from a stock at 1 pmol/mL – see Notes 6 and 7).

3.1.3. Posthybridization 1. Wash the sections 4 × 15 min each in SSC 1× at 55°C, then for and Detection 30 min in SSC 1× at RT. 2. Incubate in buffer A/BSA (1%) pH 7.5 for 30 min at RT. 3. Incubate with the alkaline phosphatase-conjugated anti-digoxigenin antibody 1:5,000 in buffer A/BSA for 30 min at RT. 4. Wash for 10 min in buffer A and then for 10 min in buffer B pH 9.5 at RT. 5. Detect alkaline phosphatase activity with BCIP/NBT in 5 mL developing solution, pH 9.5 for few hours (see Note 8). 6. Wash thoroughly in water. 7. Air-dry the sections for at least 2 h. 8. Mount in PBS–glycerol (v/v) solution.

3.2. Chromogenic 1. Synthesize the riboprobe using SP6 (T7 for sense riboprobe) Detection of RNA polymerase. Reporter molecules (digoxigenin or biotin) Riboprobes with a TSA are incorporated, respectively, by adding 11 digoxigenin-dUTP Enhancement Protocol into the medium or by using a Brightstar Psoralen Kit (BioRad, (mRNA) Hercules, CA) according to the manufacturer’s instructions, for 45 min under 365 nm UV light (3). 3.2.1. Preparation of Labeled RNA Probes

3.2.2. Single-Labeling 1. Equilibrate the sections to RT. In Situ Hybridization 2. Fix in 4% paraformaldehyde for 20 min (see Note 4). 3. Deproteinize with proteinase K 10 mg/mL for 15 min (see Note 5). Stop reaction with 0.5 M EDTA in 1 M Tris–HCl, pH 8 (v/v). 4 RNA Localization 81

4. Dehydrate through graded ethanols (see step 6 in Subheading 3.1.1). 5. Delipidate the sections in chloroform for 5 min. 6. Rehydrate in 70% ethanol and air-dry. 7. Hybridize the sections overnight at 65°C with 10 ng of digoxigenin- or biotin-labeled probes diluted in hybridization buffer. 8. Rinse slides 2 × 5 min each in 1× SSC. 9. Treat with RNAse A for 30 min at 37°C. 10. Wash for 5 min in SSC 1×, 5 min in SSC 0.5× at RT, 30 min in SSC 0.1× at 65°C two times each, and 15 min in SSC 0.1× at RT (see Note 9). 11. Transfer the sections to buffer A containing 0.5% BSA for 30 min.

3.2.3. Detection 1. Incubate the sections overnight at 4°C with sheep HRP- conjugated anti-digoxigenin, or anti-biotin antibody at (1/1,1000) dilution. 2. Rinse in TNT containing 0.5% blocking reagent (TNB). 3. Incubate in biotinylated tyramide diluted 1:500 in the provided diluent for 10 min at 37°C (see Note 10). 4. Rinse in TNT. 5. Incubate in TNB with alkaline phosphatase-conjugated strepta- vidin diluted 1:5,000 for 30 min. 6. Rinse 3 × 10 min each in buffer A and then for 10 min in buffer B. 7. Detect alkaline phosphatase with NBT/BCIP (see Note 8). 8. Rinse and mount the sections as described above (steps 6–8 in Subheading 3.1.3).

3.3. Double 1. Synthesize digoxigenin and biotin labeled probes (see Chromogenic Subheading 3.2.1). Detection 2. Perform section pretreatment as in the single labeling protocol of Riboprobes (steps 1–6 in Subheading 3.2.2). (mRNA) 3. Hybridize the sections with a mixture of both probes (see step 7 in Subheading 3.2.2). 4. Carry out posthybridization rinsing (see steps 8–11 in Subheading 3.2.2). 5. First detect the biotin-labeled probe was first detected using HRP-conjugated streptavidin (1/200, 2 h in TNB). 6. Visualize the biotin-labeled probe with diaminobenzimide. 7. Block unreacted biotin with the avidin/biotin blocking kit prior to the second detection. 82 M. Landry et al.

8. Incubate the sections overnight at 4°C with sheep HRP conjugated anti-digoxigenin, or anti-biotin antibody diluted 1,000. 9. Rinse in TNT containing 0.5% blocking reagent (TNB). 10. Incubate in biotinylated tyramide diluted 1:500 in the provided diluent for 10 min at 37°C. 11. Rinse in TNT. 12. Incubated in TNB with alkaline phosphatase-conjugated streptavidin diluted 1:5,000 for 30 min. 13. Rinse 3 × 10 min each in buffer A and then for 10 min in buffer B. 14. Perform colorimetric detection with NBT/BCIP. This double detection results in brown (for TSA) and purple (for alkaline phosphatase) staining, respectively. 15. Rinse and mount the sections as described above (steps 6–8 in Subheading 3.1.3).

3.4. Fluorescent 1. Thaw the Eppendorf tubes containing frozen sections for ISH Detection of LNA (see Note 11). Pour thawed floating sections in a 12-well tissue Probes with a TSA culture plate containing a plastic cuvette with a perforated Enhancement Protocol bottom. (miRNA) 2. Rinse the sections with 0.01 M PBS twice. 3. Prepare the acetylation medium. Care should be taken not to add the acetic anhydride until the last minute before incubation. 4. Incubate the sections in the acetylation medium for 10 min at RT. 5. Rinse 3 × 5 min each with 0.01 M PBS. 6. Add 2 mM LNA probe to the hybridization buffer and incubate the mixture for 5 min at 70°C. 7. Transfer the tubes on ice for 3 min to unwind the probe. 8. Transfer the floating sections into a sterile 24-well tissue culture plate and incubate with the probe hybridization buffer over- night at 55°C. 9. Rinse the sections in a 12-well plate 2 × 20 min each with rinsing solution 1 at 65°C. 10. Rinse for 10 additional minutes with rinsing solution 2 at 65°C. 11. Rinse again 3 × 10 min with TNT at RT. 12. Block sections for 30 min in the TNB blocking solution in a 24-well culture plate. 13. Incubate the floating sections with the anti-digoxigenin HRP- conjugated antibody diluted 1:5,000 in TNB. 4 RNA Localization 83

14. Rinse in a 12-well plate 3 × 10 min each with TNT. 15. Incubate in a 24-well culture plate with tyramide FITC diluted 1:50 in its amplification diluent for 10 min at RT. 16. Rinse 3 × 10 min each in a 12-well plate with TNT at RT. 17. Transfer the sections on slides and let them dry for 30 min in a 37°C incubator. 18. Mount them with coverslips using an antifading agent. 19. Store the slides at −20°C in dark until examination.

3.5. Double 1. Repeat steps 1–11 in Subheading 3.4. Fluorescent Labeling 2. Dilute primary antibody at optimal titer in TNT + 1% BSA. Combining ICC 3. Incubate the sections overnight with the primary antibody in a and ISH with LNA 24-well plate at 4°C. Probes (miRNA) 4. Rinse 3 × 10 min each with TNT in a 12-well culture plate. 5. Block the sections for 30 min in the TNB blocking solution in a 24-well culture plate. 6. Incubate floating sections with the anti-digoxygenin HRP- conjugated antibody diluted 1:100 and Alexa Fluor 568 anti- species diluted 1:500 in TNB for 3 h at RT (in dark). 7. Rinse in a 12-well plate 3 × 10 min each with TNT. 8. Incubate in a 24-well culture plate with Tyramide FITC diluted 1:50 in its amplification buffer for 10 min at RT. 9. Rinse 3 × 10 min each in a 12-well plate with TNT at RT. 10. Transfer the sections on slides and let them dry for 30 min in a 37°C incubator. 11. Mount them with coverslips using an antifading agent. 12. Store the slides at −20°C in dark until examination.

3.6. ISH at the Electron 1. Use cryoprotected and cryocut chemically fixed cells or tissue Microscope Level sections. with Silver-Enhanced 2. Hybridize digoxigenin labeled oligoprobes or LNA probes Gold Particles (mRNA according to the above methods. and miRNA) 3. Carry out posthybridization washings. 4. Preincubate in a blocking solution (PBS-BSA, gelatin, normal goat serum) 2 h at RT. 5. Incubate with 1 nm gold-conjugated anti-digoxigenin antibody diluted in the blocking solution for 30 min at RT. 6. Rinse in PBS. 7. Postfix in paraformaldehyde 4% in PBS for 10 min at RT. 8. Rinse in PBS. 9. Perform silver enhancement according to the manufacturer instructions, rinse in water and PB (see Note 12). 84 M. Landry et al.

10. Postfix in OsO4 1% diluted in PB for 10 min at RT. 11. Embed in resin. 12. Cut ultrathin sections and counterstain with uranyl acetate. 13. Observe under a TEM.

4. Notes

1. Most commonly, ISH is carried out on tissue sections from paraffin-embedded material or on frozen sections. Paraffin embedding leads to unsurpassed histological preservation. It also allows storage at room temperature at low cost. The main dis- advantage is a reduced sensitivity as compared with frozen ­sections. The latter are suitable for chemically unfixed material, lack of fixation offering an increase in sensitivity. Moreover, frozen tissue provides better probe access to target RNA that further increases when using oligoprobes (8). Altogether, frozen sections make ISH more sensitive than with any other type of sections. It is of course possible to use frozen sections of fixed material. In this case, animals are perfused with4% paraformaldehyde, tissues are dissected out, postfixed, cryo- protected with 10% glycerol and 25% saccharose, and embedded in Tissue-Tek. Use a cryostat to cut sections (14 mm thickness), thaw-mount them on Super Frost Plus Gold slides, and, if necessary, store at −80°C. 2. Either DNA or RNA probes can be used. The former type is made of oligoprobes usually ranging between 25 and 50 nucle- otides. The latter corresponds to riboprobes whose length depends on the cDNA that has been subcloned. The detection of miRNA relies on shorter probes, between 17 and 25 nucle- otides. The short length of miRNA makes traditional oligo- probes unsuitable for ISH because of the low melting temperature (Tm) of formed hybrids. LNA is a modification that dramatically increases the Tm of duplexes, allowing their use even in stringent conditions. To increase sensitivity, LNA probes are double labeled at both 5¢ and 3¢ ends, thus improv- ing the detection of low abundance miRNA. The signal-to- noise ration benefits from a cooperative effect of the two digoxigenin labeled and makes the detection of lowly expressed targets possible. 3. To label oligoprobes, use twenty-five 100 pmoles of probe and nonradioactive (digoxigenin, biotin, fluorescein) dNTP (Roche Diagnostics). For riboprobes 1 g template is transcribed, and reporter molecule can be inserted by using nonradioactive NTP or by performing chemical addition. 4 RNA Localization 85

4. Fixation is the first and a crucial step for ISH and ICC, since it must allow both a good morphological preservation and a good efficiency of the histochemical reactions, two criteria being most often contradictory. Two kinds of fixative can be considered: chemical and physical fixatives. Among the former, precipitating fixatives give a nice morphology but are generally not well suited for RNA. Nonprecipitating aldehydic fixatives have been introduced in ISH studies in the eighties, and 4% paraformaldehyde is now the most common fixative for both ICC and ISH and their combination. The fixation by the form- aldehyde or its derivatives is largely reversible. Thus, the chemical properties of nucleic acids are not modified by the fixation and hybrids Tm remains unchanged upon aldehyde fixation. Despite these favorable properties, it is preferable to use slight concentrations of formaldehyde on nervous tissue to allow a better accessibility of the probes to the target. The glutaralde- hyde is known to be of a poor interest in ISH studies since it creates a dense network of double bonds making difficult the probe penetration. Finally, the use of picric acid should be banned since it modifies chemical properties of the probe and can alter it. Physical fixations have also been used and proved to be of a great interest at least for double ISH. Thus, drying is a very easy way to fix nervous tissues and air-dried cryostat sections (8) or cell cultures (9) are more sensitive to mRNA detection than fixed material. Also, microwave heating has been reported to improve hybridization efficiency without altering tissue morphology (10). 5. Tissue section pretreatments are performed to facilitate probe penetration and access to the target. Actually, RNA can be asso- ciated to many RNA-binding proteins that make the access difficult. Protease treatment allows exposing cellular RNAs to the probe. However, overdigestion makes morphology subopti- mal and hinder cellular, and of course subcellular, localization. 6. To increase the sensitivity of ISH with oligoprobes, a cocktail of several oligoprobes, all complementary to the same TNA species, can be applied. Every probe hybridizes to a specific sequence of the target and contributes to the signal (11). 7. Various controls should be conducted when performing ISH to test the specificity of probes, antibodies and detection systems. Positive controls should monitor the quality of sample preparation and tissue; they also confirm that all reagents are properly prepared. A positive control could be a probe or antibody that is highly expressed in the cell type considered. Probes against ribosomal RNA are good positive controls for ISH. Negative controls are used to assess the level of nonspecific background. The best control for ISH is to use probes that are known to work but not expressed in the tissue being analyzed. 86 M. Landry et al.

For LNA probes against miRNA, control probes include probes with few mismatches, probes with scrambled sequence, and pretreatment of the samples with RNase to ensure that probes hybridize to RNAs. 8. Incubation time in NBT/BCIP must be adjusted according to the probe used and the RNA targeted. Typically, incubation time ranges between 2 and 3 h, but it can increase up to36 h. One of the major problems of long incubation time is a strong background. To reduce background, and therefore to increase signal-to-noise ratio, levamisole is used to block endogenous alkaline phosphatases before applying digoxigenin-conjugated reagents (levamisole solution: 1 mM levamisole-containing buffer A). 9. Posthybridization rinsing is important, especially when using riboprobes or LNA probes, and rinsing that are described in protocol are only indicative. An alternative washing protocol for riboprobes can be as follows: two times 30¢ at 70°C (50% for- mamide; 5× SSC, 1% SDS), two times 30¢ at 65°C (50% forma- mide; 2× SSC). 10. It is important for reducing background when using TSA to block endogenous peroxidase by preincubating the sections in

H2O2 at 2% in PB or Tris buffer. 11. When using chemically fixed tissue, ISH is preferably performed on free-floating sections rather on slide attached sections, to further increase sensitivity. The protocols described here are intended for free-floating sections, but they can be easily adapted to thaw-mounted sections. In this case, incubations are done by applying drops of solution (typically 200 mL per slide) on sections surrounded by a repellant medium. ISH can be also applied on adherent cell types including primary cell cultures (9) with no or minimal modifications as compared to tissue sections. 12. Overamplification is another source of nonspecific staining when using the silver enhancement procedure. Silver enhance- ment time should then be adapted to every experiment.

References

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sections without protease treatment. Nat. 9. Kerekes, N., Landry, M., and Hökfelt, T. Protoc. 5, 1061–1073. (1999) Leukemia inhibitory factor regulates 6. van Gijlswijk, R.P., Zijlmans, H.J., Wiegant, J., galanin/galanin message-associated peptide Bobrow, M.N., Erickson, T.J., Adler, K.E., expression in cultured mouse dorsal root Tanke, H.J., and Raap, A.K. (1997) ­ganglia; with a note on in situ hybridization Fluorochrome-labeled tyramides: use in immu- methodology. Neuroscience 89, 1123–1134. nocytochemistry and fluorescence in situ hybrid- 10. Lan, H.Y., Hutchinson, P., Tesch, G.H., ization. J. Histochem. Cytochem. 45, 375–382. Mu, W., and Atkins, R.C. (1996) A novel 7. Vila-Porcile, E., Xu, Z.Q., Mailly, P., Nagy, F., method of microwave treatment for detec- Calas, A., Hökfelt, T., and Landry, M. (2009) tion of cytoplasmic and nuclear antigens by Dendritic synthesis and release of the neuro- flow cytometry. J. Immunol. Methods. 190, peptide galanin: morphological evidence from 1–10. studies on rat locus coeruleus neurons. J. Comp. 11. Trembleau, A., Roche, D. and Calas, A. (1993) Neurol. 516, 199–212. Combination of non-radioactive and radioac- 8. Dagerlind, A., Friberg ,K., Bean, A.J., and tive in situ hybridization with immunohis- Hökfelt, T. (1992) Sensitive mRNA detection tochemistry: a new method allowing the using unfixed tissue: combined radioactive and simultaneous detection of two mRNAs and non-radioactive in situ hybridization histochem- one antigen in the same brain tissue section. istry. Histochemistry 98, 39–49. J. Histochem. Cytochem. 41, 489–498.

Chapter 5

Intron-Specific Neuropeptide Probes

Harold Gainer, Todd A. Ponzio, Chunmei Yue, and Makoto Kawasaki

Abstract

Measurements of changes in pre-mRNA levels by intron-specific probes are generally accepted as more closely reflecting changes in gene transcription rates than are measurements of mRNA levels by exonic probes. This is, in part, because the pre-mRNAs, which include the primary transcript and various splicing intermediates located in the nucleus (also referred to as heteronuclear RNAs, or hnRNAs), are processed rapidly (with half-lives <60 min) as compared to neuropeptide mRNAs, which are then transferred to the cytoplasm and which have much longer half-lives (often over days). In this chapter, we describe the use of exon-and intron-specific probes to evaluate oxytocin (OT) and vasopressin (VP) neuropeptide gene expres- sion by analyses of their mRNAs and hnRNAs by quantitative in situ hybridization (qISH) and also by using specific PCR primers in quantitative, real-time PCR (qPCR) procedures.

Key words: Pre-mRNA, Heteronuclear RNA, hn RNA, Real-time qPCR, Oxytocin, Vasopressin, Gene expression

1. Introduction

The idea that measurements of changes in pre-mRNA levels by intron-specific probes more closely reflect changes in gene tran- scription rates than measurement of mRNA levels by exonic probes is based on the observation that excision of the intron from the primary transcript and the subsequent degradation of the intron is very rapid, typically with half-times <30–60 min (1–4). By contrast, neuropeptide mRNAs can have turnover rates over days, and hence changes in their mRNA levels are as likely to reflect degradation rates as well as transcription rates. The first evaluation of neuropep- tide gene expression by the use of intron sequence-specific probes and quantitative in situ hybridization (qISH) procedures was reported by Roberts and coworkers (5, 6) for the study of the

Adalberto Merighi (ed.), Neuropeptides: Methods and Protocols, Methods in Molecular Biology, vol. 789, DOI 10.1007/978-1-61779-310-3_5, © Springer Science+Business Media, LLC 2011 89 90 H. Gainer et al.

­proopiomelanocortin (POMC) gene primary transcript in individual neurons of the hypothalamus. Consequently, many assays of gene transcription changes using intron-specific probes and qISH have been used in studies of a wide variety of genes in the CNS (7–15). The use of intron-specific probes to study neurohypophysial peptide gene expression in the hypothalamus by qISH was first accomplished for vasopressin (VP), and clearly showed that the intron 1-directed riboprobe that was used detected changes in the VP pre-RNA with much more rapid kinetics than exonic probes did for changes in mRNA (9). Experiments have also been done using intron-specific probes and qISH to evaluate oxytocin (OT) gene expression in the hypothalamus with similar success (16–19). More recently, intronic and exonic primers have also been success- fully utilized in quantitative, real-time PCR assays for the study of OT gene expression in the rat hypothalamus (20). In this chapter, we illustrate the use of exon- and intron-specific riboprobes (see Notes 2 and 3) or PCR primers to evaluate oxytocin and vasopres- sin neuropeptide gene expression by both qISH and quantitative, real-time PCR procedures, respectively (18–22).

2. Materials

2.1. Quantitative 1. DNA polymerase, platinum Taq polymerase (Invitrogen, In Situ Hybridization: Carlsbad, CA), which includes 10×PCR buffer containing see Note 1 Tris–Cl (pH 8.4), KCl, 50 mM MgCl2. 2.1.1. Preparation of 2. 100 mM dNTP set (PCR grade, dATP, dGTP, dCTP, dGTP, Intronic and Exonic OT 100 mM each, Invitrogen). and VP Riboprobes 3. Primers (custom-ordered from Sigma Genosys, St. Louis, MO). for qISH T3 CGCGCAATTAACCCTCACTAAAGG T7 GCGCGTAATACGACTCACTATAGGG SP6 CATACGATTTAGGTGACACTATAG 4. PCR tubes, MicroAmp Autoclaved Reaction Tube with cap (Perkin Elmer Life Sciences, Waltham, MA). 5. PCR thermocycler (e.g., Gene Amp PCR system 9700, Applied Biosystems, Foster City, CA). 6. Agarose for electrophoresis (available from Invitrogen). 7. Tris–Cl Borate EDTA (available from Quality Biological Inc., Gaithersburg, MD). 8. Ethidium bromide. 9. Ready-load 100 bp DNA ladder (available from Invitrogen). 10. QIA quick gel extraction kit – contains columns, binding ­buffer, and washing buffer (Qiagen, Hilden, Germany). 11. TE buffer pH 8.0 (Quality Biological Inc.). 5 Intron–Specific Probes 91

12. Tris EDTA buffer pH 8.0 (Quality Biological Inc.). 13. PCR primers to generate the OT intronic riboprobes (see Note 4). For intron 1: 5¢-ATGAAGCTTCCGTTCTGGCAA GGCTAAG-3¢ (sense), and 5¢-ATCTGCAGCACAATCCATA TCGGGACTG-3¢ (antisense). For OT intron 2: 5¢-ATGAAGC TTGTGAGCAGGAGGGGGCCTA-3¢ (sense), and 5¢-ATG CTGCAGCTGCAAGAGAAATGGGTCAGT-3¢ (antisense). 14. 1× PCR buffer: 20 mM Tris–HCl, pH 8.4, 50 mM KCl. 15. dNTPs. 16. Platinum Taq DNA polymerase (Invitrogen). 17. PCR thermocycler (e.g., GeneAmp PCR System 9700 – Applied Biosystems). 18. Agarose gel. 19. DNA electrophoresis apparatus. 20. Spectrophotometer (e.g., Ultrospec2100 pro, Amersham Pharmacia Biotech., Piscataway, NJ). 21. MinElute Gel Extraction Kit (Qiagen). 22. pBlueScript II SK (+) for subcloning PCR fragments (Stratagene, La Jolla, CA). 23. Modified T7 (GCGCGTAATACGACTCACTATAGGG) and T3 (CGCGCAATTAACCCTCACTAAAGG) primers (see Note 5).

2.1.2. 35S Labeling 1. MAXIscript T7/T3 or MAXIscript SP6/T7 labeling kit, and Purification ­[contains10× buffer, dNTP (mixture of dATP, dCTP, dGTP), of Riboprobes RNA polymerase (T7, T3, SP6), DNAseI, nuclease-free water] (Ambion, Austin, TX). 2. [a-35S]-uridine 5¢-triphosphate (35S UTP) and [a-35S]-citosine 5¢-triphosphate (35S CTP) (Perkin Elmer Life Sciences). 3. Dithiothreitol (DTT). 4. RNeasy mini kit (for RNA purification: contains columns, lysis buffer, washing buffer, Qiagen). 5. ETOH 100% (Quality Biological Inc.). 6. TE buffer pH 8.0 (Quality Biological Inc.). 7. Tris EDTA buffer pH 8.0 (Quality Biological Inc.). 8. LS6500 Multipurpose Scintillation counter (Beckman-Coulter Co, Fullerton, CA).

2.1.3. Tissue Preparation 1. Cryostat. and Hybridization 2. Poly-l-lysine coated slides (Fisher Scientific Company, Newark, DE). 3. Slide warmer. 4. 4% formaldehyde in 1×PBS (to fix frozen sections). 92 H. Gainer et al.

5. 1×PBS: 0.017 M KH2PO4, 0.05M Na2HPO4, 1.5 M NaCl, pH 7.4. 6. Solution for hybridization sheet, 4×SSC 50% formamide. 7. For TEA buffers: acetic anhydride (Sigma Chemicals), TEA [triethanolamine (98%), Sigma Chemicals]. 8. For nucleic acid mix: salmon sperm DNA (available from Sigma Chemicals or Invitrogen), Yeast total RNA type XI (ribonucleic acid – Sigma Chemicals), Yeast tRNA (Invitrogen). 9. Hybridization buffers: Tris–Cl, pH 7.4, EDTA pH 8.0, NaCl, Formamide, Dextran sulfate, Denhardt’s solution, DTT, sodium dodecyl sulfate (SDS), sodium thiosulfate (NTS – Sigma Chemicals), nuclease-free water. 10. SSC buffer: 0.3 M sodium citrate, 3 M NaCl, pH 7.0. 11. TNE buffer [10 mM Tris–Cl pH 8.0, 0.5 M NaCl, 0.25 mM EDTA pH 8.0] containing 20 mg/mL ribonuclease A (Sigma Chemicals). 12. Ethanol wash solutions (EtOH: 70, 80, 90, 95, and 100%). 13. Dry incubator. 14. Coverslips. 15. X-ray Cassettes. 16. Microscope Slide Holder, Peel-A-Way (Polysciences Inc., Warrington, PA). 17. NTB-3 autoradiography emulsion (Kodak, Rochester, NY). 18. Ilford Nuclear research Emulsions K5D (Polysciences Inc.). 19. Rack and dish for dark storage (Research Products International Corp., Mt. Prospect, IL). 20. Dektol (developer for normal dipping – Kodak). 21. D-19 (developer for digoxigenin double label – Kodak). 22. Fixer (VWR International, West Chester, PE).

2.1.4. Quantitation 1. Storm 860 Phosphoimager (Amersham Bioscience, Amersham, of Hybridization UK). 2. Image Quant software version 5.2 (Amersham Biosciences). 3. Phosphorscreen, low energy (LE), unmounted (Amersham Biosciences).

2.2. Quantitative 1. Primers for qPCR (see Note 6). Real-Time PCR 2. Single Step quantitative real-time PCR (qRT-PCR) kit (Qiagen). The kit contains reverse transcriptase along with a master mix solution containing a hot-start Taq polymerase. 3. Absolutely RNA miniprep kit (Stratagene). The kit is used for RNA extraction and includes lysis buffer, beta mercaptoethanol, 5 Intron–Specific Probes 93

prefilter spin cups, RNA binding low and high salt wash buffers, 70% ethanol, and DNAse I solutions. 4. Qiagen RNase-Free DNase in RNA Easy kit (Qiagen – see Note 7). 5. Spectrophotometer (e.g., Nano Drop, Wilmington, DE). 6. Heraus Biofuge Fresco centrifuge (DJB Labcare Ltd, Buckingshire, England). 7. Vortex. 8. Air incubator. 9. Real-time PCR apparatus [e.g., Smart Cycler (Cepheid, Sunnyvale, CA), or Step One Plus PCR apparatus, 96 wells (Applied Biosystems)].

3. Methods

3.1. Quantitative In situ hybridization histochemistry (ISH) is an important method In Situ Hybridization to detect and measure quantities of specific RNAs in tissue sections. Its principal value when compared to RT-PCR, which is a more sensitive assay (see Subheading 3.2), is the higher spatial resolution (even to the cellular level) of ISH. There are many ways to detect RNAs by ISH, e.g., by nonradioactive digoxigenin or biotin-labeled probe hybridization followed by immunodetection using either alkaline phosphatase or fluorescence-coupled antibodies or by qualitative/quantitative assays with 35S labeled oligonucleotide or RNA probes (riboprobes). We focus here only on qISH assays using 35S labeled riboprobes directed at exonic and intronic sequences. An example of such an ISH assay using 35S-labeled probes of VP hnRNA and VP mRNA hybridized to sections at two different ana- tomic levels of hypothalamus is shown in Fig. 1. It is notable that the VP hnRNA levels appear to be similar between the supraoptic (SON) and suprachiasmatic nucleus (SCN), whereas the mRNA levels in the SON appear to be substantially greater than in the SCN. Quantitative in situ hybridization (qISH) (23) and quanti- tative PCR (20) confirm these impressions and show that the SON/ SCN VPmRNA ratio was equal to about 15, whereas the SON/SCN VPhnRNA ratio of 2.8 is much lower (and this difference is not statistically significant). Thus, quantitative conclusions drawn from qISH and qPCR methods appear to be comparable.

3.1.1. Preparation of 1. The rat VP intron 1 (735 bp), VP exonic (229 bp), and OT Intronic and Exonic OT exonic (487 bp) DNA fragments are subcloned into pGEM-3 and VP Riboprobes for qISH vectors (see Note 3). The OT intronic fragments were obtained and subcloned into pBlueScript II SK (+) as described from steps 2 to 5 below. 94 H. Gainer et al.

Fig. 1. Photomicrograph of ISHH of VP mRNAs and hnRNAs in rat brain sections at two different anatomic levels of ­hypothalamus. Two top panels illustrate VP mRNAs by hybridization with VP exon-specific probe. The VP mRNA levels in the SON appear to be similar as in the PVN, but significantly greater than in the SCN.Two bottom panels illustrate VP hnRNAs by hybridization with VP intron 1-specific probe. The VP hnRNA levels appear to be quantitatively similar among all these three hypothalamic nuclei. Note that the signal to background ratio is much better for the abundant mRNAs than that for the much less abundant mRNAs (from ref. 19).

2. The PCR primers used to generate the OT intronic fragments are described in Note 4. 3. To obtain the the OT intronic fragments, the PCR is carried out in a 50-mL reaction volume, containing 0.8 mg rat genomic DNA, 1× PCR buffer (20 mM Tris–HCl, pH 8.4, 50 mM KCl), 200 mM each of dNTPs, 200 nM each of the primers, and 2.5 U Platinum Taq DNA polymerase (Invitrogen). 4. PCR is performed on a GeneAmp PCR System 9700 Thermal Cycler (Applied Biosystems) and consists of an initial 2 min, 94°C denaturation, followed by 30 cycles of denaturing (94°C, 30 s), annealing (60°C, 30 s), and extension (72°C, 45 s), fol- lowed by a final extension of 7 min at 72°C. 5. The PCR products are loaded on the 1.5% agarose gel and purified by the MinElute Gel Extraction Kit (Qiagen). Then, the purified PCR fragments were subcloned into pBlueScript II SK (+) (Stratagene, La Jolla, CA). 5 Intron–Specific Probes 95

6. Modified T7 and SP6 primers are used to PCR-amplify all of the three fragments, VP intron1, VP and OT exonic fragments that are subcloned into pGEM-3 vectors and the OT intronic frag- ments that are subcloned into pBlueScript II SK (+). The PCR is carried out in a 50-mL reaction volume, containing 20–50 ng plasmid, 1× PCR buffer (20 mM Tris–HCl, pH 8.4, 50 mM KCl), 200 mM each of dNTPs, 0.2 pM each of the primers, and 2.5 U Platinum Taq DNA polymerase (Invitrogen). PCR is per- formed on the same conditions as described in step 4. 7. All the PCR products are loaded on the 1.5% agarose gel and purified by the MinElute Gel Extraction Kit, and the resulting PCR products are used as templates to synthesize the riboprobes. 8. The T7 RNA polymerase was used to synthesize the antisense of VP intronic and exonic probes, the SP6 polymerase is for the antisense of the OT exonic riboprobe and T3 polymerase is for the antisense of OT intronic riboprobes. SP6 RNA poly- merase was used to synthesize the sense of VP intronic and exonic probes, T7 polymerase is for the sense OT exonic ribo- probe and the sense OT intronic riboprobes.

3.1.2. 35S Labeling 1. Label intronic and exonic riboprobes with 35S by using and Purification 30–40 ng of PCR product, 50 mCi of 35S UTP , and 10 mM of Riboprobes DTT along with the reagents in the MAXIscript in vitro tran- scription kit (see Notes 8 and 9). 2. Adjust 35S RNA sample to a volume of 100 mL with 1×TE pH 8.0. Add 350 mL Buffer RLT and mix thoroughly. 3. Add 250 mL ethanol (96–100%) to the diluted RNA and mix thoroughly by pipetting. Do not centrifuge. Continue imme- diately with step 4. 4. Apply the sample (700 mL) to an RNeasy mini column placed in a 2-mL collection tube (supplied). Close the tube gently and centrifuge for 15 s at ³8,000 × g. 5. Take the flow-through with a pipette and Repeat step 4. 6. Discard the flow-through and collection tube. 7. Transfer the RNeasy column to a new 2-mL collection tube (supplied). Pipette 500 mL Buffer RPE onto the RNeasy ­column. Close the tube gently and centrifuge for 15 s at ³8,000 × g to wash the column. Discard the flow-through. Reuse the collection tube in step 7 (see Note 10). 8. Add another 500 mL Buffer RPE to the RNeasy column. Close the tube gently and centrifuge for 2 min at ³8,000 × g to dry the RNeasy silica-gel membrane. Continue directly with step 6, or, to eliminate any chance of possible Buffer RPE carryover (see Note 11). 96 H. Gainer et al.

9. To elute, transfer the RNeasy column to a new 1.5-mL collection tube (supplied). Pipette 50 mL 1× TE (pH 8.0) directly onto the RNeasy silica-gel membrane. Close the tube gently, incubate for 1 min, and centrifuge for 1 min at ³8,000 × g to elute. 10. Discard the column and measure the radioactivity using a scin- tillation counter. Take out 1 mL of labeled probe and mix into 10 mL of scintillant in a scintillation vial. If the count is less than 300,000 cpm, it is suggesting that it is an inefficient label- ing (see Note 12).

3.1.3. Tissue Preparation 1. Cut serial 10-mm brain sections in a cryostat and place them and Hybridization onto poly-l-lysine coated slides. 2. Dry sections on a slide warmer for 10–30 min at 37°C and store at −80°C until further use. 3. Before hybridization, fix the sections in 4% formaldehyde for 10 min at room temperature, rinse once in PBS, and wash twice for 5 min each in 1× PBS. 4. Put into 0.1 M triethanolamine–HCl (pH 8.0) containing 0.25% acetic anhydride for 10 min at room temperature. 5. Rinse with 2× SSC buffer, transfer through graded ethanol (EtOH:70% – 1 min, 80% – 1 min, 95% – 1 min, 100% – 1 min, 95% – 1 min.), and finally air-dry. 6. Mix probes and nucleotide mix, heat to 65°C, and cool on ice for 5 min. Then, add it to hybridization solution (20 mM Tris–Cl pH 7.4, 1 mM EDTA pH 8.0, 300 mM NaCl 50% formamide, 10% dextran sulfate, 1× Denhardt’s solution, 100 mg/mL salmon sperm DNA, 250 mg/mL yeast total RNA, 250 mg/mL yeast tRNA, 0.0625% SDS, and 0.0625% sodium thiosulfate). 7. Carry out hybridization in 80 mL hybridization solution con- taining approximately 106 cpm denatured S35-labeled ribo- probe for each section. 8. Coverslip the slide and use a 4× SSC/50% formamide solution to saturate 3MM paper to humidify the hybridization chamber. 9. After an approximately 24 h (overnight) incubation at 55°C, remove the coverslips in 4× SSC/1 mM DTT at room tem- perature and wash with the same solution for 5 min. 10. Incubate the sections in TNE buffer [10 mM Tris–Cl pH 8.0, 0.5 M NaCl, and 0.25 mM EDTA pH 8.0] containing 20 mg/mL ribonuclease A for 30 min at 37°C. 11. Wash twice (5 min/wash) in 2× SSC/1 mM DTT, once in 1× SSC/1 mM DTT and then in 0.5× SSC/1 mM DTT (5 min each) at 65°C. 5 Intron–Specific Probes 97

12. Let the sections cool to room temperature in 0.1× SSC/1 mM DTT. 13. Rinse the sections in graded ethanol solutions (1 min each in 70, 80, 90, 95, and 100% ethanol) and then air-dry.

3.1.4. Quantitation 1. Before use, erase the screen to be employed with the Storm of Hybridization 860 Phospho Imager System. Put the phosphor screen facing the surface on the eraser and switch on the eraser. Leave till the lights go off automatically. 2. Align the slides on the cassette. Use a liquid tape or tape to fix the slides so that the slides will not move during the exposure. 3. Lift the screen from the eraser and place it with the surface on the slides. Do not move the screen once you place it on the slides because the screen is sensitive enough to pick up signals at the time you start exposing. Also, do not place any material (wraps etc.) between the slides and the screen, since it will interfere with the exposure. 4. Place a spacer over the screen and shut the cassette at once. Be careful not to move the screen while closing the cassette. 5. Label the cassette with the exposure starting date and time. Store the cassette at room temperature. It is not needed to store in a dark place or −80°C. 6. Expose the screen for the required time. Usually, the phosphor screens are ten times more sensitive than regular autoradiography films, so you can estimate the days as 10% of the required time in autoradiography films. To get a better quality in the image (e.g., for making figures), longer exposure is better (see Note 13). 7. For scanning, open the “Storm Scanning Control” software (see Note 14). 8. Open the lid of the scanner and clean the glass surface with a Kimberly-Clark tissue wipe (or any kind of paper that can be used on microscopic glasses). Use distilled water if necessary. Do not use any detergent. 9. Mark the area that you are going to scan on the left panel of the “Storm Scanning Control” window. Then, go to the right panel and make sure that the acquisition mode is storage phos- phor. Choose the pixel size of 50 mm. 10. Choose the image analysis to Image Quant if you want that the Image Quant software is opened automatically after the scan- ning is finished. 11. Click the scan button. This will prompt you to save your file before scanning. Name you file and save and then the scanning will start. It usually takes 40 min for a large screen and 20 min for a small screen to complete the scanning (see Note 15). 98 H. Gainer et al.

12. Start the “Image Quant 5.2” software and open the scanned files that you are going to analyze. The software enables you to evaluate the levels of mRNA or hnRNA in the brain nucleus/ nuclei of interest (e.g., the SON) by measuring the average densities and unit areas per SON of phosphor screen in the sec- tions from each experimental animal. 13. To optimize the display, first click the “Gray/color adjust” button on the top tool bar and check if the image is not satu- rated. The levels showing in the high should be less than 100,000, if it is exceeding 100,000 the image is saturating, and you should scan the image with a shorter exposure time. Then adjust the image contrast by dragging the slider above the his- togram. Adjust the image till you see the first saturated signals (shown in red on the screen). If necessary, you can enlarge the image by using the “creating frame” option in the top tool panel, before adjusting the contrast. The quantitation results do not change by changing the contrast. 14. To select the region to quantitate, scroll to the section you would like to quantitate, enlarge it by using the “creating frame” option. Use the “region” tool in the left tool bar to select the area of interest that you need to quantitate. Each selected area will be numbered in the order you selected. For the background normalization, use the “ellipse” tool in the left tool bar to select an area that shows background hybridization. 15. After selecting the areas, go to analysis/background correc- tion. Under the objects, you will see the numbered regions and ellipses that you selected. Select the regions that you would like to correct and select “object average” under the methods. After that, you will see the ellipses that you selected under the object average. Select the ellipse that you would like to use as the background and click the “set” button. Now you should see the regions marked as “OA” which means that it is cor- rected using certain background areas. After you finish the cor- rection, close the inspector window. 16. Go to analysis/volume review. Your quantitation results will show up. Double-click the results and this will lead you to the results in an excel format. Save the data. The values shown in the volume columns are your corrected data (average intensity × area). 17. Determine statistical significance of differences between groups by one-way ANOVA followed by Fisher’s protected least sig- nificant difference (PLSD) test or Student’s test. Differences between groups are considered statistically significant when P values were less than 0.05. Experiments are routinely repeated on four animals and the results expressed as means ± SEMs. 5 Intron–Specific Probes 99

3.2. Quantitative The use of real-time reverse transcription PCR (aka real-time Real-Time PCR RT-PCR, or qRT-PCR) is a valuable alternative to qISH to do quantitative studies of pre-mRNAs. Because of its greater sensitiv- ity, qRT-PCR can often detect intron-containing pre-mRNAs when qISH fails to do so. In a study of OT gene expression, we found that the short introns in the OT gene did not permit the genera- tion of effective riboprobes to detect the intron 1 bearing hnRNAs by qISH (18). However, these pre-mRNAs could be readily stud- ied by in qRT-PCR using primers directed against either intron1 or intron 2 (20). Real-time PCR can be done using either a one-step or two-step reaction. In the one-step method, the entire PCR ­procedure is performed in a single tube, whereas in the two-step procedure the reverse transcription and PCR amplification reac- tions occur in separate tubes. While a comprehensive comparison between these two PCR methods is beyond the scope of this article (but see (24, 25) for more discussion), we can state that we have studied both OT and VP hnRNAs and mRNAs by both of these approaches and have found both to be equally effective (19–22). However, because the single-step PCR method is obviously ­simpler to perform, we focus on this method here.

3.2.1. RNA Extraction 1. Dissect a 6–10 mg tissue sample (approx. 106 cells) and place the sample into 250 mL of Lysis Buffer containing an addi- tional 1.8 mL of beta mercaptoethanol (see Note 16). 2. Homogenize the sample by repeatedly pipetting (more than 20 times) and vortexing for about 15 s. Continue pipetting, if necessary, to completely homogenize the solution. A slightly opaque solution void of flocculent debris should result. 3. Transfer the homogenate to a Prefilter Spin Cup (blue), which is placed in a 2-mL receptacle tube, and spin the tube in a cen- trifuge at maximum speed (17,900 × g for 10 min). 4. Discard the blue spin cup but retain the filtrate. Add an equal volume (approx. 200 mL, as some of the original solution is retained by the Prefilter Spin Cup) of 70% ethanol to the fil- trate and vortex for 5 s. 5. Transfer the mixture to an RNA Binding Spin Cup (clear), which should be placed in a new 2-mL receptacle tube. 6. Centrifuge the spin cup for 1 min at 17,900 × g. 7. Retain the spin cup, aspirate/discard the filtrate, and replace the spin cup in the receptacle tube. 8. Add 550 mL of 1× Low-Salt Wash Buffer and centrifuge for 1 min at 17,900 × g. 9. Retain the spin cup, aspirate/discard the filtrate, and replace the spin cup in the receptacle tube. 100 H. Gainer et al.

10. Centrifuge for another 2 min at 17,900 × g to completely dry the binding cup. 11. Prepare the DNase solution (see Appendix D in Qiagen RNeasy mini handbook, p. 70) by mixing 70 mL of RDD (buffer) with 10 mL of reconsitituted RNase-Free DNase. Gently mix by pipetting three to four times. 12. Add DNase solution directly onto the matrix of the spin cup. 13. Incubate the solution at 37°C for 30 min, in an air incubator. 14. Add 550 mL of 1× High-Salt wash buffer to the spin cup and then centrifuge for 1 min at 17,900 × g. 15. Retain the spin cup, aspirate/discard the filtrate, and replace the spin cup in the receptacle tube. 16. Add 550 mL of 1× Low-Salt wash buffer to the spin cup and then centrifuge for 1 min at 17,900 × g. 17. Retain the spin cup, aspirate/discard the filtrate, and replace the spin cup in the receptacle tube. 18. Add 300 mL of 1× Low-Salt wash buffer to the spin cup and then centrifuge for 2 min at 17,900 × g. 19. Transfer the spin cup to a 1.5 mL microcentrifuge tube and discard the 2 mL receptacle tube with the filtrate. 20. Add 40 mL of pure room-temperature water directly onto the center of the matrix inside the spin cup and wait for 2 min. 21. Centrifuge the tube for 1 min at 17,900 × g. 22. Immediately place the tube (with the eluted RNA) on ice. 23. Determine RNA concentration and purity using a spectrophoto­ meter. 24. Acceptable purity: 260/280 ratio should be between 1.8 and 2.1.

3.2.2. qRT-PCR 1. To prepare the qRT-PCR reaction tubes, prepare 25 mL master (see Note 17) mix/tube containing the following: 0.6 mM of total primers (1.5 mL of a 100 mM F + R primer mixture), 12.5 mL of 2×-Taq polymerase-SYBR Green buffer, 0.25 mL Reverse Transcriptase, 10–20 ng of template RNA/reaction (see Note 18), and pure water to a final volume of 25 mL (approximately an additional 9.75–10 mL) (see Note 19). 2. Aliquot from the master mix to a reaction tube that has been placed in the cold (4°C) tube-holder rack that comes with the SmartCycler. 3. Annealing temperatures were 60°C for all primers shown in Table 1 for the qRT-PCR reactions. 4. Measure and calculate cycle threshold (Ct) values by the real- time PCR apparatus’ software (see Note 20). 5 Intron–Specific Probes 101

Table 1 PCR primers used to assay c-fos, vasopressin (VP), oxytocin (OT), and GAPDH RNAs by single-step, real-time, quantitative PCR

Amplicon a Primers Sequence length TM (°C) c-fos mRNA primer pairs 5¢-AGCATGGGCTCCCCTGTCA (forward) 134 bp 83.4 5¢-GAGACCAGAGTGGGCTGCA (reverse) VP mRNA primer pairs 5¢-TGCCTGCTACTTCCAGAACTGC (forward) 77 bp 80.7 5¢-AGGGGAGACACTGTCTCAGCTC (reverse) VP hnRNA primer pairs 5¢-GCCCTCACCTCTGCCTGCTA (forward) 100 bp 81.5 5¢-CCTGAACGGACCACAGTGGT (reverse) OT mRNA primer pairs 5¢-GGCATCTGCTGTAGCCCG (forward) 62 bp 84.5 5¢-AAGGCAGACTCAGGGTCG (reverse) OT hnRNA primer pairs 5¢-CCGTCGGGTTTGCTGCTCA (forward) 87 bp 84.3 5¢-AAGGCAGACTCAGGGTCG (reverse) GAPDH mRNA primer 5¢-CAGAGCTGAACGGGAAGCT (forward) 125 bp 81.2 pairs 5¢-CTTCACCACCTTCTTGATGTCA (reverse)

TA, annealing temperature and time for each primer pair is 60°C, and 30 s All the sequences shown in this Table were designed using GeneFisher software (26) a T M, PCR product melting temperature is shown for each primer

5. Run each individual qRT-PCR reaction in duplicate or triplicate, and run together samples from all animals in a given experiment (e.g., experimental and control samples). 6. At the end of every run, produce a melting curve (50–95°C with 0.1°C/s) to confirm the homogeneity of the PCR product. 7. Calculate an average Ct value for each PCR primer pair and each experimental condition. 8. If hnRNA are to be assayed, perform an additional reaction using the VP hnRNA primer pair in the absence of reverse transcriptases (−RT) to control for DNA contamination in the RNA template. 9. Include only templates with Ct differences >5 for the +RT ­versus −RT experiments, representing little if any DNA con- tamination, are included in the analysis (see Note 21).

3.2.3. Data Analyses 1. Calculate average Ct values ± SEMs for each experiment. Perform and Calculations control reactions with the PCR primers but without RT reac- of Fold Changes tions for each PCR run to monitor the assays for DNA contami- nation and primer artifacts. Typical fluorescent “growth” curves for high-abundance VP mRNA and low-abundance c-fos mRNA, and VP hnRNA, are illustrated in Fig. 2. 102 H. Gainer et al.

500 c-fos mRNA

VP mRNA 400 VP hnRNA (a.u.) 300 Value Value

200 Fluorescence Fluorescence 100

0 14710 13 16 19 22 25 28 31 34 37 40 PCR cycles

Fig. 2. Representative fluorescence “growth curves” produced by quantitative real-time (qRT) PCR of c-fos mRNA, VP mRNA, and VP hnRNA in the SON in control rats. Locations of the qRT-PCR primers in the RNAs are shown in Fig. 2 (from ref. 22).

2. Calculate relative quantification of changes in the specific RNA’s levels by the comparative Ct method using the formula, 2-∆∆Ct (27–29) (see Note 22 for discussion about the appro- priate method for the statistical analysis of Ct values). The comparative Ct method assumes that the efficiency of the ­target gene of the PCR is close to one, and that the efficiencies of the PCRs of the gene of interest and the control gene are similar. In one study of the efficiencies of the PCRs of VP mRNA and VP hnRNA, this proved to be the case (21). Hence, we use the simplified formula for the fold change in hnRNA or mRNA in any sample as the fold change or ratio = 2.0[(the Ct of gene of interest minus the Ct of the control gene in the experimental RNA sample) − (the Ct of gene of interest minus the Ct of the control gene in the control RNA sample)], and this is expressed as percent change from control. Analyses of data obtained from in vivo in studies of the effects of acute and chronic salt loading on the changes in expression of OT and VP hnRNAs and OT and VP mRNAs in rat SONs are illustrated in Figs. 3 and 4, respectively. Note that in the acute salt loading (Fig. 3a) the Cts of the VPmRNAs did not change, and so the VP mRNA was used as the control gene to calculate the per- cent change in the c-fos mRNA and VP hnRNA (Fig. 3b). By con- trast, in the chronic salt loading study in which the VPmRNA which, as expected, did undergo significant change (Fig. 4a), we used the GAPDH mRNA, which did not change, as the control gene to calculate the percent changes in the other genes’ expres- sion after salt loading (Fig. 4b). ab

35 400 Control * * 32 Hypertonic * 300 29 * 26 200 23 % of Control

Average Ct Value NS 100 20

0 0 VP mRNA c-fos mRNA VP hnRNA c-fos mRNA VP hnRNA

Fig. 3. Changes in Ct values for c-fos mRNA, VP mRNA and VP hnRNA in rat SONs after acute osmotic stimulation as ­measured by the qRT-PCR assay. (a) Average ± SEM Cycle threshold (Ct) values from control SONs in rats injected with 0.15 M NaCl (black bars) versus SONs in rats injected with 2 M NaCl (stripped bars) are shown. (b) Calculated percent changes in c-fos mRNA and VP hnRNA in the SON 30 min after the injection of hyperosmotic 2 M compared to rats injected with isotonic 0.15 M NaCl injection. Statistically significant differences between Ct values in the 0.15 M and 2 M injected rats are shown by asterisks where P < 0.05 (unpublished data).

a c 30 300 Control ** Hypertonic

25 l 200

Ct Value 20 NS ** 100 % of Contro erage 15 Av

0 0 GAPDH mRNA VP mRNA VP hnRNA VP mRNA VP hnRNA b d 30 300 ** e 25 200

Ct Valu 20 NS ** 100 erage erage % of Control 15 Av

0 0 GAPDH mRNA OT mRNA OT hnRNA OT mRNA OT hnRNA

Fig. 4. Changes in Ct values for GAPDH mRNA, VP mRNA, and VP hnRNA in rat SONs after chronic osmotic stimulation as measured by the qRT-PCR assay. (a) Average ± SEM Cycle threshold (Ct) values from control SONs in rats given normal drinking water (black bars) versus SONs in rats given 2.1% NaCl solution ad lib as their only drinking fluid for 5 days (stripped bars) are shown. (b) Changes in Ct values for GAPDH mRNA, OT mRNA, and OT hnRNA in rat SONs after chronic osmotic stimulation as measured by the qRT-PCR assay. (a) Average ± SEM Cycle threshold (Ct) values from control SONs in rats given normal drinking water (black bars) versus SONs in rats given 2.1% NaCl solution ad lib as their only drinking fluid for 5 days (stripped bars) are shown. In (c) are the calculated percent changes in VP mRNA and VP hnRNA in the SON in rats given 2.1% NaCl solution ad lib as their only drinking fluid for 5 days compared to rats given normal drinking water. In (d) the calculated percent changes in OT mRNA and OT hnRNA in the SON in rats given 2.1% NaCl solution ad lib as their only drinking fluid for 5 days compared to rats given normal drinking water. Statistically significant differences between Ct values in the control and 2.1% NaCl given rats are shown by asterisks where P < 0.01 (unpublished data). 104 H. Gainer et al.

4. Notes

1. The materials described here are directed at studies using RNA probes (riboprobes). These probes provide the greatest sensi- tivity for ISH. The typical low abundance of intron-containing RNAs in cells usually requires the most sensitive ISH methods for detection and quantification. 2. DNA sequences in intron and exons of the rat genes are used to design the PCR primers for the intronic and exonic probes using DNASIS-Mac v3.5 software (Hitachi Software Engineering Co., Ltd. Tokyo, Japan). In all cases, BLAST searches of the nucleotide sequences in GenBank™ are done to reveal any nonspecific matches for these probes, and to con- firm that the primers are specific for the gene. In addition, it is advisable to sequence the resultant PCR amplicons to confirm the annotation. The PCR products are used to prepare both sense and the antisense probes ranging in size preferably from 500 to 650 bp in length. Hybridization of the sense probes were performed as negative controls. 3. The rat VP intronic riboprobe used here was obtained from Dr. Thomas Sherman, Georgetown University, Washington, DC and was a 735 bp fragment of intron 1 of rat VP gene subcloned into pGEM-3 vector (Promega, Madison, WI) (9). The rat VP exonic probe was a 229 bp fragment of the rat VP gene and the rat OT exonic probe was a 487 bp fragment obtained from Dr. W. Scott Young (NIMH). Both were sub- cloned into a pGEM-3 vector. Modified T7 and SP6 primers were used to PCR amplify all of three fragments to make tem- plates that were used to synthesize the riboprobes. The T7 RNA polymerase was used to synthesize the antisense of hnVP and exonic VP probes and the SP6 the antisense of the OT exonic riboprobe. 4. The PCR primers used to generate the OT intronic riboprobe for intron 1 of OT are: 5¢-ATGAAGCTTCCGTTCTGGCAAGG CTAAG-3¢ (sense), and 5¢-ATCTGCAGCACAATCCATATCG GGACTG-3¢ (antisense). Using these primers, one obtains a 162 bp fragment of OT intron 1 after PCR. The primers used to obtain an OT intronic probe for intron 2 of OT are: 5¢ATGAAGCTTGTGAGCAGGAGGGGGCCTA-3¢ (sense), and 5¢-ATGCTGCAGCTGCAAGAGAAATGGGTCAGT-3¢ (antisense). Using these primers, one obtains a 84-bp fragment of OT intron 2 after PCR. Because both OT introns are very short, the resulting OT riboprobes are below optimal length (around 500 bp) for ISH. This can be compensated for, in part, by using more than one radioactive nucleotide for the labeling of the riboprobes (see Subheading 3). 5 Intron–Specific Probes 105

5. These primers are used to PCR amplify the fragments and the resulting PCR products are used as templates for the synthesis of riboprobes. Here, T7 and T3 RNA polymerases are used to obtain both sense and antisense labeled probes, respectively. As for the VP probes, hybridization of sense probes is performed as negative controls. 6. All gene-specific PCR primers discussed in this chapter and illustrated in Fig. 5, and with sequences shown in Table 1, were designed using GeneFisher software (26). Nonetheless, alternative software is available for this pur- pose. A good, free program called Autoprime that specializes in qPCR primers for mRNA is now available. It has the addi- tional benefit of having access to the genomes of over 50 com- mon model organisms. The Autoprime Web site is: http:// www.autoprime.de/AutoPrimeWeb. Another valuable software program for designing PCR primers is called Primer3, which can be found at http://frodo.wi.mit.edu/primer3/. The latter is a more general primer designing tool that is useful for all kinds of PCR, including qPCR.

Fig. 5. Locations of qRT-PCR primers in the c-fos mRNA, VP mRNA, VP heteronuclear (hn)RNA, OT mRNA, OT hnRNA and GAPDH mRNA species used for quantitative real-time PCR (qRT-PCR). The arrows depict the positions of forward and reverse PCR primer binding sites (See Table 1 for specific sequences). In single step qPCR, the reverse PCR primer also serves as the primer for reverse transcription. 106 H. Gainer et al.

The primers in Table 1 are shown for two intronic assays, (i.e., for measurements of intron 1 in VP hnRNA, of intron 2 in OT hnRNA) and for three exonic measurements (i.e., for c-fos, VP, OT, and GAPDH mRNAs, see Fig. 5 for primer loca- tions). A typical PCR reaction mixture contains 0.6 mM of primers and 10–20 ng of template RNA. Reaction volumes are 25 mL for the Smart Cycler system or 15 mL for the Step One Plus. A very useful validation of the PCR primers is to electro- phorese the resulting PCR reaction products on an agarose gel to confirm that the lengths of the amplicons are as expected (see Fig. 6).

Fig. 6. (a) Diagram of VP RNA species and PCR primer binding sites. Top, Pre-mRNA containing both introns and exons. Only the intronic primer pair can bind to this species. Bottom, following the removal of both introns, the exonic regions are joined, thereby allowing the binding of the exonic primer pair. (b) Gel analysis of exonic and intronic PCR products gener- ated from VP mRNA and VP hnRNA from rat . PCR triplicates were subjected to gel electrophoresis on a 2.5% agarose gel containing ethidium bromide. The products are the predicted sizes for the amplicons and are reproduc- ible (modified from ref. 21). 5 Intron–Specific Probes 107

7. DNase treatment is necessary when qRT-PCR is done on ­heteronuclear RNA. We find that the DNase in the RNA Easy kit from Qiagen is superior to that supplied in the Stratagene Absolutely RNA miniprep kit. 8. More specifically, labeling of the VP intronic, VP exonic and OT exonic riboprobes of optimal length used 40 ng, 60 ng, and 100 ng of the templates, respectively, and 50 mCi of 35S UTP, 10 mM DTT, and a MAXI script in vitro transcription adequate kit. 9. The shorter length riboprobe sequences such as for the OT intron 2 probe which contains only 13 uridines requires modi- fication of the above labeling procedure for detection of het- eronuclear RNA by ISH. Noting that the probe contained 31 cytidine nucleotides, versus the 13 uridine nucleotides, we found that use of both 35S-UTP and 35S-CTP together to label the OT intron 2 probe results in a very robust signal. Consequently, effective OT intron 2 riboprobe labeling was performed as described above but using 40 ng of the PCR product, 50 mCi of 35S UTP, and 50 mCi of 35S-CTP. 10. Buffer RPE is supplied as a concentrate. Ensure that ethanol is added to Buffer RPE before use. 11. Optional: Place the RNeasy column in a new 2 mL collection tube (not supplied), and discard the old collection tube with the flow-through. Centrifuge in a microcentrifuge at full speed for 1 min. It is important to dry the RNeasy silica-gel mem- brane, since residual ethanol may interfere with downstream reactions. This centrifugation ensures that no ethanol is carried over during elution. Following the centrifugation, remove the RNeasy mini column from the collection tube carefully so that the column does not contact the flow-through as this will result in carryover of ethanol. 12. The protocol for ethanol precipitation ends at 100 mL, so the minimum count of radioactivity using the ethanol precipita- tion method will be 150,000 cpm. 13. Half of the time required to expose an autoradiography film in the standard procedure is a good rule of thumb for selecting an exposure time using a phosphoimager. See Subheading 2.1.4 for materials. 14. When scanning the large screens, it is mostly necessary to restart the computer; when scanning small (20 × 40) screens, it is mostly unnecessary. 15. If the scanning stops in the middle of the scanning for any reason (e.g., crashes), a border line will be created in your image and this will remain even though one starts a new scan. It is better to reexpose image if this happens. 108 H. Gainer et al.

16. If storing for extended period of time, keep solution in the −80°C freezer. 17. This protocol is based on Qiagen kit QuantiTect SYBR Green RT-PCR Handbook and a 25-mL reaction volume in the Smart Cycler System. 18. Volume of template solution is usually £2 mL. The exact ­volume should be calculated based on concentration from spectropho- tometer reading. 19. This volume can be scaled up for performing duplicate or trip- licate reactions. 20. Ct values indicate a PCR cycle number at which the measured fluorescence of the indicator dye (SYBR green) corresponding to the quantity of amplified products, is increasing in an expo- nential linear fashion above background. 21. The −RT control is critical for PCR measurements using intronic primers, since in many cases the hnRNAs are of very low abundance. 22. An important issue in qPCR is how to evaluate variation in the data and how to determine whether there are statistically sig- nificant differences between the control and experimental data. The exponential nature of PCR argues against using the raw Ct values in standard statistical tests to determine statistical sig- nificance between the control and experimental samples. According to several experts in the qPCR field variation in the data should not be calculated using raw Ct values since these are logarithmic units, and the data used for calculation of coef- ficients of variations must be linear values (25, 30). Therefore, it is recommended that first a log transformation of normalized relative values should be done, followed by mean centering and autoscaling to remove outliers before statistical analysis (for details about these methods, see (30)).

Acknowledgments

We wish to thank various past and present members in our LNC, NINDS, NIH laboratory, Noriko Mutsuga, Ray Fields, Shirley House, Daniel Lubelski, and Madison Stevens, and colleagues in other NIH institutes, Eva Mezey, Sharon Key and Zuszanna Toth in the NICDR, and W. Scott Young in NIMH for their help in establishing the qISH and qPCR methods in our laboratory. This work was supported by the intramural program of the NIH, NINDS. 5 Intron–Specific Probes 109

References

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PCR using superscript III J. Biomolecular 2(−Delta Delta C(T)) Method. Methods 25: Techniques 16: 266–271. 402–408. 25. Wong, M.L., and Medrano, J.F. (2005) Real 28. Pfaffl, M. W. (2001) A new mathematical model time PCR for mRNA quantitation. Biotechniques for relative quantification in real-time RT-PCR. 39: 75–85. Nucleic Acids Res. 29: e45. 26. Giegerich, R., Meyer, F., and Schleiermacher, 29. Schmittgen, T. D., and Livak, K. J. (2008) C. (1996) GeneFisher – software support for Analyzing real-time PCR data by the compara- the detection of postulated genes. Proc. Int. tive C(T) method. Nat. Protoc. 3: 1101–1108. Conf. Intell. Syst. Mol. Biol. 4: 68–77. 30. Willems, E., Leyns, L., and Vandesompele, J. 27. Livak, K. J., and Schmittgen, T. D. (2001) (2008) Standardization of real-time PCR Analysis of relative gene expression data expression data from independent biological using real-time quantitative PCR and the replicates. Anal. Biochem. 379: 127–129. Chapter 6

Direct In Situ RT-PCR

Laura Lossi, Graziana Gambino, Chiara Salio, and Adalberto Merighi

Abstract

In situ polymerase chain reaction (PCR) is a histological technique that exploits the advantages of PCR for detection of mRNA directly in tissue sections. It somehow conjugates together PCR and in situ hybridiza- tion that is more traditionally employed for mRNA localization in cell organelles, intact cells, or tissue sections. This chapter describes the application of in situ PCR for neuropeptide mRNA localization. We provide here a detailed protocol for direct in situ reverse transcription (RT) PCR (RT-PCR) with nonra- dioactive probes after fixation and paraffin embedding or cryosectioning. Digoxigenin-labeled nucleotides (digoxigenin-11-dUTP) are incorporated in the PCR product after RT and subsequently detected with an anti-digoxigenin antibody conjugated with alkaline phosphatase. The procedure can be modified for use with fluorescent probes and employed in combination with enzyme/fluorescence immunocytochemical labeling.

Key words: In situ PCR, mRNA, Reverse transcription, Digoxigenin, In situ hybridization, Alkaline phosphatase, Histology, Neuropeptides, CGRP

1. Introduction

The detection of mRNAs directly in tissue sections by the use of in situ hybridization (ISH) techniques represents the histological counterpart to Northern blotting in molecular biology. In the sev- enties and eighties of the last century, ISH has had been developed in parallel with immunocytochemistry (ICC) for detection of pro- teins and, mostly relevant to the topic of this book, small peptides. Under this perspective, ICC parallels, in histology, Western blot analysis in molecular biology. All these tools have been and still are

Adalberto Merighi (ed.), Neuropeptides: Methods and Protocols, Methods in Molecular Biology, vol. 789, DOI 10.1007/978-1-61779-310-3_6, © Springer Science+Business Media, LLC 2011 111 112 L. Lossi et al.

of notable value for investigation of neuropeptide localization and function in central and peripheral neurons (1–3). ICC has known a more widespread application and fortune than ISH, mainly because the latter has low detection sensitivity to a point that the threshold for mRNA detection is approximately 10 copies per cell. The development of polymerase chain reaction (PCR) produced a technological revolution in solution nucleic acid detection, given its high sensitivity in comparison to Northern and Southern blotting. Therefore, it is not surprising that histolo- gists have tried to join together in a single technique PCR and ISH, aiming to obtain substantial nucleic-acid amplification in intact cells and/or tissue sections. Nevertheless, the potential of in situ PCR has not been fully exploited yet, likely because the tech- nique requires very careful handling to avoid false-negative/posi- tive results (4, 5). The first application of in situ PCR was described about 20 years ago for amplification of lentiviral DNA in infected cells (6). Since then, the technique has been mainly applied for detection of viruses in human (pathological) samples and different mRNAs in cells with low levels of expression (4, 7–9). Two variants of the technique are available: direct and indi- rect in situ PCR. In the first, a label is incorporated in the ampli- con during PCR of cell DNA. To this step detection follows, by the use of different reporter molecules to localize the amplified product. In the indirect technique, the amplicon is produced by PCR of cell DNA without label incorporation and is then detected by standard ISH. Therefore, four different types of in situ amplification techniques can be described: (1) direct in situ PCR and (2) indirect in situ PCR can be used for DNA localiza- tion, whereas (3) direct in situ reverse transcription (RT) PCR (RT-PCR) and (4) indirect in situ RT-PCR can be used for RNA localization (9). Several excellent reviews have been recently published on general applications of in situ PCR in biomedical studies (10–13). We describe here a specific protocol for direct in situ RT-PCR of neuropeptide mRNAs with nonradioactive probes after fixation and paraffin embedding (Fig. 1). In direct in situ RT-PCR, mRNA sequences in cells and tissues are amplified by first creating a complementary DNA (cDNA) template by RT and then by amplification of the newly synthesized cDNA template by PCR with labeled nucleotides. The labeled PCR amplicon is then detected by ICC. To this purpose, we employ digoxigenin-labeled nucleotides (digoxigenin-11-dUTP) directly incorporated in the PCR product after RT and subsequently detect them with an anti-digoxigenin antibody conjugated with alkaline phosphatase. The procedure can be modified for use with fluorescent probes and employed in combination with enzyme/fluorescence 6 In Situ Localization of Neuropeptide Messengers 113

Fig. 1. Schematic representation of the direct in situ PCR procedure. See http://extras.springer.com/ for the color version of this figure. 114 L. Lossi et al.

ICC labeling. Given that direct in situ RT-PCR is the only nucleic acid amplification technique that allows target-specific incorpora- tion of the reported nucleotide, its main advantage lies in elimi- nation of the need for a hybridization step. This is at the same point the more important advantage and drawback of the proce- dure: from one side, elimination of the ISH step reduces the numerous problems related to the additional use of another technically demanding procedure (5), and from another side, how- ever, one must be aware that the direct incorporation of nonisoto- pically labeled nucleotides into the amplification products may lead to significant nonspecific incorporation, thus requiring a careful planning of controls (13). We have successfully used the protocol described in this Chapter for localization of neuropeptide-related receptors in cen- tral and peripheral neurons (14, 15). As an example for the use of direct in situ RT-PCR in neuropeptide mRNA detection, we show here the localization of the calcitonin gene-related peptide (CGRP) mRNA in tissue sections from mouse spinal cord and dorsal root ganglia (DRGs). In rodents and primates two CGRP isoforms have been described and referred to as aCGRP and bCGRP. aCGRP is produced by alternative RNA splicing of the CALCA (calcitonin/ calcitonin-related polypeptide, alpha) gene. bCGRP is produced from the CALCB (calcitonin/calcitonin-related polypeptide, beta) gene. aCGRP has been involved in several biological processes (16–18), and is one of the most widely studied neuropeptides in sensory systems. In particular, the peptide is synthesized in small to medium-sized DRG neurons and transported along their periph- eral and central projections to tissues and the spinal cord dorsal horn, respectively. In spinal cord, aCGRP is also synthesized by motor neurons (Fig. 2). A very recent description of CGRP tran- script in mouse tissues at various developmental stages and of their tissue expression sites has been provided by conventional ISH (19). We do not discuss the type of primary fixation for samples to be processed in the in situ RT-PCR procedures. As mentioned, most of the initial work in developing these procedures has been carried out by pathologists, with the aim of being able to apply the techniques for diagnostic purposes in retrospective studies. Therefore, most protocols have been developed for use with for- malin (or formaldehyde) fixed, paraffin embedded samples. Many of these protocols, which include the one described here, have proven to be reliable enough for consistent localization of the nucleic acid(s) of interest. Nevertheless, depending on the intracel- lular component to be studied, formaldehyde-based fixatives may not always be the most-optimal fixatives for nucleic acid preserva- tion (20–26). 6 In Situ Localization of Neuropeptide Messengers 115

Fig. 2. Localization of aCGRP mRNA in the mouse spinal cord and DRGs after direct in situ RT-PCR and alkaline phosphatase immunolocalization. (a–c) Localization of aCGRP transcripts in spinal cord under different conditions of tissue pretreat- ments: (a) nonspecific DNA synthesis results in massive aspecific nuclear staining (arrows) in a sample that has not been treated with DNase; (b) a too long proteinase K treatment destroys structure and large areas of complete tissue (asterisks) can be easily seen under the microscope; (c) optimal localization of signal specifically in the cytoplasm of ventral horn motor neurons. (d) Localization of mRNA in medium to large DRG neurons (arrows). Original magnifications: a, b = 20×; c = 40×; d = 10×.

2. Materials

2.1. Buffers 1. DEPC water: Add 1 mL of 0.1% diethylpyrocarbonate (DEPC) and Solutions to 1,000 mL of MilliQ-purified water. Mix for 1 h at 37°C and autoclave the solution (see Note 1). 2. DEPC-phosphate buffer (DEPC-PB): Prepare a stock solution

“A” with 0.2 M NaH2PO4∙H2O and a stock solution “B” with

0.2 M Na2HPO4. Prepare working solutions by adding 19 mL “A” and 81 mL “B” solutions to 100 mL of DEPC water. Adjust to pH 7.4 if necessary. Store at room temperature. 116 L. Lossi et al.

3. DEPC-phosphate-buffered saline (DEPC-PBS): Prepare a 9‰ NaCl solution in 900 mL DEPC water and add 100 mL 0.2 M DEPC-PB. Adjust to pH 7.4 if necessary. Store at 4°C. 4. Fixative – 4% paraformaldehyde in DEPC-PBS: Prepare a 4% (w/v) paraformaldehyde solution (available from Sigma, St. Louis, MO), in DEPC-PBS. Dissolve the solution by carefully heating it with a stirring hot plate in a fume hood, then cool at room temperature and finally store at 4°C. 5. 0.1 M HCl: Prepare a 10× stock solution (1 M) by diluting 8.33 mL of 37% HCl to 100 mL with DEPC water. For use, dilute to 1:10 with DEPC water. 6. Proteinase K: Prepare a 10 mg/mL Proteinase K solution in DEPC water. 7. Triethanolamine stock solution: Dissolve 1.85 g trietha- nolamine in 100 mL DEPC water. Adjust pH to 8.0. 8. Alkaline phosphatase (AP) buffer: To 10 mL distilled water add 0.12 g Tris (100 mM), 0.058 g NaCl (100 mM), and 0.002 g MgCl (1 mM).

2.2. Tissue Preparation 1. Cryostat or microtome. 2. Water bath (for paraffin sections only). 3. SuperFrost®Plus microscope slides 25 × 75 mm (Menzel, Braunschweig, Germany – see Note 2). 4. Vertical Coplin jars or staining dishes (available from Ted Pella, Redding, CA). 5. Slide oven (available from Electron Microscopy Sciences, Hatfield, PA).

2.3. RT 1. Reverse Transcription System (Promega Corporation, Madison, WI). 2. Gene Frame® slide chamber coverslips (ABgene Plastics, Epsom, UK). 3. PAP Pen (available from Abcam, Cambridge, UK).

2.4. PCR 1. PCR Master Mix (Promega Corporation). 2. Forward and reverse primers. For aCGRP amplification: Fw TGGTTGTCAGCATCTTGCTC Rv CAACACGATGCACAATAGGC 3. Digoxigenin-11-dUTP (Roche Diagnostics, Mannheim, Germany). 4. Omnislide Thermal Cycler (Thermo Fisher Scientific, Waltham, MA). 6 In Situ Localization of Neuropeptide Messengers 117

2.5. Immunodetection 1. Anti-digoxigenin antibody alkaline phosphatase conjugated and Visualization (Roche Diagnostics) or anti-digoxigenin antibody fluorescein conjugated (available from Sigma Chemicals). 2. 5% (v/v) normal serum (NS, available from Vector Labora­ tories, Burlingame, CA), made in the same species of the anti- digoxigenin antibody and diluted in PBS. In alternative, use 6% (w/v) bovine serum albumin (BSA), fraction V (Sigma Chemicals) in PBS. 3. 4-Nitroblue tetrazolium (NBT, Sigma Chemicals): Prepare a NBT stock solution by dissolving 100 mg NBT in 2 mL N,N dimethylformamide 70% in distilled water (see Note 3). 4. 5-Bromo-4-chloro-3-indolyl phosphate (BCIP, Sigma Chemicals): Prepare a BCIP stock solution by dissolving 100 mg BCIP in 4 mL N,N dimethylformamide 50% in dis- tilled water (see Note 3). 5. DPX mounting medium (available from Electron Microscopy Sciences). 6. Prolong® Gold Antifade Reagent mounting medium for fluo- rescence microscopy (Invitrogen, Carlsbad, CA).

3. Methods

Like any other in situ molecular procedure, direct in situ RT-PCR for mRNA localization is associated with the risks of a false-positive background and/or a lack of positive signal in the presence of the target molecule. Fortunately, the false-positive nonspecific signal is localized to the cell nucleus, whereas target-specific mammalian mRNAs are localized to the cytoplasm. Direct in situ RT-PCR utilizes direct incorporation of the reported nucleotide during PCR amplification. It is obvious that the labeled nucleotide will be incorporated into any type of DNA molecule that is synthesized during PCR, irrespectively of the fact that it is derived from the target-specific cDNA or nonspecific DNA inside the cell. Primer- independent DNA synthesis (27) and mispriming (28) are the two forms of nonspecific DNA synthesis that can be operative inan intact cell. Nicks in DNA induce primer-independent DNA ­synthesis. They can be generated from several sources, among which paraffin embedding is of notable importance to the present discussion. To eliminate primer-independent DNA synthesis one can either degrade the naïve cellular DNA using DNase digestion or avoiding the DNA nicks to form, an impossible task in paraffin- embedded material. As an alternative, one can use cryostat sections in association with hot-start PCR (29) to eliminate mispriming. A preliminary step to DNase digestion is protease digestion that is 118 L. Lossi et al.

essential for DNase and reverse transcriptase to access cellular DNA and RNA, respectively. Protease digestion has the primary purpose to break the protein–DNA and protein–protein cross-links that are formed after aldehyde fixation. Therefore, not only protease diges- tion is essential to the elimination of false-positive nuclear staining after DNase treatment but also it is of paramount importance to allow for optimal RT, thus avoiding false-negative results. Central nervous system (CNS) samples are very resistant to protease diges- tion, and the optimal digestion time must be determined empiri- cally in each set of experiments (30). Overdigestion, on the contrary, results in loss of cell and tissue integrity, with poor borders of individual cells and poor nuclear counterstaining (when applied).

3.1. Tissue Preparation 1. Distend paraffin sections in a water bath filled with DEPC water. 3.1.1. Preparation ® of Paraffin Sections 2. Collect the sections on SuperFrost Plus microscope slides and for In Situ RT-PCR allow them to dry protected from dust (see Note 4). 3. Bring the sections to DEPC water (see Note 5) in a vertical Coplin jar by progressive passages in xylene and a graded series of ethanol. 4. Fix for 10 min at room temperature in 4% paraformaldehyde in DEPC-PBS. 5. Wash two times for 10 min each at room temperature in DEPC-PBS. 6. Incubate for 10 min at room temperature in 0.1 M HCl (see Note 6). 7. Wash thoroughly in DEPC water. 8. Incubate for 10–30 min at room temperature or at 37°C in the proteinase K solution (see Note 7). 9. Wash two times for 10 min each at room temperature in DEPC-PBS. 10. Fix for 10 min at room temperature in 4% paraformaldehyde in DEPC-PBS. 11. Wash two times for 10 min each at room temperature in DEPC-PBS. 12. Incubate for 10 min at room temperature in acetic anhydride 0.3% in 0.1 triethanolamine. Dilute to 1:10 the triethanolamine stock solution in DEPC. In a vertical Coplin jar, add 750 mL acetic anhydride to 300 mL ready-to-use triethanolamine solution (see Note 8). 13. Wash two times for 10 min each at room temperature in DEPC-PBS. 14. Wash thoroughly in DEPC water. 15. Dry slides in oven at 37°C. 6 In Situ Localization of Neuropeptide Messengers 119

3.1.2. Preparation 1. Collect sections on SuperFrost®Plus microscope slides and of Cryostat Sections allow to dry protected from dust (see Note 4). for In Situ RT-PCR 2. Fix for 10 min at room temperature in 4% paraformaldehyde in DEPC-PBS. 3. Wash two times for 10 min each at room temperature in DEPC-PBS. 4. Incubate for 10 min at room temperature in 0.1 M HCl (see Note 6). 5. Wash thoroughly in DEPC water. 6. Dry slides in oven at 37°C.

3.2. RT 1. Prepare 20 mL of Reverse Transcription System solution for each section (see Note 9). The solution is prepared according to

the manufacturer instructions as follows: 4 mL 25 mM MgCl2 (see Note 10), 2 mL Reverse Transcription 10× Buffer, 2 mL 10 mM dNTP Mixture, 0.5 mL Recombinant RNasin® Ribonuclease Inhibitor, 0.75 mL AMV Reverse Transcriptase (High Conc.), 1 mL Oligo(dT)15 Primer (see Note 11), Nuclease-Free Water to a final volume of 20 mL (9.75 mL if the

amount of MgCl2 is unchanged). 2. Fix the Gene Frame® slide chamber coverslips over sections or use a PAP Pen to circle sections (see Note 12). 3. Put a 20 mL drop of Reverse Transcription System solution over each section. To avoid evaporation put the Gene Frame® coverslip on the top of slide chamber by simply lowering it over the sections, but without sealing the chamber. If you are not using the Gene Frame® slide chamber, simply put a clean coverslip over the solution drop, being very careful to avoid air bubbles and not to damage sections (see Note 13). 4. Program the thermal cycle as follows: step 1, 10 min at room temperature; step 2, 30 min at 42°C (see Note 14). 5. Wash thoroughly in DEPC water (see Note 15). 6. Dry slides in oven at 37°C.

3.3. PCR 1. Program the thermal cycle according to the required PCR ­protocol. In the example, amplification of retrotranscribed CGRP cDNA is carried out as follows (see Note 16):

1×: 95°C 5 min 35×: 95°C 1 min 60°C 40 s 72°C 50 s 1×: 72°C 5 min 120 L. Lossi et al.

2. Prepare 25 mL of PCR Master Mix solution for each section (see Note 9). The solution is prepared according to the ­manufacturer instructions as follows: 12.5 mL PCR Master Mix 2×, 0.5 mL 1 mM digoxigenin-11-dUTP (see Note 17), ­0.25–2.5 mL 10 mM Upstream Primer (see Note 18), 0.25– 2.5 mL 10 mM Downstream Primer (see Note 18), Nuclease- Free Water to a final volume of 25 mL. 3. Wash thoroughly in distilled water (see Note 19).

3.4. Immunodetection 1. Wash two times for 10 min each at room temperature in PBS and Visualization or distilled water (see Note 15).

3.4.1. Detection 2. Incubate the sections overnight in a humid chamber with drops of Amplicons with Alkaline of the anti-digoxigenin antibody alkaline phosphatase conju- Phosphatase ICC gated (1:1,000 in NS-PBS). 3. Develop the alkaline phosphatase reaction as follows: Prepare a working solution by adding 7 mL of NBT stock solution to 1 mL alkaline phosphatase buffer, mix well and then add 7 mL of BCIP stock solution. Put drops of 20–25 mL directly on the tissue sections. Development time is usually around 7 min (see Note 20). 4. Stop the reaction by incubating the sections in alkaline phos- phatase buffer. 5. Counterstain in nuclear Fast red for 5 min (optional). Dehydrate and mount the sections in DPX.

3.4.2. Detection 1. Wash two times for 10 min each at room temperature in PBS of Amplicons with or distilled water (see Note 15). Immunofluorescence 2. Incubate the sections with drops of the anti-digoxigenin anti- body fluorescein-conjugated (1:50). 3. Wash thoroughly in PBS and distilled water. 4. Mount the sections in fluorescence-free medium.

4. Notes

1. DEPC is an inhibitor of RNase. Since RNA is a very labile molecule, it is mandatory that all procedures preceding RT are carried out under RNase-free conditions. To this purpose, use sterile glassware (baked overnight in a 250–300°C oven) or disposables, and wear gloves in all steps of the procedure. It is strongly advisable to maintain these precautions also for PCR. 2. It is of paramount importance that sections adhere perfectly to glass slides, since they tend to detach from support during the preparative steps and the RT-PCR procedure. SuperFrost®Plus 6 In Situ Localization of Neuropeptide Messengers 121

microscope slides have strong adhesion capacity due to electric charge interactions with tissue. In some protocols, the use of slides coated with different types of adhesives (white glue, albumin, chrome-gelatine, poly-l-lysine, silane) is recom- mended (13). However, we strongly discourage their use. Sections should be less than 10 mm thick to permit better adhesion and reagents to reach mRNAs. 3. NBT and BCIP stock solutions are stable for about 1 year at 4°C in the dark. 4. Sections are better distended and dried in a slide oven at 37°C protected from dust. Carefully inspect slides for the presence of air bubbles below sections. If bubbles are present, discharge sections, since adhesion to slide may be compromised. 5. Hydration of sections must be carried with series of alcohols at decreasing concentrations (100, 80, and 70%) prepared with DEPC. 6. HCl pretreatment is used to denaturate cellular DNA and to improve reagent penetration in tissue sections. This treatment can be omitted if unnecessary. 7. The proteinase K pretreatment has the purpose to degrade protein–protein and protein–DNA networks to facilitate pen- etration of reagents into tissue sections. The treatment may be detrimental for tissue structure. Therefore, it may be necessary to vary the proteinase K concentration and length of incuba- tions according to the type of tissue employed. As an alterna- tive to proteinase K, it is possible to use pepsin. For CNS samples, pepsin should be used at high concentration (2 mg/mL in 0.2 N HCl) for 60 min at 37°C (5). To determine the optimal protease digestion time, it is recommended to digest three sections on the same slide for 20, 40 and 60 min respec- tively, followed by direct incorporation of the reporter nucle- otide for 30 min at 55°C. The protease digestion time that gives the strongest signal in the greatest number of nuclei is the optimal digestion time. Once the latter has been deter- mined, use another slide from the same sample and subject at least one section to overnight DNase digestion (10 U RNase- free DNase per tissue section: add 10 mL of PCR buffer to 80 mL of RNase-free water to 10 mL of RNase-free DNase). Under these conditions, the signal after direct incorporation of the reporter nucleotide should be completely eliminated. If some signal persists, try a longer protease digestion time. An additional alternative to proteinase K treatment is the use of microwaves (31). It must be stressed out here that very differ- ent opinions exist among authors as regard the best treatment for degrading genomic DNA. Therefore, each laboratory has to empirically establish the most reliable protocols for the need of its research. 122 L. Lossi et al.

8. Treatment with acetic anhydride results in tissue acetylation and prevents nonspecific sticking of primers to sections. This treatment can be omitted if unnecessary. 9. The amount indicated (20 mL) is enough for small sections. Large sections such as coronal sections of an adult rat or mouse brain require a higher amount of fluid (40–50 mL). The volume of the reaction mixture applied to each section should be enough to completely cover it and to avoid complete evapora- tion during tissue heating.

10. The concentration of MgCl2 may vary depending on the template, primer, and dNTP concentrations in the amplifica- tion reaction. The suggested magnesium concentration may be optimized for any given sequence to achieve better yields. To do so, it is preferable to run a tube RT-PCR experiment prior

to the in situ procedure to determine the optimal MgCl2 con- centration for the amplification reactions (Fig. 3). 11. Reverse transcription may be primed with either Oligo(dT)15 or random primers. Choose Oligo(dT)15 when priming at the 3¢ poly(A) region is desired. Choose random primers when priming throughout the length of the RNA is desired. Oligo(dT)15 is frequently used when cDNA will be used for cloning and RT-PCR. Random primers are sometimes pre- ferred for cDNA that will be used in RT-PCR, especially when the PCR primers target a region near the 5¢-end of the RNA.

Fig. 3. Tube RT-PCR detection of aCGRP mRNA in mouse spinal cord and DRGs. The pres- ence of aCGRP transcripts is confirmed by the band of approximately 500 bp, the pre- dicted size of the amplifieda CGRP cDNA (555 bp). The other band corresponds to the size of the amplified HPRT-1 (hypoxanthine phosphoribosyl-transferase 1) that was used as a normalization gene to correct for RNA concentration and RT efficiency. Lanes: A = mw marker; B, E = DRGs; C, F = spinal cord; D, G = spinal cord organotypic cultures; H = nega- tive control. 6 In Situ Localization of Neuropeptide Messengers 123

Specific downstream primers 3¢ to the gene of interest may be substituted for the Oligo(dT)15 or random primers. 12. The Gene Frame® slide chamber coverslips are made by an adhesive frame with different strength adhesives on the two sides. The side that adheres to the glass slide has an easy release adhesive, whereas the side adhering to the coverslip forms a stronger bond. Be careful not to invert sides during assembly. PAP Pen is a special marking pen that provides a thin film-like hydrophobic barrier when a circle is drawn around a specimen on a slide. This water repellent barrier is insoluble in alcohol and acetone and keeps staining reagents localized on the tissue sections, thus preventing mixing of reagents when differentially staining two sections on the same slide. It allows for use of fewer reagents per section. PAP Pen markings can be removed with xylene after the staining pro- cedure is complete. 13. The use of the Gene Frame® slide chamber coverslips is some- how preferable to that of the PAP Pen since markings tend to dissolve during heating in the PCR procedure. However, it may be difficult to place different chambers on the same glass slide when multiple sections are collected on a single slide, as it is often the case since it is advisable to directly compare efficacy of different tissue pretreatments. Moreover, it is suggested that during the RT step the coverslip is simply put on top of the frame without removing the protecting film from the adhesive surface. By this means, it will be easily removed during subse- quent wash. If instead the chamber is sealed, it will be neces- sary to remove it and use a new chamber during the PCR amplification step. As an alternative it is possible to employ the patented Self-Seal reagent (BioRad, Hercules, CA) that provides an effective method for sealing slides for high temperature thermal cycling (32). 14. Heat inactivation of reverse transcriptase, which is commonly employed for tube RT-PCR, is not necessary, as the enzyme is removed during the subsequent washing of the slides. 15. Be very careful at this stage not to damage sections while removing coverslips. The washing is better performed by first immersing slides with coverslips in a vertical Coplin jar and then, after a few minutes of gentle agitation, by carefully lifting coverslips from sections while still immersed in DEPC water. 16. It is advisable to run a tube RT-PCR experiment prior to the in situ procedure to choose the optimal conditions for amplifica- tion (Fig. 3). This type of experiment is useful for determining the initial cycling parameters to start with for use during the in situ procedure. We suggest that these parameters are not altered for the in situ PCR unless strictly necessary: increase in 124 L. Lossi et al.

number of cycles below the point of saturation of Taq DNA polymerase is not associated with any amelioration of the signal- to-background ratio. In the example given, the in-tube proce- dure was carried out as follows:

1×: 95°C 5 min 35×: 95°C 1 min 60°C 1 min 70°C 50 s 1×: 72°C 5 min

Differences with the in situ procedure in the example have been introduced to reduce non specific background on sections. Specifically, they consisted in the reduction in the duration of the annealing step, and increase in the temperature of the elon- gation step of cycle. 17. The reaction solution for in situ PCR is very similar to that for the solution-phase reaction. In some protocols, it is suggested to add 2% BSA stock solution at the final concentration of 0.06% (4). Nonetheless, the most important difference is the addition of digoxigenin-11-dUTP as a reporter molecule in the PCR mixture. Initial work was done at a 20 mM final con- centration. This parameter cannot be obviously tested under liquid-phase conditions. We have carried out a series of experi- ments to establish the optimal digoxigenin-11-dUTP molarity in the in situ PCR amplification. Dilution to 10 mM signifi- cantly reduces background staining and is not associated with any apparent decrease in specific stain. 18. It is advisable to start with 1.5 mL and, if necessary, to further adjust primer concentration. Again, the preliminary experi- ments with solution-phase amplification should give useful information as regarding the need to increase primer concen- tration. In our conditions, the final primer concentration is 0.6 mM, but certain protocols use a concentration up to 20 mM (13). 19. It may be of help to wash slides at 60°C in 0.1× SSC/2% BSA for 10 min to remove any labeled primer dimers that may have formed in the PCR solution and weakly binding to proteins and/or nucleic acids in sections. 20. The mix must be used within 1 h. Carefully check for absence of precipitates. If incubation time becomes longer or the reac- tion product tends to be pinkish rather than blue, discard the solution and prepare new stocks. 6 In Situ Localization of Neuropeptide Messengers 125

Acknowledgments

This work was supported by grants of the Italian MiUR (Fondi PRIN 2007 and 2008) and Compagnia di San Paolo Torino to LL and AM. We are greatly indebted to Gianfranco Zanutto for his excellent artwork.

References

1. Dagerlind, A., Friberg, K., Bean, A. J., and 13. Bagasra, O. (2008) In situ polymerase chain Hökfelt, T. (1992) Sensitive mRNA detection reaction and hybridization to detect low- using unfixed tissue: combined radioactive and abundance nucleic acid targets. Curr.Protoc. non-radioactive in situ hybridization histochem- Mol.Biol. Chapter 14:Unit 14.8. istry. Histochemistry 98, 39–49. 14. Vergnano, A. M., Ferrini, F., Salio, C., Lossi, 2. Penschow, J. D. and Coghlan, J. P. (1994) L., Baratta, M., and Merighi, A. (2008) The Hybridization histochemistry using radiola- ghrelin modulates beled oligodeoxyribonucleotide probes. Methods inhibitory neurotransmission in deep laminae Mol.Biol. 33, 277–292. of mouse spinal cord dorsal horn. Endocrinology. 3. Penschow, J. D. and Coghlan, J. P. (1994) 149, 2306–2312. Subcellular location of mRNA by electron 15. Ferrini, F., Salio, C., Lossi, L., Gambino, G., microscope hybridization histochemistry. and Merighi, A. (2010) Modulation of inhibi- Methods Mol.Biol. 33, 345–358. tory neurotransmission by the vanilloid receptor 4. Nuovo, G. J. (2000) In situ localization of type 1 (TRPV1) in organotypically cultured PCR-amplified DNA and cDNA. Methods Mol. mouse substantia gelatinosa neurons. Pain. Biol. 123, 217–238. 150, 128–140. 5. Nuovo, G. J. (2001) Co-labeling using in situ 16. Seybold, V. S. (2009) The role of peptides in PCR: a review. J.Histochem.Cytochem. 49, central sensitization. Handb.Exp.Pharmacol. 1329–1339. 451–491. 6. Haase, A. T., Retzel, E. F., and Staskus, K. A. 17. Benemei, S., Nicoletti, P., Capone, J. G., and (1990) Amplification and detection of lentiviral Geppetti, P. (2009) CGRP receptors in the DNA inside cells. Proc.Natl.Acad.Sci.U.S.A 87, control of pain and inflammation. Curr.Opin. 4971–4975. Pharmacol. 9, 9–14. 7. Komminoth, P. and Long, A. A. (1993) In-situ 18. Todd, K. J. and Robitaille, R. (2006) Neuron- polymerase chain reaction. An overview of glia interactions at the neuromuscular synapse. methods, applications and limitations of a new Novartis.Found.Symp. 276, 222–229. molecular technique. Virchows Arch.B Cell 19. Rezaeian, A. H., Isokane, T., Nishibori, M., Pathol.Incl.Mol.Pathol. 64, 67–73. Chiba, M., Hiraiwa, N., Yoshizawa, M., and 8. Long, A. A. and Komminoth, P. (1997) In situ Yasue, H. (2009) alphaCGRP and betaCGRP PCR. An overview. Methods Mol.Biol. 71, transcript amount in mouse tissues of various 141–161. developmental stages and their tissue expres- sion sites. Brain Dev. 31, 682–693. 9. Broholm, H. and Gammeltoft, S. (2002) In Cellular and molecular methods in neuroscience 20. Wester, K., Asplund, A., Backvall, H., Micke, P., research (Merighi, A. and Carmignoto, G., Derveniece, A., Hartmane, I., Malmstrom, P. U., eds.), pp. 145–159, Springer-Verlag, New York and Ponten, F. (2003) Zinc-based fixative improves preservation of genomic DNA and 10. Bagasra, O. and Harris, T. (2006) In situ PCR proteins in histoprocessing of human tissues. protocols. Methods Mol.Biol. 334, 61–78. Lab Invest 83, 889–899. 11. Bagasra, O. and Harris, T. (2006) Latest devel- 21. Dotti, I., Bonin, S., Basili, G., Nardon, E., opments in in situ PCR. Methods Mol.Biol. Balani, A., Siracusano, S., Zanconati, F., 334:221–40., 221–240. Palmisano, S., De, M. N., and Stanta, G. (2010) 12. Bagasra, O. (2007) Protocols for the in situ Effects of formalin, methacarn, and fineFIX PCR-amplification and detection of mRNA and fixatives on RNA preservation. Diagn.Mol. DNA sequences. Nat.Protoc. 2, 2782–2795. Pathol. 19, 112–122. 126 L. Lossi et al.

22. Boon, M. E. and Kok, L. P. (2008) Theory and 27. Nuovo, G. J., MacConnell, P., and Gallery, F. practice of combining coagulant fixation and (1994) Analysis of nonspecific DNA synthesis microwave histoprocessing. Biotech.Histochem. during in situ PCR and solution-phase PCR. 83, 261–277. PCR Methods Appl. 4, 89–96. 23. van, M. F., de, W. M., Morsink, F., Musler, A., 28. Kher, R. and Bacallao, R. (2001) Direct in situ Weegenaar, J., and van Noesel, C. J. (2008) reverse transcriptase-polymerase chain reaction. Effects of processing delay, formalin fixation, Am.J.Physiol Cell Physiol. 281, C726–C732. and immunohistochemistry on RNA Recovery 29. Kramer, M. F. and Coen, D. M. (2001) From Formalin-fixed Paraffin-embedded Tissue Enzymatic amplification of DNA by PCR: stan- Sections. Diagn.Mol.Pathol. 17, 51–58. dard procedures and optimization. Curr.Protoc. 24. Lykidis, D., Van, N. S., Armstrong, A., Spencer- Immunol. Chapter 10:Unit 10.20. Dene, B., Li, J., Zhuang, Z., and Stamp, G. W. 30. Casoli, T., Stefano, G. D., Fattoretti, P., Solazzi, (2007) Novel zinc-based fixative for high qual- M., Delfino, A., Biagini, G., and Bertoni-Freddari, ity DNA, RNA and protein analysis. Nucleic C. (2003) GAP-43 mRNA detection by in situ Acids Res. 35, e85. hybridization, direct and indirect in situ 25. Cox, M. L., Schray, C. L., Luster, C. N., RT-PCR in hippocampal and cerebellar tissue Stewart, Z. S., Korytko, P. J., KN, M. K., sections of adult rat brain. Micron. 34, Paulauskis, J. D., and Dunstan, R. W. (2006) 415–422. Assessment of fixatives, fixation, and tissue pro- 31. Tesch, G. H., Lan, H. Y., and Nikolic-Paterson, cessing on morphology and RNA integrity. D. J. (2006) Treatment of tissue sections for in Exp.Mol.Pathol. 80, 183–191. situ hybridization. Methods Mol.Biol. 326, 1–7. 26. Vincek, V., Nassiri, M., Nadji, M., and Morales, 32. Sullivan, D. E., Bobroski, L. E., Bagasra, O., A. R. (2003) A tissue fixative that protects mac- and Finney, M. (1997) Self-seal reagent: evapo- romolecules (DNA, RNA, and protein) and ration control for molecular histology proce- histomorphology in clinical samples. Lab Invest. dures without chambers, clips or fingernail 83, 1427–1435. polish. Biotechniques. 23, 320–325. Chapter 7

Laser Capture Microdissection and Quantitative-PCR Analysis

Sarah J. Paulsen and Leif K. Larsen

Abstract

Laser capture microdissection (LCM) provides an efficient and precise method for the sampling of single cells or subgroups of cells in heterogeneous tissues such as the brain. We have recently applied LCM coupled with microarray analysis for the examination of gene expression in the hypothalamic arcuate nucleus of free fed versus fasted rats. We successfully used QPCR analysis to validate our findings and to confirm the regulation of several known neuropeptides. We show that the combination ofLCMand QPCR analysis provides a reliable, fast, and efficient alternative to semiquantitative in situ hybridization analysis of gene expression.

Key words: Laser capture microdissection, Quantitative PCR, Stereological sampling

1. Introduction

The power of LCM lies in the combination of direct light ­microscopic visualization of cells and laser-based microdissection. Two types of systems have been developed for LCM, each with its own advantages and possible limitations (1). Importantly, both types of systems provide efficient and precise methods for the sam- pling of single cells or subgroups of cells in heterogeneous tissues such as the brain. Here, we describe the use of the “Veritas LCM system.” The Veritas (and other Arcturus Microdissection Systems) uses an ultraviolet (UV) laser for cutting and an infrared (IR) laser for capturing. The UV laser beam is used to cut around relevant tissue. Using the IR laser, the system captures cells onto a thermo- plastic membrane attached to a “cap.” The IR laser transiently melts the thermoplastic membrane at the cap onto the cells of interest. When the plastic cools and solidifies, the cells are­embedded

Adalberto Merighi (ed.), Neuropeptides: Methods and Protocols, Methods in Molecular Biology, vol. 789, DOI 10.1007/978-1-61779-310-3_7, © Springer Science+Business Media, LLC 2011 127 128 S.J. Paulsen and L.K. Larsen

into the plastic membrane and can be removed together with the cap. Single cells can be captured using the IR laser alone, while larger areas are collected using the two lasers in combination. For identification of cells, staining is most often necessary. It is essential to prevent RNA degradation during tissue processing and staining. We have tested various staining protocols and have found staining with cresyl violet to be superior for the staining of brain tissue. Cresyl violet binds anionic macromolecules (DNA and RNA) and produces an efficient staining of the neurons of the ­central nervous system. Cresyl violet is soluble in ethanol, and it is, therefore, not necessary to hydrate the tissue prior to staining. The application of stereological sampling techniques during tissue sampling can be extremely useful when preparing samples for LCM in the central nervous system. Stereological sampling techniques enable systematic uniform random sampling of relevant brain nuclei or areas, thus eliminating sampling errors (2) and ­minimizing the workload (see Note 1).

2. Materials

2.1. Brain Removal 1. Cryostat. and Sectioning 2. −80°C freezer. 3. PEN foil-coated microscope slides (Molecular Devices, Sunnyvale, CA). 4. Tissue-Tek® O.C.T ™ Compound (Sakura Finetech, Torrance, CA).

2.2. Cresyl Violet 1. Cresyl Violet Acetate (Sigma Chemical St. Louis, MO). Staining 2. Pap jars (Evergreen Scientific, Los Angeles, CA). 3. Ethanol.

2.3. LCM Cut 1. Veritas LCM system (Molecular Devices). and Capture 2. CapSure Macro LCM Caps (Molecular Devices).

2.4. RNA Extraction 1. PicoPure™ RNA Isolation Kit (Molecular Devices). and cDNA Synthesis 2. DNA-free™ DNase (Ambion, Austin, TX).

2.5. Quantitative 1. QPCR apparatus. PCR Analysis 2. Brilliant® II SYBR® Green QPCR Master Mix (Stratagene, La Jolla, CA). 3. Primers. 7 LCM and QPCR 129

3. Methods

The following methods describe QPCR gene expression analysis with SYBR Green on tissue collected using a Veritas™ Micro­ dissection Instrument. For an introduction to other microdissec- tion systems, please refer to ref. 1. Figure 1 shows an overview of the process from tissue cutting to RNA purification. The proce- dure involves the need to work with a very delicate molecule such as RNA. Therefore, the cryostat and other relevant work stations must be cleaned appropriately for RNA work. Always wear gloves and use filter tips for RNA work. When working with RNA, it is crucial to focus on achieving a high RNA quality. However, when working with few cells and under demanding conditions as during LCM, it is often impossible to obtain RNA samples of superb quality. Thus, under these cir- cumstances, it may be necessary to limit the demand to achieving samples of comparable RNA quality. The quality of small size RNA samples can be determined using a microfluidics-based platform (Agilent’s Bioanalyzer or Bio-Rad’s

Fig. 1. Method overview. (a) Tissue sampling followed by storage at −80°C. (b, c) Cryocutting at −20°C applying stereological principles followed by storage at −80°C. (d) Cresyl-violet staining of tissue. Only tissue from one animal processed at a time. (e, f ) Laser capture microdissection of the relevant hypothalamic area. (g) Tissue extraction followed by storage at −80°C. (h) RNA purification using PicoPure columns. Following purification the RNA is ready for cDNA synthesis. Reprint from (3) with permission from Elsevier. 130 S.J. Paulsen and L.K. Larsen

Table 1 Comparison of RIN value obtained using an Agilent Bioanalyzer with 5¢-, 3¢-ratios determined for rat glyceraldehyde 3-phosphate dehydrogenase (GAPDH) using QPCR. The following primers were used: GAPDH 5¢ end L: ACATTGTTGCCATCAA CGAC. GAPDH 5¢ end R: CTTGCCGTGGGTAGAGTCAT. GAPDH 3¢ end L: TTCTTCC ACCTTTGATGCTG. GAPDH 3¢ end R: ATGTAGGCCATGAGGTCCAC

RIN 28S/18S GAPDH 5¢ end GAPDH 3¢ end GAPDH QPCR 5¢/3¢

9.15 1.55 1.17 1.17 1.00 8.50 1.30 1.87 1.93 1.03 7.70 1.05 1.77 1.81 1.02 7.00 0.90 1.48 1.55 1.05 6.60 0.65 1.10 1.18 1.07 5.35 0.20 0.89 1.25 1.40 4.60 0.10 0.79 1.35 1.72 2.80 0.00 0.17 1.02 5.97

Experion) or by measuring mRNA integrity using QPCR. In the latter method, the quantitative ratio between the 5¢ and 3¢ ends of a candidate gene is determined using QPCR analysis. For this approach, first-strand cDNA synthesis is employed using oligo(dT) primers, and the generated cDNA is then used as template in two separate QPCRs with amplicons detecting either 5¢- or 3¢-proximal sequences. We have compared the RIN value obtained using an Agilent Bioanalyzer with 5¢-, 3¢- ratios determined for rat glyceral- dehyde 3-phosphate dehydrogenase (GAPDH NM_017008). RNA of varying quality was obtained by degrading rat brain RNA by heating it to 72°C for varying lengths of time. The results as well as primer information are shown in Table 1 and may be used for similar analyses of rat RNA quality.

3.1. Brain Removal 1. Gently and immediately remove brains from sacrificed animals. and Sectioning Snap-freeze brains on powdered dry ice (see Note 2). Once frozen, store at −80°C. 2. Remove tissue from storage at −80°C, bring to the precooled cryostat and allow acclimatization for a few minutes. Never allow tissue to thaw. We always transport tissue and slides in the cold, by keeping them in boxes with dry ice. 3. Mount tissue in the cryostat with Tissue-Tek. 4. Cut 10-mm thick sections onto room-temperature PEN Foil- Coated microscope slides. If stereological sampling principles 7 LCM and QPCR 131

are applied and series of sections are cut and mounted onto individual slides, keep slides in the cryostat during the entire cutting procedure until all sections are collected onto that par- ticular slide. It may be necessary to heat the area of the slide onto which subsequent sections are placed. This can be done by pressing a finger onto the glass side (back) of the slide. 5. After the cutting, store the sections in the cryostat for at least 5 min to allow the tissue to dry (important as it promotes a better adhesion of the tissue to the slide). 6. Store the slides at −80°C until LCM.

3.2. Cresyl Violet Process one slide at a time just prior to LCM. Staining 1. Remove the sections from −80°C freezer and allow them to thaw at +4°C for a couple of minutes. 2. Place the sections in the cresyl violet stain solution (see Notes 3 and 4) at +4°C for approximately 5 min. 3. Remove the sections from the stain, drain excess stain on filter paper, and dehydrate by 1× 96% ethanol and 2× 100% ethanol (approximately 30 s in each). The ethanol is stored at −20°C prior to dehydration. Preferably, this step should take place at +4°C. Alternatively, keep the liquids on ice during staining. 4. Dry the dehydrated sections for a couple of min in a fume hood at room temperature (see Note 5).

3.3. LCM Cut 1. Turn on the Veritas Microdissection Instrument. Preferably, and Capture this should be done a few minutes before use to allow the machine to warm up. After the warm-up, turn on the com- puter and open the Veritas software. 2. Load caps into the cap loading tray, and slides into the slide holders. The instrument now automatically adjusts focus and light intensity and prepares a “roadmap” of the slide. Use this roadmap for navigation in your tissue. 3. Prepare a suitable capture group. The idea is to have the instru- ment automatically placing spots for the capture laser. For rather large areas, enable tabs and set the number of tabs to for example 6 with a spacing of 750 and a size of 2. Choose for example three spots per tab (settings can be stored). 4. Place the cap on the slide ensuring that the cap covers both tissue and blank slide. On the blank part of the slide, make sure to focus the cutting laser and the capture laser. Use the 10× objective. 5. Adjust the capture laser. Use of the capture laser should result in the appearance of a clear round “doughnut” image on the slide view. As the capture laser does not harm the tissue, it can be advantageous to use quite high settings, e.g., a power of 132 S.J. Paulsen and L.K. Larsen

70 mW, a pulse of 2,500 ms, two hits with a delay of 10 ms, and an intensity of 200 mV. 6. Move to an area of the slide with tissue and adjust the cutting laser. The cutting laser obviously destroys the tissue, so here the power should be as low as possible while still cutting nicely through the tissue. In the menu “Microdissection” choose “Cut and Capture.” Use the knife or circle tool to draw a figure (not your region of interest!) and cut this out using the “Go – Cut and Capture” tool. During this step, it can also be checked that the capture laser settings work well on the actual tissue. 7. Move the cap to a blank part of the slide to examine that the tissue was actually placed on the cap during step 6. This can easily be done by visualization with a 2× objective. If success- ful, offload the cap to the offload station and begin your actual sampling (see Note 6). When both the UV and IR lasers are used for sampling, use the IR first “Go – capture and cut.” 8. Once all relevant tissue has been collected, move the cap to the offload station, and open the door. Immediately place the cap onto a 0.5-mL tube with extraction buffer (available from the PicoPure™ RNA Isolation Kit, see below) and place this at 42°C for 30 min to lyse the cells. After the incubation time, the buffer can be spun down and stored at −80°C until RNA extraction.

3.4. RNA Extraction 1. Perform RNA extraction using the PicoPure™ RNA Isolation and cDNA Synthesis Kit as recommended by manufacturer, except for a decreased incubation time for DNase treatment (7.5 min). The RNA can be eluted in as little as 11 mL elution buffer. 2. Synthesize cDNA with the AffinityScript QPCR cDNA Synthesis Kit as recommended by the manufacturer. Use random nonamer primers or a mixture of random nonamer and oligo(dT)primers. 3. Dilute the cDNA 20 times prior to QPCR analysis.

3.5. Quantitative In the following section, we describe QPCR using SYBR Green II. PCR Analysis SYBR Green is a DNA binding dye which increases its fluorescence signal intensity when bound to double stranded DNA. Thus, as PCR amplification increases the amount of dsDNA in a reaction, the SYBR Green signal is increased proportionally. SYBR Green is simple to use and cost-effective and thus represents a first starting point for optimization of QPCRs (see Note 7) as well as a good alternative to the use of more advanced probe based QPCR. 1. Primer design: Primers can be designed using primer design software (e.g., http://frodo.wi.mit.edu/primer3). The prim- ers should preferably be designed to give rise to products of around 60–150 bp, and to be intron-spanning, to ensure that cDNA and not genomic DNA is amplified during the PCR. 7 LCM and QPCR 133

A BLAST search (http://blast.ncbi.nlm.nih.gov/Blast.cgi) should be ­performed to ensure that the primers represent a unique sequence. 2. QPCR mixture: 12.5 mL SYBR Green II Master Mix, ROX reference dye (the optimal reference dye concentration depends on the QPCR instrument, 30 nM is recommended if a sensi- tive ROX filter is available), primers (optimal concentration to be determined), 5 mL diluted cDNA, and water to 25 mL. 3. QPCR settings: One cycle at 95°C for 10 min for QPCR enzyme activation. 40 or more cycles with (1) denaturation – 30 s at 95°C, (2) annealing – 30 s at 55°C (or a couple of °C

below primer Tm), and (3) elongation – 10–20 s at 72°C (time based on product length. Enzyme efficiency ~1,000 bp/min). Melting curve. 4. Primer sets are tested, and an optimal primer concentration is determined. The primers may be tested in a concentration of, e.g., 100, 200, 250, and 300 nM. Choose a concentration that

gives a low Ct value, where the melting curve shows only a single product, and where there is no product in a negative control (i.e., no primer–dimer formation). For this test, there is no need to work with cDNA from precious LCM samples. 5. Check the integrity of the PCR product based on visual exami- nation on an agarose gel and the SYBR Green dissociation curve (see Note 8). 6. Standard curve: Prepare a standard curve with 5–6 dilutions (e.g., four times dilutions). After QPCR the standard curve must have an R-square of the linear fit (R2) ³ 0.985, the slope must be between −3.6 and −3.3 and the PCR efficiency must be between 90 and 100%. An efficiency of 100% indicates a perfect doubling of the template in each cycle. Efficiency above 100% indicates the formation of primer dimers. The standard curve should be made with triplicates, but for subsequent work on precious LCM samples duplicates may be sufficient.

4. Notes

1. See for an example Paulsen et al. (3) for a schematic view of stereological sampling of the hypothalamic arcuate nucleus. 2. By freezing brains on powdered dry ice, the morphology is well preserved. We have tested the effect of various freezing techniques (e.g., liquid nitrogen and 2-methylbutane–dry ice bath) on RNA quality and found the use of powdered dry ice to be as good as any of the others. 3. We highly recommend the use of cresyl violet stain, as it ­produces a high and stable RNA quality downstream. If other 134 S.J. Paulsen and L.K. Larsen

stains are used, remember to test the effect of the stain on the RNA quality not only immediately after staining, but also after storage at room temperature for a period of time comparable to that needed for LCM. Pap jars are excellent for the staining procedure. 4. Preparation of cresyl violet stain: Dissolve 0.1% cresyl violet acetate in 70% ethanol (prepared with RNase free water). The stain dissolves best if the solution is heated up almost to a boil. Filter the stain when dissolved. This may conveniently be done using a filter attached to a syringe. 5. For LCM using a Veritas, the sections need to dry sufficiently to allow the local fusion with the film on the cap. Furthermore, dehydration limits RNase activity. To achieve complete dehy- dration, the use of xylene is often recommended. However, we found that xylene dehydration changes the tissue morphology to a remarkable degree. Using the method described herein (in particular limiting the tissue thickness to 10 mm), there is no need for xylene dehydration. 6. When placing the cap, be aware that the cap is centered at the spot where it is asked to be centered. Once a piece of tissue is attached to the cap, this area of the cap cannot be used to cap- ture any more tissue. Thus, for collection of multiple pieces of tissue onto the same cap it can be an advantage to first draw around the region of interest, and next, in an ordered fashion, move up, down, or to the sides from where the drawing is before the cap is placed. Figure 2 shows LCM of the hypothalamic paraventricular nucleus from rat tissue, and how the tissue from a full stereological sampling of the nuclei is placed on a cap.

Fig. 2. Laser capture microdissection of the hypothalamic paraventricular nucleus (PVN) in rat. (A) The PVN has been located. (B) The PVN has been cut and captured. (C) The PVN has been placed on the cap. The PVN was stereologically sampled during cryocutting, so the 2 × 7 PVN sections provide full coverage of the entire PVN. See http://extras.springer.com/ for the color version of this figure. 7 LCM and QPCR 135

7. For an excellent introduction to QPCR please refer to Stratagene’s “Introduction to Quantitative PCR” (4). 8. The first time primers are tested, the integrity of the PCR product should be checked by visual examination on a 2% aga- rose or a 10% PAGE gel and by analysis of the SYBR Green dissociation curve. The agarose gel should show only one band of the specific length – this product may be sequenced to ensure that it is indeed the right product. The SYBR Green dissociation curve should give one sharp peak, and the melting

point (Tm) for the product should be noted. For subsequent PCR analysis, the SYBR Green dissociation curve is sufficient

as integrity check. Small variations in a product’s Tm are to be expected.

References

1. Murray, G.I., and Curran, S. (2005) Laser 3. Paulsen, S.J., Larsen L.K., Jelsing, J., Janssen, U., Capture Microdissection: Methods and Gerstmayer, B., Vrang, N. (2009) Gene expression Protocols. Meth. Mol. Biol. Vol. 293. Humana profiling of individual hypothalamic nuclei from Press, Totowa, New Jersey. single animals using laser capture microdissection 2. Gundersen, H.J., and Jensen, E.B. (1987) The and microarrays. J. Neurosci. Meth. 177, 87–93. efficiency of systematic sampling in stereology 4. Introduction to Quantitative PCR, Methods and its prediction. J. Microscopy 147, and Applications Guide, Stratagene. Freely 229–263. available from http://www.agilent.com.

Chapter 8

Visualization of Peptide Secretory Vesicles in Living Nerve Cells

Joshua J. Park and Y. Peng Loh

Abstract

Analysis of real-time movements of peptidergic vesicles in live neurons provides insight into molecular mechanism(s) supporting the activity-dependent secretion of neurotrophins and neuropeptides. We exam- ined the effect of overexpression of exogenous peptides comprising of the cytoplasmic tail sequence of vesicular carboxypeptidase E (CPE), proposed to be involved in the mechanism of trafficking of peptider- gic secretory vesicles, in live hippocampal neurons. E16 rat hippocampal neurons were transfected with the peptidergic vesicle markers, CPE C-terminally tagged with red or green fluorescent protein, or brain- derived neurotrophic factor (BDNF) tagged with green fluorescent protein, and grown on dishes special- ized for real-time live cell visualization. Movements of peptidergic vesicles were imaged in a temperature-controlled chamber on a confocal inverted microscope and analyzed with respect to their velocity, displacement distance, and processivity.

Key words: Carboxypeptidase E, Brain-derived neurotrophic factor, Hippocampal neurons, Peptidergic secretory vesicles, Vesicle trafficking

1. Introduction

Post-Golgi anterograde transport of peptidergic vesicles to the secretion sites at the nerve terminals is a prerequisite for the activity- dependent secretion of neurotrophins and neuropeptides for neu- rotransmission or neuromodulation in various areas of the brain, such as the hippocampus (1–3). Conversely, retrograde transport of the peptidergic vesicles is also important for delivery of neurotrophin- based survival signals to the cell body, as well as for clearance and recycling of membrane proteins at the synaptic terminals (3). Our previous studies have shown that peptidergic vesicles in neuroendo- crine cells and hippocampal neurons contain a transmembrane form

Adalberto Merighi (ed.), Neuropeptides: Methods and Protocols, Methods in Molecular Biology, vol. 789, DOI 10.1007/978-1-61779-310-3_8, © Springer Science+Business Media, LLC 2011 137 138 J.J. Park and Y.P. Loh

of the prohormone processing enzyme, carboxypeptidase E (CPE), with a cytoplasmic tail that interacts with microtubule-based motors to effect vesicle transport (4). The methods for examining the molecular mechanism(s) by which neurons mediate peptidergic vesicle transport, involve conducting live neuron imaging using rat hippocampal neurons to visualize real-time movement of peptider- gic vesicles, in the absence, and in the presence of peptides compris- ing of the CPE cytoplasmic tail amino-acid sequence that is expected to interfere with transport of vesicles containing both CPE and brain-derived neurotrophic factor (BDNF). In this chapter, we describe in detail the methods used for the live neuron imaging (4). Briefly, E18 rat hippocampal neurons were transfected with the peptidergic vesicle markers, CPE tagged with either GFP (CPE- GFP), red fluorescent protein (CPE-RFP), or BDNF tagged with green fluorescent protein (BDNF-GFP). Along with the peptider- gic vesicle markers, we transfected the cytoplasmic tail of CPE

N-terminally tagged with either GFP or RFP (GFP-CPEC25 or RFP-

CPEC10), which was expected to affect real-time trafficking of CPE/ BDNF vesicles (4). Transfected live neurons were seeded on the delta TPG dish specialized for live cell imaging and imaged for ~6.7 min using confocal microscopy. The neuron images were analyzed with respect to the velocity, and the total distance and steps each pepti- dergic vesicle traveled in live neurons. The methodology described in this chapter may be used for following vesicle transport in any neuron transfected with any fluorescent-tagged vesicle protein of interest.

2. Materials

2.1. Neuron 1. E18 rat primary hippocampal neurons (Gene Therapy Systems, Transfection San Diego, CA) (see Note 1). Cultures should be ordered and Culture 1 week before transfection.

2.1.1. Hippocampal 2. 0.25 M Borate solution (Sigma Chemicals, St. Louis, MO): Neuron Culture Dilute in deionized water to make 0.1 M Borate buffer. 3. 0.01% Poly-l-lysine (Sigma Chemicals): Dilute in 0.1 M Borate buffer to make 0.001% poly-l-lysine prior to use (see Note 2). 4. 0.17-mm black delta TPG dishes (Bioptechs, Inc., Butler, PA) (see Note 3). 5. Hepes-based saline buffer (HBS). 6. Phenol red-plus/free Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (see Note 4). 7. Neurobasal/B27 medium (GIBCO-BRL, Life Technologies, Bethesda, MD) (see Note 5). 8 Imaging of Peptide Vesicle Movement in Live Neurons 139

2.1.2. DNA Constructs 1. A 5¢-XhoI-PstI-3¢ digest of PCR product of CPE tail fragments

(CPEC25 or CPEC10) amplified from full-length CPE cDNA is subcloned into pEGFP-C1 or pDsRed2 (BD Bioscience, San

Diego, CA) to generate GFP-CPEC25 or RFP-CPEC10. 2. Full-length CPE cDNA is excised from pEGFP-N1 and inserted into pDsRed-Express-N1 (BD Bioscience Clontech Co.) to generate CPE-RFP. 3. BDNF-GFP is a gift from Dr. Bai Lu (Glaxo Smith Kline, China) (see Note 6).

2.1.3. Electroporation 1. Rat Neuron Nucleofector® kit (Amaxa Inc., Gaithersburg, MD) (see Note 7). 2. Electroporation device (Amaxa Nucleofector, Amaxa Inc.). 3. 2-mm-gap electroporatable cuvettes (Amaxa Inc.). 4. 2-mL plastic spoid pipets (Amaxa Inc.).

2.2. Live Neuron 1. Zeiss Axiovert 200 M inverted microscope (Carl Zeiss, Imaging and Analysis Thornwood, NY) equipped with 63× Zeiss planapochromat oil, 1.4 NA, DIC and 100× Zeiss alpha plan fluor oil, 1.45 NA, DIC objectives. The microscope is also equipped with a closed chamber that is connected to a hot air blower controlled by a temperature sensor. 2. “LSM 510 Meta” software version 3.5 (Carl Zeiss). 3. “Metamorph” software (Molecular Devices, Downingtown, PA).

3. Methods

Compared to real-time imaging of fibroblasts and epithelial cells that are easy to transfect and manipulate, live cell imaging of neurons should be executed in a timely and punctual manner. Moreover, intra- cellular events including trafficking of peptidergic secretory vesicles in neurons are very sensitive to the environment, such as changes in tem- perature and air composition; any delay in preparation and execution should be avoided during the whole procedure. To obtain good reso- lution for individual vesicle movement, the levels of expression of fluo- rescent protein should be optimized by adjusting the amount of DNA used for transfection based on the strength of the promoter for fluo- rescent protein expression. It is also important to allow enough time after transfection of the neurons for neurite outgrowth, while post- transfection time should not be too long to over-saturate the capacity of the neurons with exogenous proteins. To achieve vesicle move- ments in the environment closest to physiological condition, only one live neuron should be imaged from one dish that is just removed from the incubator to a pre-warmed microscope. 140 J.J. Park and Y.P. Loh

During image analysis of real-time movies, it is hard to track a complete set of movements of single vesicle using an automated program such as “Single Particle Tracking” of “Metamorph” soft- ware because the tracking program loses its target easily when other vesicles cross over the region of interest on a target vesicle. The way to track a target vesicle from the beginning to the end of the movement is to follow the movement of a single vesicle by marking each movement using the method described by King et al. (5).

3.1. Neuron 1. One day before neuron delivery, coat the bottom of the delta Transfection TPG dish with 1 mL of 0.001% poly-l-lysine in 0.1 M borate and Culture buffer overnight (see Note 8). 2. Remove poly-l-lysine from the dish. 3. Wash the dish surface with 1 mL of HBS buffer twice. 4. Add 1 mL of DMEM plus 10% FBS into the dish and incubate

in 37°C + 5% CO2 incubator before use. 5. Upon neuron delivery, transfer the neurobasal medium con- taining E18 rat hippocampal neurons (2 × 106 neurons) into a 15-mL conical tube (see Note 9). 6. Completely dissociate neurons by gentle pipetting (see Note 10). 7. Spin down dissociated neurons in a bench top centrifuge for 10 min at 700 × g. Carefully remove the supernatant over ­neuron pellets by gentle pipetting (see Note 11). 8. Resuspend pelleted neurons in 200 mL of rat neuron nucleofec- tor solution for electroporation and split into two 1.5-mL tubes (100 mL per tube). Each tube will contain 1 × 106 neurons. 9. 10 mg/10 mL of control vector DNA (5 mg/5 mL) (pEGFP or pDsRed alone) or peptide inhibitor-encoding DNA (GFP-

CPEC25 or RFP-CPEC10) along with DNA (5 mg/5 mL) for vesicle marker (CPE-RFP or BDNF-GFP) is added into 100 mL of neuron suspension. 10. Transfer 110 mL of the mixture of DNA and neurons to a 2-mm-gap electroporatable cuvette (see Note 12). 11. Perform electroporation of neurons using the program C-13 in the Amaxa nucleofector device (see Note 13). 12. Immediately after electroporation, slowly and gently drop ­neurons onto 1 mL of pre-warmed 10% FBS + DMEM medium in a delta TPG dish using a 2-mL plastic spoid pipet (see Note 14). 13. Incubate neurons within the TPG dish in DMEM plus 10% FBS for 2 days. This incubation step is for recovery of neurons from the electric shock of electroporation (see Note 15). 14. Replace DMEM with neurobasal/B27 medium and incubate neurons for an additional 2 days. Transfection efficiency of <10% is expected. 8 Imaging of Peptide Vesicle Movement in Live Neurons 141

3.2. Live Neuron 1. Pre-warm the closed chamber on the Zeiss Axiovert 200 M Imaging and Analysis inverted microscope to 37°C 30 min prior to live neuron imaging. 3.2.1. Imaging 2. After temperature inside the chamber reaches 37°C, place a delta TPG dish containing transfected neurons on the stage of the microscope (see Notes 16 and 17). Figure 1 shows the best example of a neuron that has both an optimum level of fluores- cent protein-containing peptidergic vesicles and well-devel- oped neurites for live neuron imaging.

3. Image neurons expressing CPE-RFP and GFP/GFP-CPEC25

or BDNF-GFP and RFP/RFP-CPEC10 using “LSM 510 Meta” software at 1.97-s exposure/shot for 200 shots. Supplemental movie S1 shows bi-directional movements of CPE-RFP con- taining peptidergic vesicles along the neurites in live hippocam- pal neurons expressing GFP alone. 4. After taking 200 images of vesicle movements for 394 s, turn on the hot air blower connected to microscope chamber for the next live neuron imaging.

Fig. 1. Confocal image of a hippocampal neuron expressing CPE-RFP in peptidergic ­vesicles. E18 rat hippocampal neurons were transfected with CPE-RFP via electropora- tion, seeded on a poly-l-lysine-coated coverslip, and incubated in DMEM plus 10% FBS for 2 days for recovery. Then, the DMEM was replaced with neurobasal medium and incu- bated for 2 more days for neuron differentiation. Neurons on the coverslip were fixed in 3.5% paraformaldehyde and mounted on a slide for confocal microscopy (reproduced from ref. 5 with permission from Elsevier Science). Scale bar = 5 mm. 142 J.J. Park and Y.P. Loh

3.2.2. Analysis 1. Import a set of 200 images into “Metamorph” software. 2. Use Pixels in the scale bar on the image generated in “LSM 510 Meta” software to calculate mm/pixel using “Metamorph” software (4, 6). 3. Determine the displacement of CPE-RFP/BDNF-GFP vesicle on the trajectory route on the basis of x- and y-coordinates. The total number of pixels per each displacement is converted to micrometer. 4. Calculate the velocity of each vesicle movement by dividing the length (mm) of each displacement with an interval of 1.97 s. 5. Calculate the average speed of movements of each vesicle by averaging all the instant velocities shown by a single vesicle. 6. Calculate the total distance from the summation of all the dis- placements each vesicle moved. Figure 2 shows the summary of average speeds and total distances of movements of CPE- RFP-containing peptidergic vesicles in hippocampal neurons

expressing either GFP or GFP-CPEC25. Peptidergic vesicles containing CPE-RFP move at different speeds in the presence

of GFP or GFP-CPEC25. 7. Count the total step (interval) numbers from all the displace- ments of each CPE-RFP-containing peptidergic vesicle in the

presence of GFP or GFP-CPEC25. Figure 3 shows the summary of the total step (interval) numbers counted from all the dis- placements of CPE-RFP-containing peptidergic vesicles in the

presence of GFP or GFP-CPEC25.

80

60

40

20

m m) ( distance Total

0 -1 -0.5 0 0.5 1

Cell body Neurite-tip

Mean velocity (mm/s)

Fig. 2. Graphs showing analyses of live in vivo movement of CPE-RFP vesicles in ­hippocampal neurons. The velocity of vesicle movement was calculated from each dis- placement per 1.97 s, while the total distance was the summation of all the displace- ments each vesicle moved. Vesicles moving in either anterograde or retrograde direction with respect to the total number of time intervals (1.97 s) each vesicle moved were clas- sified separately (reproduced from ref. 5 with permission from Elsevier Science). 8 Imaging of Peptide Vesicle Movement in Live Neurons 143

Anterograde 16

14 GFP 12 GFP-CPEC25 10

8

6

No of vesicles 4

2

0 135791113151719212325272931333537 time intervals (x1.97sec)

Fig. 3. Bar graphs showing the number of vesicles moved in each time interval (1.97 s × number of steps). Vesicles moving in either anterograde or retrograde direction with respect to the total number of time intervals (1.97 s) each vesicle moved were classified separately (reproduced from ref. 5 with permission from Elsevier Science).

8. Sort separately among each other vesicles moving in either anterograde or retrograde direction with respect to the total number of time intervals (1.97 s) each vesicle moved. 9. Calculate the average speed, total distance, and total step num- bers of movements of BDNF-GFP-containing vesicles in RFP

or RFP-CPEC10-expressing neurons in the same way as above. 10. Carry out statistical analysis using the Student t-test (two tailed, non-paired) and report the mean values ± SEM for the average of total distances, total steps, and mean velocities that the vesicles move.

4. Notes

1. The company dissects hippocampal neurons from rat on Wednesday and delivers the neurons on Thursday. 2. Borate buffer is recommended by many different neuroscience research laboratories for coating the plate/coverslip for neu- rons. Neurons are easily detached from the dish coated with 0.001% poly-l-lysine in PBS or HBS during procedure. 3. Black delta dish is recommended rather than transparent dish because the fluorescence in live cells is more easily bleached by any light source. 4. Phenol red-free DMEM is required for acquisition of red fluores- cence to avoid interference by the red color of the phenol red. 144 J.J. Park and Y.P. Loh

5. Neurobasal medium usually comes with delivery of rat ­hippocampal neurons so that it is not necessary to order it separately. 6. Red fluorescence yields a stronger signal than green fluores- cence, but offers less resolution in live cells. This may be due to a stronger innate intensity of red fluorescence than green fluorescence. 7. Mouse Neuron Nucleofector kit® also works for transfection of rat neurons. 8. The capacity of delta TPG dish is 1 mL. 9. Delivered neurons can be kept at 4°C for several hours, but longer storage decreases the survival rate of neurons after electroporation. 10. While dissociation solution containing papain is recommended for neuron dissociation by the company, it should not be used for neuron dissociation. Use of the solution results in death of more than 90% of the neurons after electroporation. It may be due to influx of proteases in the dissociation buffer into neu- rons upon electroporation. 11. The size of neuron pellet is very small so that the pellet is easily sucked up by rough pipetting. 12. The electroporation cuvette with DNA and neuron mixture should be gently tapped to cause the mixture to settle to the bottom of the cuvette. 13. Other electroporation programs do not yield better transfec- tion efficiency than C-13. 14. White debris (dead neurons) floating on the top of electropo- rated neurons should be removed before transferring live neu- rons into a delta T dish. 15. The incubation in FBS-DMEM for longer or shorter than 2 days does not enhance the transfection efficiency. 16. The time for transfer of the dish containing live neurons from incubator to microscope and finding a neuron that expresses both GFP and RFP proteins at optimum levels, and has well- developed neurites should be minimized to less than 4 min. 17. Before starting acquisition of real-time images, the hot air blower should be turned off to prevent effect of air current on the oil between the top of the objective and the bottom of the dish. 8 Imaging of Peptide Vesicle Movement in Live Neurons 145

Acknowledgements

We thank the lab members in SCN, NICHD for technical ­assistance and helpful discussions. We thank Dr. Vincent Schram in the NICHD Microscopy Imaging Core for technical support. This research was supported by the Intramural Research Program of the NICHD, NIH. JJP is supported by NICHD K22 and ARRA grants.

References

1. Altar, C. A., and DiStefano, P. S. (1998) transport mechanism controls BDNF vesicle Neurotrophin trafficking by anterograde trans- homeostasis in hippocampal neurons. Mol Cell port. Trends Neurosci 21, 433–437. Neurosci 39, 63–73. 2. Poo, M. M. (2001) Neurotrophins as synaptic 5. King, S. J., and Schroer, T. A. (2000) Dynactin modulators. Nat Rev Neurosci 2, 24–32. increases the processivity of the cytoplasmic 3. Goldstein, L. S., and Yang, Z. (2000) dynein motor. Nat Cell Biol 2, 20–24. Microtubule-based transport systems in neu- 6. Kwinter, D. M., Lo, K., Mafi, P., and Silverman, rons: the roles of kinesins and dyneins. Annu M. A. (2009) Dynactin regulates bidirectional Rev Neurosci 23, 39–71. transport of dense-core vesicles in the axon and 4. Park, J. J., Cawley, N. X., and Loh, Y. P. (2008) dendrites of cultured hippocampal neurons. A bi-directional carboxypeptidase E-driven Neuroscience 162, 1001–1010.

Chapter 9

Fluorescence Imaging with Single-Molecule Sensitivity and Fluorescence Correlation Spectroscopy of Cell-Penetrating Neuropeptides

Vladana Vukojevic´, Astrid Gräslund, and Georgy Bakalkin

Abstract

Neuropeptide–plasma membrane interactions in the absence of a corresponding specific receptor may result in neuropeptide translocation into the cell. Translocation across the plasma membrane may repre- sent a previously unknown mechanism by which neuropeptides can signal information to the cell interior. We introduce here two complementary optical methods with single-molecule sensitivity, fluorescence imaging with avalanche photodiode detectors (APD imaging) and fluorescence correlation spectroscopy (FCS), and demonstrate how they may be applied for the analysis of neuropeptide ability to penetrate into live cells in real time. APD imaging enables us to visualize fluorescently labeled neuropeptide molecules at very low, physiologically relevant concentrations, whereas FCS enables us to characterize quantitatively their concentration and diffusion properties in different cellular compartments. Application of these meth- odologies for the analysis of the endogenous dynorphin A (Dyn A), a ligand for the kappa- (KOP), demonstrated that this neuropeptide may translocate across the plasma membrane of living cells and enter the cellular interior without binding to its cognate receptor.

Key words: Dynorphin A, Cell-penetrating peptides, Fluorescence correlation spectroscopy, APD imaging, single-molecule detection

1. Introduction

1.1. Background Cell-surface receptors and their corresponding endogenous ligands on Dynorphin form the basis of neurotransmission in animal organisms (1, 2). Neuropeptides However, not all information is transmitted through the cell-surface receptors. Actions of several neuropeptides including substance P and dynorphin A (Dyn A) may also be mediated via alternative, evolutionary ancient mechanisms, which might have operated before the specific receptors for these peptides had evolved 3( , 4).

Adalberto Merighi (ed.), Neuropeptides: Methods and Protocols, Methods in Molecular Biology, vol. 789, DOI 10.1007/978-1-61779-310-3_9, © Springer Science+Business Media, LLC 2011 147 148 V. Vukojevic´ et al.

The proinflammatory actions of substance P involve a receptor- independent mechanism that is apparently based on direct interaction of this peptide with the a subunit of heterotrimeric G proteins, following its translocation across the plasma membrane (5). Dyn A, a -derived opioid peptide that is an endogenous ligand for the kappa-opioid receptor (KOP) (6), may induce several pathological processes including pain, paralysis, and neuronal loss that are not mediated through the KOP (6–9). Because of their property to cross the plasma membrane barrier without binding to their specific receptor, these neuropeptides are termed membrane- permeant or cell-penetrating neuropeptides (CPnP) by analogy to cell-penetrating peptides (CPP). The mechanism of CPnP/CPP translocation and their actions on cellular processes are not well known. To this point, several translocation mechanisms have been proposed (10–13): 1. Cellular uptake mediated through macropinocytosis, 2. Cellular uptake through clathrin- or caveolin-mediated endocytosis, 3. Direct translocation through transiently or stably formed pores, and 4. Direct translocation through the formation of inverted micelles. Several mechanisms of CPP action on cells have been described (10–13): 1. Interaction with intracellular targets, 2. Disruption of the plasma membrane through pore formation, and 3. Cell-surface receptor activation through local perturbation of the mechanical properties of the plasma membrane. In order to gain better understanding of the translocation mechanism and its dynamics, interactions between CPnP and the plasma membrane need to be studied nondestructively, in the living cell. The complexity of the problem, due to low number of reporter molecules (0.1–100 nM concentrations) in comparison to the surrounding cellular molecules, fast molecular interactions (on-off rates of the order 105–108 M−1s−1), short life time of transient molecular complexes (10−6–10−3 s), and rapid molecular movement (diffusion coefficients of the order 10−13–10−10 m2/s), requires ultrafast methods with high spatial (submicrometer) and temporal (submillisecond) resolution and single-molecule detection sensitivity. In this chapter, we introduce two such methods, fluorescence imaging with avalanche photodiode detectors (APD imaging) (14) and fluorescence correlation spectroscopy (FCS) (15–22). These complementary methods enable us to visualize neuropeptide 9 Imaging Cell Penetrating Neuropeptides 149

molecules on the plasma membrane, in the cytoplasm and the nucleus, measure their local concentrations, and characterize quan- titatively their local mobility in the plasma membrane, cellular interior, and the surrounding medium.

1.2. APD Imaging Fluorescence imaging with avalanche photodiode detectors (APD imaging) was performed using an individually modified instru- ment for confocal laser scanning microscopy (CLSM). As com- pared to photomultiplier tubes (PMTs), which are typically used in commercially available instruments, avalanche photodiodes (APDs) are characterized by superior quantum yield and collection effi- ciency (about 70% in APDs as compared to 15–25% in PMTs), higher gain, faster response time, negligible dark current and bet- ter efficiency in the red part of the spectrum. Because of their exceptional properties, these detectors are routinely used for FCS measurements, but their application for imaging was rather restricted. The considerably improved detection efficiency achieved by a simple replacement of the PMTs by APDs increases signifi- cantly the signal-to-noise ratio (SNR) and enabled us to perform imaging with single-molecule sensitivity (14).

1.3. Fluorescence Principles of FCS were formulated about 40 years ago (23–27) in Correlation the frame of a more general method called fluctuation correlation Spectroscopy analysis. Pioneers in the FCS field developed a theoretical basis and built state-of-the-arts equipment to perform fluorescence fluctua- tion measurements, setting cornerstones not only for FCS but also for fluorescence photobleaching recovery (28–33), multiphoton FCS (34, 35), and multiphoton microscopy (36–38). The early use of FCS after its introduction in the seventies involved large excitation volumes and long correlation times, causing photodestruction. Only after introducing technological improvements (27, 29, 30) did FCS acquired its present characteristics – single-molecule detection sensitivity and short measurement times, allowing studies of fast dynamic processes in living cells even if reporter molecules are present at low levels. Recent advances and development of new techniques that enable protein labeling within the cells by genetic fusion with fluorescent proteins or by small organic fluorophores (31, 32, 39, 40) further speeded up the rapid expansion of FCS applications in live cell studies. In FCS, the time course and the amplitudes of fluorescence intensity fluctuations occurring in a very small, diffraction-limited volume element are analyzed using statistical methods to derive molecular numbers, conventional diffusion transport coefficients, and chemical rate constants. For more comprehensive reviews on FCS, see for example (15–21). We describe here briefly the most important elements of an FCS instrument and the most important steps in FCS analysis. 150 V. Vukojevic´ et al.

Fig. 1. Schematic presentation of the instrumentation for APD imaging and FCS. (a) To induce fluorescence, the sample is illuminated by incident laser light. The irradiating laser beam is reflected by a dichroic mirror and is sharply focused by the objective to form a diffraction limited volume element. A confocal aperture, set in the image plane to reject the out-of-focus light, further reduces the volume from which fluorescence is detected. This is crucial for providing an elliptical observation volume element (magnified in the inset) and enabling submicrometer resolution and quantitative analysis. After the absorp- tion of energy, fluorescent molecules lose energy through photon emission. Light emitted by fluorescing molecules passing through the confocal volume element is separated from the exciting radiation and the scattered light by a dichroic mirror and barrier filter and transmitted to the detector. The number of pulses originating from the detected photons, recorded during a specific time interval, corresponds to the measured light intensity. a( ) Fluorescence intensity fluctuations recorded in real time. (b) Normalized autocorrelation curve G(t ) indicating the presence of a single molecular species, characterized with an average diffusion times tD = 1 ms. The experimental autocorrelation curve is fitted using the autocorrelation function derived for an underlying model, in this case a free three-dimensional diffusion of a single component with intersystem crossing (transition to the triplet state), Eq. (3a). (b) Schematic drawing of a PC12 cell with sites at which FCS measurements were performed.

To induce fluorescence, the sample is illuminated by incident light of a certain wavelength delivered by a laser (Fig. 1). The laser beam is reflected by a dichroic mirror and sharply focused by the objective in the sample. Following the absorption of energy, fluorescent molecules lose energy through photon emis- sion. The emitted light is separated from the exciting radiation and the scattered light by a dichroic mirror and barrier filter, and trans- mitted to the detector, which responds with an electrical pulse to each detected photon. The volume from which fluorescence is detected is further reduced by a pinhole (confocal aperture) in the image plane, to reject out-of-focus light. This is crucial to achieve a small and very well-defined observation volume element (typically about 2 × 10−16 L – roughly the size of an E. coli bacterium, Fig. 1), as well as quantitative and low background analysis. The number of pulses originating from the detected photons, recorded during a 9 Imaging Cell Penetrating Neuropeptides 151

specific time interval, corresponds to the measured light intensity (Fig. 1a). Thus, in one experimental run changes in fluorescence intensity in time are registered (Fig. 1a). Statistical methods of data analysis are applied to detect non- randomness in the data. Typically, this is done by temporal auto- correlation analysis, but other ways of data analysis such as higher order autocorrelation functions (41, 42), fluorescence intensity distribution analysis (FIDA) (43, 44), photon-counting histograms (PCH) (45, 46), and the recently developed fluorescence cumulant analysis (FCA) (47) can also be applied. In temporal autocorrelation analysis, we first derive the intensity autocorrelation function C(t). C(t) gives the correlation between the fluorescence intensity, I(t), measured at a certain time, t, and its intensity measured at a later time t + t, I(t + t). The intensity autocorrelation function may be defined as an ensemble average: C()tt=á ItIt () ( + ) ñ (1a) or, alternatively, as a time average of the product I(t)I(t + t) mea- sured over a certain accumulation time T:

1 T C(tt )=+ limItIt ( ) ( )d t . (1b) T ®¥ T ò0

Since the unprocessed data in FCS are essentially fluorescence fluc- tuations over the mean fluorescence intensity , it is also possible to express the autocorrelation function through fluctuations of light intensities dI(t) = I(t) − and dI(t + t) = I(t + t) − , at times t and t + t, respectively. In this way, the intensity autocorrelation function is defined as follows: = á ñ2 + ád d + ñ C(tt ) I It () It ( ). (1c) Regardless of the form of expression (1a)–(1c), as they are all equivalent, it is not convenient to use the intensity correlation function in practice because its value depends on properties of the applied experimental setup (18). Therefore, instead of using the intensity autocorrelation function, it is more convenient to use the so-called normalized autocorrelation function, G(t), defined as:

ádIt() d It ( +t ) ñ G()t =+ 1 , (2) áñI 2

which is independent of the properties of the experimental setup, such as laser intensity, detection efficiency of the instrumentation, and fluorescence quantum yield of the fluorophore. For further analysis, the normalized autocorrelation function G(t) has to be calculated over many autocorrelation times (t) and plotted for different t values. This is how an autocorrelation curve, as shown in Fig. 1b, is built from the fluorescence intensity fluctuations shown in Fig. 1a. In molecular systems undergoing only stochastic 152 V. Vukojevic´ et al.

fluctuations, we observe random variations ofG (t) around the value G(t) = 1. For processes that are not random, an autocorrelation curve is determined (Fig. 1b). Typically, one observes a maximal limiting value of G(t) as t → 0, decreasing to the value of G(t) = 1 at long times, indicating that correlation between the initial and the current value has been lost. Very often, only the so-called nonuniform part of the normalized autocorrelation function is analyzed. In this case, one observes a maximal limiting value of G(t) as t → 0 that decreases to the value of G(t) = 0 at long times. The limiting value of G(t), as t → 0, is then inversely proportional to the absolute concentration of the fluorescing molecules, as we show later. Thereafter, the experimentally obtained autocorrelation curve has to be compared to an autocorrelation function that is derived for a corresponding model system (18). For example, if we are looking at molecules in the medium, we are expecting two processes that may contribute to the fluorescence intensity fluctuations, intersys- tem crossing (transition to the triplet state) and free three-dimen- sional diffusion in the medium. We would, therefore, use an autocorrelation function that takes into account these processes:

11éùT æöt G(t )=+ 1 êú1+ exp - (3a) NT2 1 - èøç÷t æöttwxy ëûT ++ ç÷112 èøttDw zD

In Eq. (3a), N is the average number of ligand molecules in

the observation volume; wxy and wz are radial and axial radii of the ellipsoidal observation volume element (Fig. 1), respectively,

related to spatial properties of the detection volume element, tD is the characteristic decay time of the autocorrelation curve, called the average residence time or the average diffusion time of the molecule through the detection volume element. T is the average

equilibrium fraction of molecules in triplet state, tT is the triplet correlation time, related to rate constants for intersystem crossing and the triplet decay, and t is the autocorrelation time. If we are looking at molecules interacting with the plasma mem- brane, we expect that following processes can contribute to the fluo- rescence intensity fluctuations, intersystem crossing, free diffusion in the medium just above the plasma membrane, and two-dimensional diffusion in the plasma membrane. To account for these processes, the autocorrelation curve acquires the following form: æö ç÷ 11ç÷- y yTéùæöt G(t )=+ 1 + êú1+ exp - (3b) NTç÷2 æö1 - ç÷èøt æöttwxy t ëûêúT ç÷11++ 1 + ç÷ç÷2 ç÷èøt èøèøttD11wzD D 2 9 Imaging Cell Penetrating Neuropeptides 153

In Eq. (3b), tD1 and tD2 are characteristic decay times of the autocorrelation curve, corresponding to the average lateral transition time of Dyn A in the medium and the cell surface, respectively. Spatial properties of the detection volume, represented in Eqs. (3a) and (3b) by the square of the ratio of the radial and axial 2 parameters (wxy / wz) , are determined experimentally in calibration measurements (48). The calibration is performed in vitro, by using an aqueous solution of a dye for which the diffusion coefficient (D)

is known. The average diffusion time (tD) is related to the transla- tion diffusion coefficient D:

w2 t = xy , (4) D 4D

The autocorrelation curve derived in the calibration experi- ments is fitted by the autocorrelation function (3a) using the known diffusion coefficient (D), of the dye as constant in the fitting 2 procedure, and the value of (wxy / wz) is determined from the best fitting curve.

The diffusion time, tD, of the investigated molecules is deter- mined from autocorrelation functions (3a) or (3b) that matches best the actual, experimentally determined, autocorrelation curve 2 using the value for (wxy / wz) determined from the calibration experiments. As can be seen from Eqs. (3a) and (3b), the limiting value of G(t) as t → 0 is related to the average number of molecules in the observation volume (N), and it can be used to determine the absolute concentration (c) of the fluorescing molecules. For example, if G(t) = 2.25 at t = 0 (Fig. 1b) and the observation volume element is V = 2.0 × 10−19 m3, the concentration of the fluorescent molecules in the sample is c = 6.6 × 10−9 mol/dm3. In the example shown in Fig. 1b, the inflection point at the autocorrelation curve indicates

that the average diffusion time is tD = 1 ms. Although the measured fluctuations are utterly stochastic by themselves, their average rate of relaxation to the equilibrium value is not stochastic. Rather, it is constrained by macroscopic proper- ties of the sample. And it is exactly this interrelation that makes it possible to apply fluctuation analysis to obtain information about mobility coefficients, local concentration, apparent hydrodynamic radius, chemical reaction constants and rates, association, dissocia- tion, and equilibrium binding constants. In cases other than the analyzed example of free diffusion of a single fluorescent species, the autocorrelation function attains forms different from the one given in Eqs. (3a) and (3b) because all processes leading to statisti- cal fluctuations in the fluorescence signal will generate a character- istic decay time in the autocorrelation curve. 154 V. Vukojevic´ et al.

2. Materials

2.1. Cell Culturing 1. Rat pheochromocytoma PC12 cells (American Type Culture Collection (ATCC), obtained through LGC Promochem, Teddington, UK). 2. RPMI 1640 medium supplemented with 5% fetal bovine serum (FCS – Invitrogen, Carlsbad, CA) (see Note 1). 3. 10% heat-inactivated horse serum (Invitrogen). 4. 100 U/mL penicillin and 100 mg/mL streptomycin (Invitrogen) with and without phenol red. 5. 8-well chambered coverslips (Nalgene Nunc, Thermo Scientific, Rochester, NY). 6. Collagen-coated tissue culture flasks (Sarstedt, Germany). 40 mg/mL Bovine Collagen I (Invitrogen) was used for coating. 7. Deionized water with conductivity of W = 0.02 mS/cm (Milli-Q ultrapure water system, Bedford, MA, USA). 8. Water bath. 9. Centrifuge.

10. CO2 incubator.

2.2. Peptide Solutions 1. Aqueous solution of the fluorescently labeled Dynorphin A 1–17 (Dyn A; Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-

Lys-Leu-Lys-Trp-Asp-Asn-Gln, C99H155N31O23, Mr = 2147.50). The peptides where synthesized according to the procedure described in Marinova et al. (4).

2.3. APD Imaging 1. Custom order modified ConfoCor3 instrument (Carl Zeiss and FCS MicroImaging GmbH, Jena, Germany) consisting of: (1) inverted microscope for transmitted light and epifluorescence (Axiovert 200 M); (2) the VIS-laser module comprising the Ar-ion (458, 477, 488, and 514 nm), HeNe 543 nm and HeNe 633 nm lasers; (3) scanning module LSM 510 META modified to enable detection in the imaging mode using silicone ava- lanche photodiodes (SPCM-AQR-1X; PerkinElmer, USA), and (4) FCS module with three detection channels (see Note 2). 2. C-Apochromat 40×/1.2 W objective. This water immersion objective is equipped with a correction collar to compensate for spherical aberrations arising due to differences in refractive indices and layer thicknesses, and corrected for chromatic aberrations. The high numerical aperture, i.e., large opening angle of the objective and relatively high refractive index of the immersion medium (water) enable efficient gathering and transmission of light, yielding a strong signal and high- resolution images. 9 Imaging Cell Penetrating Neuropeptides 155

3. Programs for image examination and online FCS data analysis (a part of the running software package). 4. Rhodamine 6 G (Rh6, Invitrogen) standard solution for instru- ment alignment and calibration. 5. Heated microscope stage (Heating insert P), incubator box (Incubator S), atmosphere controlling device (CTI-Controller 3700) equipped with a humidifier and a temperature-controlling device (Tempcontrol 37–2 digital HemoGenix®, Colorado Springs, CO) (see Note 3).

3. Methods

The most important factor for successful cell culturing is working 3.1. Cell Culturing under aseptic conditions. To ensure aseptic conditions, environmen- 3.1.1. Aseptic Techniques tal control, personal hygiene, and equipment and media sterilization procedures must be routinely performed. Therefore, cell culturing and manipulation should be performed in hoods with laminar flows, in specially designed laboratories for cell culturing. Before using the hood, sterilize the interior by wiping down all accessible surfaces with 70% ethanol, followed by a 10 min exposure to UV irradiation. Do not look directly at the UV lamp, since UV radiation can seri- ously damage your cornea even if you are wearing safety goggles. For the purposes of our experiments, the cells need to be taken out from the cell culturing laboratory and taken to the microscopy room. To reduce the possibility for contamination during trans- portation, the chambered coverslips are placed in a Petri dish and transported between laboratories. During imaging and FCS measurements, the chambered coverslips are kept closed. Cells that were subjected to imaging can be returned to the incubator. Return the chambered coverslips to the Petri dish, wipe it with 70% ethanol on the outside, air-dry, and place it in the incubator. By following these simple routines, we were able to use one chambered coverslip for one working week (5 days).

3.1.2. Thawing and 1. Remove a vial with frozen PC12 cells from liquid nitrogen and Recovering PC12 Cells immediately place it into a 37°C water bath. To enable fast thawing, gently agitate the vial. 2. Wipe the vial with 70% ethanol and air-dry before opening. 3. Transfer the cell suspension into a sterile centrifuge tube con- taining 2 mL warm complete medium with 20% FBS. 4. Centrifuge for 10 min at about 200 × g, at room temperature. 5. Discard the supernatant, gently resuspend the cell pellet in 1 mL of warm complete medium, and transfer to culture flasks containing 5–20 mL of the medium. 156 V. Vukojevic´ et al.

6. Culture cells for 5–7 days in medium with 20% FBS until the cell line is reestablished. Replace the medium if the pH indicator changes color. The PC12 cells are semisuspended, and small clusters of cells are frequently observed.

3.1.3. Passaging PC12 1. Remove the flask from the incubator, swirl gently, and tap the Cells in Culture flask firmly two to three times, until the PC12 cells are evenly distributed in the medium. 2. Remove aseptically half of the volume of cell suspension and place into a new flask. 3. Add the required amount of RPMI complete medium with 10% FBS to the new and the original flask. Replace the medium every 2–3 days. In the next splitting round, remove the whole content from the original flask and split into two new flasks. Never use the same flask for more than 2 splitting cycles. 4. Follow the same procedure to plate the cells in 8-well chambered coverslips. If needed, the cell density can be increased by 5 min centrifugation at 200 × g. 5. Discard the supernatant and resuspend the cells in 2.5 mL warm, phenol-red-free medium. Distribute 300-mL aliquots in each well. Average cell density at plating is about 1 × 105 cells/cm2, in 300 mL medium. 6. Place the cells in the incubator and allow them to rest for 2–3 days before observation. Since you are no longer having the phenol red indicator, you cannot observe pH changes in the medium. Therefore, do not forget to replace regularly the medium every 2–3 days.

3.2. Peptide Solutions 1. Keep peptide dry and store at 4°C, since fluorescently labeled Dyn A dissolves readily in water, but it is relatively unstable and cannot be preserved for a long time in solution. 2. Prepare every day 10 mL of a new concentrated stock solution of peptide (»10−5 M), just before the measurements. During the day, keep the stock solution on ice, protected from light. The concentration of the fluorescently labeled peptide is deter- mined by FCS.

3.3. APD Imaging and 1. Turn on the instrument, the Ar-ion and HeNe 543 nm lasers and FCS Measurements the mercury lamp. Allow the system to warm up and reach stable performance. This usually takes 1–2 h. Turn off the main light and perform all measurements in the dark room (see Note 4). 2. Place a 50–100-mL water droplet on the objective. Be careful not to touch the objective lens with the micropipette tip, to avoid scratching it. Check carefully for air bubbles. If an air bubble is observed, wipe off the water droplet using a Whatman Lens Cleaning Tissue 105, and reapply. Use a specially designated 9 Imaging Cell Penetrating Neuropeptides 157

micropipette for this purpose. Do not use this micropipette for fluorescent dyes to avoid accidental contamination of the objective. 3. Place a chambered coverslip on the holder and select an empty well. To position the detection volume correctly, i.e., to “find the focus,” bright field laser reflections from the lower and upper side of the coverslip are used. To do this, you must con- figure the beam path first. A convenient setting, recommended by the instrument producer, uses the 488 nm line of the Ar-ion laser at 2% of the power, the 80/20 splitter in the major beam splitter position and a short pass emission filter (KP 685) in front of the detector. Choose line scan, the highest scan speed, image size 512 × 512 pixels, and zoom 1, open the pinhole to maximum, and set the detector gain between 250 and 300. As you start scanning, a line with a hill, which is the result of reflected light not yet focused on the lower glass surface, appears on the imaging display. Move the objective upward by turning slowly the corresponding turret on the microscope stand. As you approach the lower surface of the coverslip, the line will shift to higher values because the laser light is more and more reflected from the lower surface. As you continue moving the objective the line will start declining, indicating that you have “crossed” the lower surface of the coverslip and the laser light is no longer reflected from it. If you continue moving the objective upward, the line will start rising again, as you are approaching the upper side of the coverslip. A second maximum indicates that the upper surface of the coverslip has been found. If you now store this position, you can orient and position the detection volume into the sample in a well-defined way. In addition, you have measured the thickness of your cover- slip, which corresponds to the difference between the objective position at the second and first maximum (see Note 5). 4. Change the beam path configuration and make an appropriate optical configuration for the Rh6G and TRITC fluorophores. Select the HeNe 543 nm laser as the excitation source, the main dichroic mirror (HFT 488/543/633) and a long-pass emission filter (LP 560). Choose the APD detectors for imaging. The same optical setting is used for FCS. 5. Move the C-Apochromat water immersion objective upward 200 mm, to position the detection volume 200 mm above the upper surface and avoid disturbing interface effects. Add the previously prepared 10 nM Rh6G standard solution to the well. Select low laser intensity, for example 0.5%, and check the count rate by pressing the “Count rate button” in the data display window. Select a laser intensity that gives an average count rate that is 10–100 kHz and observe the estimated counts per molecule and second (cpm) in the “Count rate display” window (see Note 6). 158 V. Vukojevic´ et al.

6. To make a preliminary adjustment of the correction collar, note the estimated cpm value, stop the measurement and turn the correction collar in one direction. Continue the measurement and check whether the cpm value has increased or decreased. If the cpm value has increased, continue turning the correction collar in the same direction. Alternatively, change the direction. By repeating this procedure, find the best position of the cor- rection collar, where the cpm value is maximal (see Note 7). 7. To position the pinholes in the lateral (x and y) and axial (z) directions choose a pinhole size that is equal or smaller than your working pinhole size, and press the “Adjust Pinholes Coordinates” button in the data display window. By choosing a coarse alignment, an automatic procedure will be activated and the pinhole will be moved over the whole range of possible positions. The position giving the highest fluorescence inten- sity will be identified. Select this position and choose coarse alignment in the y direction. After finding the coarse pinhole positions, you may improve the signal intensity by fine-tuning the pinhole position around the identified maxima. Thereafter, try to improve the signal intensity by adjusting the collimator position in the axial (z) direction. If the signal improves by changing the z-direction, repeat the fine positioning in the x- and y-directions. 8. Repeat steps 6 and 7 until best possible alignment of the optical setting is achieved. This procedure may be sometime tedious, but it is absolutely necessary. To assure best results, check your optical setting every day. 9. Change the laser intensity and observe in the data display window the count rate and the cpm. For increasing laser inten- sities, the cpm will increase until a maximum is reached. Thereafter, the cpm decreases even though the laser intensity is increased. Two processes lead to this: dye saturation and irre- versible photobleaching. The dye becomes saturated when the number of molecules in the ground state is diminished because of the extremely high irradiation that drives the dye molecules to the excited state faster than they can revert to the ground state. In irreversible photobleaching, fluorescence is mainly lost because the triplet state of the dye interacts chemically with other molecules, primarily ambient oxygen. To select proper laser intensity for FCS and APD imaging, you should not work with excitation intensities that cause dye saturation or photobleaching, but you would still like the cpm to be as high as possible, since this is the most important determinant of the quality of your signal, i.e., the SNR. Experimentally measured cpm values depend on the photo- physical properties of the dye molecule, excitation intensity, and the instrumental setting. For aqueous solutions of Rh6G, 9 Imaging Cell Penetrating Neuropeptides 159

we readily observe cpm values that are about 40–60 kHz for excitation intensities ranging between 10 and 100 mW. Such high cpm values are seldom observed in live cells. To avoid photodamage of the cell, laser intensities should be ideally kept <10 mW. In live cell measurements, typically observed cpm ­values are 5–15 kHz for organic fluorophores and 1–5 kHz for fluorescent proteins. Cpm values that are smaller than 1 kHz should be considered with great caution. 10. In order to extract quantitative information from the experi- mental autocorrelation curves, we need to characterize first the shape of the ellipsoidal observation volume element by deter- mining the ratio between the axial (wz) and radial (wxy) radii. This property, termed structure parameter (S = wz/wxy), is determined from autocorrelation curves recorded using refer- ent solutions of known concentration of a fluorescent molecule with high quantum yield, for which the diffusion coefficient is known (49). In our case, this is Rh6G. Experimental autocor- relation curves recorded for Rh6G solutions of known concen- tration are fitted using Eq. (3a). tD and S are determined from the best fits. Typically,S has values between 4 and 6. In further analysis, the S value determined in the calibration experiments is used as a fixed parameter (see Note 8). 11. Repeat the measurements with Rh6G solution several times and compare the values for N, S, tD, T, and tT determined in consecutive runs. If similar values (within 10–15%) are obtained, the instrumentation is stable and ready for real mea- surements (see Note 9). 12. Check the autofluorescence of the medium and record an autocorrelation curve. Use several different laser intensities. Information on the background is critical for very low concen- trations and very precise quantitative measurements. 13. Prepare a fresh standard solution of TRITC-Dyn A or TRITC by dissolving a very small, barely visible amount of the powder that has attached to a dry micropipette tip in 10 mL water and place it on ice. Dilute 0.1–1 mL of this very concentrated solution in 200 mL of medium. Move the chambered coverslip to a new, empty well. Add the diluted TRITC-Dyn A solution to the well and perform FCS to determine the TRITC-Dyn A concentration. Repeat the same procedure for TRITC. 14. Remove the chambered coverslip with the solutions, wipe off the remaining water from the objective, and add a new water droplet. Place the chambered coverslip with the PC12 cells on the stage, readjust the focus, and find PC12 cells suitable for observation. Choose isolated cells or cells in smaller clusters. Select cells that are attached and do not swim in the medium. Using the same optical setting as for FCS, choose a 512 × 512 160 V. Vukojevic´ et al.

Fig. 2. Time-lapse APD imaging. (a–h) APD images (upper row ) together with the overlaid APD and transmission images (second row) show progressive accumulation of TRITC-Dyn A in the vicinity of PC12 cells, the plasma membrane and the cellular interior. (i–k) Magnified APD image i( ), the transmission image ( j ) and the overlay of these two (k) show that TRITC- Dyn A is not uniformly distributed in the plasma membrane, but rather localized in discrete patches of different brightness and size. (l) The time course of TRITC-Dyn A accumulation in the medium (blue, 2), plasma membrane (red, 3) and the cytoplasm (green, 1), followed by time-lapse APD imaging. The mean fluorescence intensity was measured in selected regions of interest, indicated in (k). The progressive increase in fluorescence intensity could be modeled as exponential x/t1 growth, y = A1⋅e + y0 (black curves). The scale bars are 10 mm. See http://extras.springer.com/ for the color version of this figure.

pixel image resolution, scanning speed between 20 and 50 ms/ pixel, single-run, zoom 4, and take an APD image of PC12 cells as control (Fig. 2, 0 min). 15. Using the same conditions as above, set a time-lapse APD imaging series collecting a new image every 5 s. Add the TRITC-Dyn A solution to achieve a concentration in the bulk of 0.5–1 mM. Start the time-lapse APD imaging to observe TRITC-Dyn A interactions with the plasma membrane and its subsequent internalization in PC12 cells (Fig. 2). 16. After imaging, perform FCS measurements in the cellular inte- rior (cytoplasm and nucleus), at the plasma membrane, in the medium immediately surrounding the cells and the bulk medium, 200 mm above the cells (Fig. 3). Fluorescence inten- sity recordings show that the local concentration of TRITC- Dyn A in the cell surroundings is gradually increasing (Fig. 3, red). This is reflected in the autocorrelation ­analysis by “tail- ing” of the autocorrelation curve, revealing the presence of 9 Imaging Cell Penetrating Neuropeptides 161

another component/process with longer characteristic time (Fig. 3, red). TRITC-Dyn A does not distribute itself evenly in the plasma membrane, but shows a rather patchy distribution. Fluorescence intensity bursts recorded at the plasma mem- brane are complex, indicating that processes with different characteristic times contribute to the observed fluorescence intensity fluctuations. This is also reflected in the autocorrela- tion correlation curve, which is bumpy and very irregular (Fig. 3, magenta). FCS data are in agreement with APD imag- ing (Fig. 2). Both methods show that TRITC-Dyn A is accu- mulating in the plasma membrane, as evident from the diminished amplitude of the autocorrelation function (Fig. 3, magenta as compared to green and red) giving rise to patchy, non uniform distribution (Fig. 2) reflected in FCS analysis by the very irregular fluorescence intensity bursts and autocorre- lation curve (Fig. 3, magenta) (see Note 10). 17. Repeat the experiments and consider changing the TRITC- Dyn A concentration. We have observed that the process is rather slow when the TRITC-Dyn A concentration in the bulk medium is low (<200 nM). At high (>3 mM) concentrations, the process is very fast and TRITC-Dyn A translocates readily inside the cells, distributes evenly in the cellular cytoplasm and even in the cell nucleus (Fig. 4). However, some cells resist the TRITC-Dyn A for a prolonged time, and internalization is observed only after prolonged exposure (Fig. 4). 18. Fitting of autocorelation curves is the final stage of FCS ­analysis. In fitting, we are comparing the experimental auto- correlation curves with theoretical curves to reveal the ­processes that are causing the fluorescence intensity fluctuations. Theoretical models are derived for different underlying mecha- nisms, molecular diffusion in 1, 2, or 3D, changes in molecular diffusion due to chemical interactions, changes in fluorescence due to chemical interactions, rotation, flow, etc., and the com- bination of these processes. Different models have recently been reviewed and can be found in reference (18). If we now look at the fluorescence intensity fluctuations recorded in the cytoplasm, we can observe high-intensity fluo- rescence bursts amid low-amplitude fluorescence intensity fluc- tuations (Fig. 5a). If we take an average over all measurements, an average autocorrelation curve shown in Fig. 5a is observed. This autocorrelation curve shows a very fast, nearly rectangular decay and cannot be fitted with a model implying free diffusion (Eq. (3b), black curve) – the black autocorrelation curve clearly deviates from the green, experimentally recorded one. The fit is not improving even if several components with different dif- fusion times are included in the model. This clearly indicates 162 V. Vukojevic´ et al.

Fig. 3. Fluorescence Correlation Spectroscopy (FCS). (a) Fluorescence intensity fluctuations generated by TRITC-Dyn A movement recorded in different locations: the bulk medium, 200 mm above the cell (orange, 2); the medium in the immedi- ate cellular vicinity (blue, 3), the plasma membrane (red, 4) and the cytoplasm (green, 5). Fluorescence intensity fluctua- tions recorded in the nucleus and the cytoplasm before TRITC-Dyna A addition are shown in the inserts. The time traces reveal that TRITC-Dyn A is gradually accumulating in the immediate vicinity of PC 12 cells, as evident from the steadily increasing fluorescence intensity (blue trace, 3). Fluorescence intensity fluctuations recorded at the plasma membrane are “bumpy,” showing complex variations in the mean fluorescence intensity (red trace, 4), in agreement with APD imaging showing that TRITC-Dyn A distribution in the plasma membrane is not uniform, but rather “patchy” (see for example Fig. 2i). Complex fluorescence intensity fluctuations, with regular fluorescence intensity fluctuations and sporadically occurring large, very intensive bursts were recorded in the cytoplasm (green trace, 5). The composite appearance of the fluorescence intensity fluctuations may hint that TRITC-Dyn A is taken up by the cells in a multiple fashion, possibly through direct translocation across the plasma membrane and occasional internalization through vesicles. (b) Auto correlation curves reflect changes in TRITC-Dyn A concentration and mobility at different locations, outside and inside the PC12 cells. TRITC-Dyn A concentration increases progressively in the immediate vicinity of the PC12 cells - the concentration of TRITC-Dyn A in the bulk medium, 200 mm above the cells is about 600 nM (orange curve, 2), about 3 mM in the medium immediately surrounding the PC12 cells (blue curve, 3) and still higher (>8 mM) at the plasma membrane (red curve, 4). −5 (c) Autocorrelation curves scaled to the same amplitude (G0(t) = 1 at t = 1 × 10 s) show that TRITC-Dyn A mobility is also different in different locations. We immediately observe that the autocorrelation curve for TRITC-Dyn A is shifted to longer characteristic times (tD, TRITC-Dyn A = (120 ± 10) ms, orange curve, 2) as compared to the fluorescent dye alone 9 Imaging Cell Penetrating Neuropeptides 163

that fluorescence intensity fluctuations are not generated by free diffusion alone, and the fitting model needs to be changed. If we look at individual measurements, we sometimes observe only regular, small-amplitude fluorescence intensity fluctuations (Fig. 5b), sporadically disrupted by high-intensity bursts (Fig. 5c). The high intensity of the fluorescence bursts indicates the large associates of TRITC-Dyn A are observed. In our study, this may reflect the occasional formation of trans- porting vesicles. Vesicle movement in the cytoplasm is complex – at short distances, the vesicles are thought to move by diffu- sion. However, their long-term movement may be propelled by cytoplasmic streaming, an electrochemical gradient or cytoskeleton-based motor proteins (50). Therefore, a model combining diffusion with flow can be described by the follow- ing function: æö ç÷2 1 11æöv G()t =+ 1 exp ç÷- ç÷t (3c) N 2 ç÷w æö æöttwxy èøxy t 11++ ç÷ç÷1 + ç÷ 2 èøèøt èøttDw zD D

where v is the velocity of the radial transport, may better describe TRITC-Dyn A dynamics in the cytoplasm over long times. In agreement with this interpretation is the observation that short-time behavior of TRITC-Dyn A can be described by diffusion (Fig. 5b, c) (see Note 11).

Fig. 3. (continued) (tD, TRITC = (90 ± 10) ms, magenta curve, 1). This reflects the difference in mass – the molecular weight of TRITC-Dyn A is about 5× that of TRITC. In the medium immediately surrounding the cell, similar characteristic times were observed for TRITC-Dyn A (the overlapping part of the orange (2) and blue (3) curves), but a long tail in the autocorrelation curve was also noted (blue curve, 3). This may indicate that TRITC-Dyn A is exposed to more interactions in the close proximity of the cell, but it may also be caused by the progressive increase in TRITC-Dyn A concentration that was observed by APD imaging (Fig. 2l, blue (2) curve) and FCS (the blue fluorescence intensity fluctuation trace shown ina ). Continuous increase of the mean fluorescence intensity is reflected in the autocorrelation curve through the appearance of a “second component,” with a long characteristic time. This is similar to photobleaching, discussed in detail in reference (21), except that the mean fluorescence intensity decreases due to photobleaching. The autocorrelation curve recorded at the plasma membrane (red curve) is complex, and extends over characteristic times that span several orders of magnitude. This suggests that multiple processes, with different characteristic times may cause the fluorescence intensity fluctuations recorded at the plasma membrane. In addition, the autocorrelation curve showed small oscillations that may reflect an underlying periodic process. To assure that this periodic behavior is not an artifact, caused by external vibrations, we repeated the measurements alternating between the medium and the membrane. The oscillations were consistently picked up in the membrane, but not the medium. The autocorrelation curve recorded in the cytoplasm (green curve, 5) is also complex. It is shifted to longer characteristic times (tD, cytoplasm = 8 ± 2 ms) and shows a very fast, nearly rectangular decay. This behavior is analyzed in detail in Fig. 5. See http://extras.springer.com/ for the color version of this figure. Fig. 4. TRITC-Dyn A uptake in PC12 cells is concentration dependent. At high TRITC-Dyn A concentrations (>1 mM), its uptake in PC12 cells is fast and may be completed within several seconds. Under such conditions, TRITC-Dyn A quickly dis- tributes inside the cell and is also observed in the nucleus. PC12 cells that are inside a cell cluster, indicated by arrows, are “shielded” by neighboring cells. Therefore, they are not immediately exposed to TRITC-Dyn A. 0 s, image taken immediately after addition of fluorescent peptide into the medium. See http://extras.springer.com/ for the color version of this figure.

Fig. 5. Analysis and fitting of autocorelation curves. a( ) Fluorescence intensity fluctuations recorded in the cytoplasm. The time traces were recorded as 10 consecutive measurements, each measurement lasting 10 s (for clarity, only 6 traces are shown). The corresponding, average autocorrelation curve is shown below. This autocorrelation curve shows a very fast, nearly rectangular decay (dots) and cannot be modeled as free diffusion (line). The disagreement between the experimental and theoretical autocorrelation curves (dots vs. line) indicates that the applied model does not represent well the underlying mechanism. (b and c) Fluorescence intensity fluctuations in the cytoplasm recorded in consecutive runs, showing small- amplitude fluorescence intensity fluctuationsb ( ) and occasional, high-intensity bursts (c). The marked difference that was observed between independent runs may suggest that the underlying mechanism is complex and that short- and long- term behavior may be governed by different processes (see Note 12). See http://extras.springer.com/ for the color version of this figure. 9 Imaging Cell Penetrating Neuropeptides 165

4. Notes

1. The RPMI 1640 medium is also applicable for cell observation under ambient conditions since it contains a dual buffering system consisting of HEPES and bicarbonate buffers. This system

maintains the medium pH at 7.4 in the CO2 incubator, but HEPES provides buffering while the cultures are being han-

dled outside of the CO2 incubator, where the CO2 concentra- tion is much lower. However, the effect of the HEPES buffer is limited, and the PC12 cell cultures should be kept in closed containers during microscopic observations in ambient condi- tions. We did not observe any major differences between mea- surements under ambient conditions, as compared to the controlled conditions. 2. The equipment should be located in a light protected and air- conditioned room, where the ambient temperature (20–22°C) and air humidity (30–50%) could be controlled and maintained at the indicated levels. The room should be classified as haz- ardous area with Class III laser danger and clearly labeled with danger signs. The room should be completely dark during the measurements. 3. The CTI-Controller 3700 supplies the cultivation chamber

with a heated mixture of CO2 and air. The CO2 concentration and the temperature can be continuously monitored and regu- lated by a digital feedback control algorithm, enabling a

­regulated dispersion of CO2 into the air stream and steady heating. 4. Place a small table lamp with a red bulb in a distant corner of the microscope room to facilitate movement in the room dur- ing experimental manipulation. 5. The correction collar allows very small deviations in the thickness of the cover glass to be compensated for. It also enables com- pensation for spherical aberrations that occur when light pass- ing through the periphery of the lens is not brought into focus at the same plane as the light traveling through the center of the lens. Spherical aberrations increase with the optical path length and the numerical aperture of the objective. They may become pronounced when working at different temperatures, because the refractive index of the immersion medium depends on the temperature. 6. The excitation maximum for Rh6G is 514 nm. Thus, the high- est cpm will be obtained when this excitation wavelength is used. However, because of its excellent photophysical proper- ties, high quantum yield (>0.9) and photostability, and well- characterized diffusion coefficient (49) this dye is frequently used in FCS studies to characterize the detection volume 166 V. Vukojevic´ et al.

element when 488 nm or 543 nm irradiation is used for excitation. It is recommendable to use the same chambered coverslips for instrument alignment and calibration as for cell culturing. Note that the thickness of the bottom of the chambered cov- erslip may vary in different wells. 7. Experienced FCS users perform this procedure under continu- ous illumination, i.e., without stopping the cpm measurement while turning the correction collar on the objective. This saves some time but increases the risk for accidently tumbling the chambered coverslips, as the space around the objective may be crowded with other objectives and narrow and ambient light is low. If you use this procedure, do not forget that you should not look directly to the laser beam coming out from the objective. 8. Standard solutions of Rh6G last long (6 months or more) if a high concentration (1 mM) solution is prepared and stored at 4°C. Some FCS users prefer to keep the fluorophore solution frozen. If so, do not freeze and thaw the solutions. In either case, store the high concentration solution in small aliquots (50–100 mL). Large volumes have larger contact surface with the test tube and the fluorescent dye molecules may adhere to the plastic walls. Low concentration (10 nM) solutions should be prepared immediately before the FCS measurements. If you encounter low signal intensity: –– Check the optical configuration. Assure that proper lasers and optical elements are used. Check that there are no unnecessary components, like a neutral density filter or a black plate in the pathway. Check whether the mirrors are correctly positioned. –– Check for air bubbles in the immersion water or in the sample. –– Check that the coverslips are placed horizontally. –– Clean the objective and the bottom of the coverslip. –– Reduce the stray light by placing the black metal cover on top of the chambered coverslips. –– Prepare a new 10 nM solution of Rh6G. –– Check the detection pathway, the pinhole position and size, and the correction collar. –– Place a paper sheet above the objective and observe the laser beam. If no light is observed, remove the objective by unscrewing it from the holder and check the laser beam using the paper sheet. Do not look directly in the laser beam! 9. If consecutive measurements give very different results, the system is most probably not warmed up properly. Allow more 9 Imaging Cell Penetrating Neuropeptides 167

time for warming up. If the problem persists, check the laser intensity. If the incident irradiation is too strong, the fluorophore may be saturated and/or subjected to photobleaching. If this is the case, both the number of molecules and the average diffusion time may change. The average number of molecules in the observation volume element (N) will be smaller and smaller, as more and more molecules are lost by irreversible photodestruction. The average diffusion time (tD) will appar- ently decrease – as the molecules are being photobleached their average residence time in the observation volume element will appear to be shorter. However, the signal is not lost because the molecules are moving faster through the observation vol- ume element, as it may be interpreted by the underlying FCS analysis, but because they lose fluorescence as they undergo transition to the “dark” triplet state while being observed. If photobleaching is extensive and the average intensity, i.e., count rate decreases continuously, photobleaching may give rise to a “tail” in the autocorrelation curve. This tail, with a long characteristic time should not be confused with a second, slowly moving component (21). In practice, it is useful to keep the triplet state fraction T <30%, and the triplet correlation time 1 ms < tT < 10 ms. Fluorescent proteins show a characteristic correlation time 1 ms < tT < 50 ms that is consistent with the transition to the triplet state. 10. FCS is generally not suitable for applications that involve high (>1 mM) concentrations. At such high concentrations, the amplitude of the fluorescence intensity fluctuations becomes too small and quantitative FCS studies become unreliable. However, APD imaging does not have this limitation and can be used for such studies. 11. Addition of more fitting parameters usually improves the fitting. However, this does not necessarily mean that the underlying model is correct. Therefore, the fitting analysis should always be taken with caution. 12. Temporal autocorrelation analysis focuses on the time course of the fluorescence intensity fluctuations and disregards differ- ences in molecular brightness. Therefore, autocorrelation analysis cannot be efficiently applied for the study of complex systems, where molecules of different brightness exist. In the presence of very bright molecules/molecular assemblies the information on the less bright molecules is simply lost in the background. An example is presented in Fig. 5b, c. In the absence of bright molecular assemblies, Fig. 5b, the autocorrelation curve reflects the behavior of individual TRITC-Dyn A molecules in the cytoplasm. In their presence, Fig. 5c, the autocorrelation anal- ysis reflects only the properties of the very bright molecular assemblies. Note also that the conditions for analyzing molecules 168 V. Vukojevic´ et al.

of different brightness are opposed – to improve the SNR ratio and “enhance” the visibility of individual TRITC-Dyn A mol- ecules in Fig. 5b, the excitation intensity should be increased. However, this will inevitably distort the signal detected from the large, slowly moving assemblies due to photobleaching.

Acknowledgments

We thank Carl-Jonas Lindskog for testing the protocol and procedures described in this work. VV acknowledges support from the Knut and Alice Wallenberg Foundation, Swedish Research Council, Swedish Brain Foundation, Ministry of Sciences and Technological Development of Serbia (Grants no. 142025 and 142019). This work was also supported by grants from the Swedish Council for Working Life and Social Research (FAS) and Swedish Science Research Council to GB.

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Analysis of Neuroimmune Interactions by an In Vitro Coculture Approach

Tadahide Furuno and Mamoru Nakanishi

Abstract

Nerve fibers innervate every organ of the body and are involved in monitoring changes of the external and internal environment. Innervation directly controls a variety of physiological responses in an adaptive manner. Today, many lines of research indicate that also the immunological response is influenced by the nervous system and that nerve and immune cells directly interact through intercellular signal trans- duction by cytokines, neurotransmitters, and neuropeptides. For instance, mast cells are often found in close proximity of nerve fibers containing substance P and calcitonin gene-related peptide, two widely studied sensory neuropeptides, in a variety of tissues. To investigate the molecular mechanism of the direct functional interplay between nerve and immune cells, we have studied their communication using an in vitro coculture system and confocal microscopy. Here, we introduce methods for the in vitro coculture of nerve and immune cells and the imaging analysis of cellular activation, and discuss soluble mediators and adhesion molecules involved in the neuroimmune interaction. Improvement of our under- standing of neuropeptide functions on these issues would lead to new therapeutic modalities for diseases based on neuroimmune interaction such as neurogenic inflammation, intestinal bowel diseases, asthma, and autoimmune disorders.

Key words: Neuroimmune interaction, Mast cell, In vitro coculture, Confocal microscopy, Calcium imaging, Adhesion molecule

1. Introduction

It is clear that nervous and immune systems interact on many aspects in both healthy and disease condition and that molecules known to act specifically in nervous or immune system play important roles in other system (1, 2). Immunohistochemical studies showed that there was the intervention of both primary and secondary lymphoid organs with sympathetic nerve fibers (3) and that mast cells were often found in the proximity of nerve fibers containing

Adalberto Merighi (ed.), Neuropeptides: Methods and Protocols, Methods in Molecular Biology, vol. 789, DOI 10.1007/978-1-61779-310-3_10, © Springer Science+Business Media, LLC 2011 171 172 T. Furuno and M. Nakanishi

neuropeptides in a variety of tissues (4). Based on electron-microscopic observation, nerve terminals are directly in contact with T cells and mast cells. These findings suggested that neuropeptides and neurotransmitters would play a role in intercellular communication form nerves to immune cells. To understand the nature of these kinds of cell association, function, and development, Blennerhassett and Bienenstock worked out a tissue culture model involving the coculture of isolated nerve cells with immune cells (5, 6). They originally studied the early events of the specific and selective association between sympathetic neurons and rat basophilic leukemia (RBL-2H3) cells (a model of mucosal mast cells). We expected that the in vitro coculture model would be very useful to investigate the molecular mechanism of direct communication between nerves and immune cells because the possibility that an intermediary cell transduces or modulates the nerve-immune cell interaction was dismissed. Using the system, we showed that nerve and immune cell interaction can occur in the absence of an intermediary transducing cell (7) and identified the soluble factors (7–11) and adhesion molecules (12, 13) involved in the interaction. The technique that we describe here can be appli- cable to investigate the direct intercellular communication between various types of cell combinations such as nerve–keratinocytes, nerve–osteoblastic cells (14), and nerve–pancreatic islet cells (15).

2. Materials

2.1. Cell Cultures 1. Animals (newborn mice: P0–P2). and Cocultures 2. Matrigel™ matrix (growth factor reduced; Becton Dickinson 2.1.1. Isolation of Nerve Biosciences, Bedford, MA): dilute with cold sterile water (8× Cells dilution) and store in 15-mL centrifuge tubes (Corning Incorp., Corning, NY) at −20°C. Matrigel must not be warmed because it is only fluid at 4°C. 3. Trypsin type II-S (Sigma-Aldrich Inc.): dissolve at 0.25% in sterile Hanks’ balanced salt solution (HBSS; Gibco™ Invitrogen Corp.) and filter using a syringe-driven filter unit (0.22 mm, Millipore Corp., Bedford). Store 2-mL aliquots of trypsin at 4°C in 15-mL centrifuge tubes (see Note 1). 4. Cytosine b-d-arabinofuranoside (Ara-C; Sigma-Aldrich Inc.): dissolved at 1 mM in sterile water and store at 4°C. 5. 35-mm plastic culture dishes (Falcon, Becton Dickinson Labware, Franklin Lakes, NJ). 6. 35-mm glass bottom culture dishes (Matsunami, Osaka, Japan). 7. Microculture dishes (Ibidi GmbH, Martinsried, Germany). 8. 60-mm plastic culture dishes (Falcon). 10 Neuro-immune interactions 173

9. 26 G needles (Terumo, Tokyo, Japan). 10. Basic surgical instrumentation (scissors, fine forceps, etc. avail- able from Natsume, Tokyo, Japan or other vendors). 11. Stereomicroscope (e.g., SteREO Discovery.V12, Zeiss, Oberkochen, Germany).

2.1.2. Establishment 1. Young mice (8–12 weeks). of Cultures of Nerve 2. Rat basophilic leukemia (RBL) cells. and Immune Cells 3. S37 cells. and Cocultures 4. F-12 nutrient mixture (Gibco™ Invitrogen Corp., Grand

Island, NY) supplemented with 14 mM NaHCO3, 0.2 mM l-glutamine, 0.3% glucose, 1% antibiotic antimycotic solution (100×) (Sigma-Aldrich Inc., St. Louis, MO), 50 ng/mL mouse (NGF, 2.5S; Upstate Cell Signaling Solutions, Lake Placid, NY), and 10% fetal calf serum (FCS; Roche Diagnostics, Indianapolis, IN) (see Note 2). 5. Dulbecco’s modified eagle medium (DMEM; Gibco™

Invitrogen Corp.) supplemented with 44 mM NaHCO3, 1% MEM nonessential amino acids solution (100×) (NEAA; Gibco™ Invitrogen Corp.), 100 U/mL penicillin, 100 mg/mL streptomycin, and 10% FCS (Roche Diagnostics). 6. Recombinant murine interleukin-3 (IL-3) (PeproTech, London, UK) and recombinant murine stem cell factor (SCF) (PeproTech): dissolve at 20 mg/mL in sterile PBS. Store 100- mL aliquots at −20°C in sterile siliconized 0.5-mL tubes (Assist, Tokyo, Japan) (see Note 3).

7. CO2 incubator. 8. Autoclave. 9. Bottle top filters (Corning Incorp.) 10. Glass Pasteur pipettes. 11. 100-mm culture dishes (Falcon). 12. 27G needles. 13. 1-mL syringes. 14. General laboratory centrifuge. 15. Rubber policeman (Ikemoto-Rika, Tokyo, Japan).

2.2. Calcium 1. Fluo 3-AM (Molecular Probes™ Invitrogen Corp.): prepare a Measurements by 1 mM solution in dimethyl sulfoxide (DMSO). Store 100-mL Confocal Microscopy aliquots at −20°C in 0.5-mL tubes. 2. HEPES buffer: 10 mM HEPES, pH 7.2, 140 mM NaCl,

5 mM KCl, 0.6 mM MgCl2, 1 mM CaCl2, 0.1% bovine serum albumin (BSA, Sigma-Aldrich Inc.), 0.1% glucose, and 0.01% sulfinpyrazone (Sigma-Aldrich Inc.) (see Note 4). 174 T. Furuno and M. Nakanishi

3. Scorpion venom (from Leiurus quinquestriatus hebraeus, Sigma-Aldrich Inc.): dissolve at 10 mg/mL in HEPES buffer and store at −20°C (16). 4. Confocal laser fluorescence microscope with heated stage (e.g., LSM-510, Zeiss).

3. Methods

Microscopic technologies have recently advanced and played an essential role in many fields of life science such as cell biology, immunology, physiology, and neurology. This set of techniques is a powerful tool to study not only the intracellular dynamic molecular organization and signaling events in individual living cells but also the molecular basis of intracellular communication. In an in vitro coculture system of nerves and immune cells, neurons isolated from mouse ganglia are cultured in the presence of NGF in matrigel- coated culture dishes. In a few days, immune cells are added into the dishes and cocultured with the nerve cells. Following another several days, associations of immune cells with neurites can be observed in large numbers. Using this coculture system, we are able to find out that a specific stimulation of neurites induces the activa- tion of immune cells attached with neurites in a dose-dependent manner (7). In superior cervical ganglia (SCG)–mast cell cocul- tures, neurite stimulation elicits not only Ca2+ mobilization but also degranulation in attached mast cells mediated by substance P (7–10). A specific activation of mast cells also induces the Ca2+ increase in SCG neurite after a lag period mediated by ATP from mast cells (11). In SCG–T cell cocultures, neurite stimulation also leads to T cell activation, which involved activation of b-adrenergic receptors on T cells (1). In addition, immunostaining analysis is able to show the accumulation of adhesion molecules such as N-cadherin and CADM1 at contact sites of immune cells to neurites (12, 13). Thus, the combination of microscopic methods and the in vitro coculture approach can illustrate the unequivocal bidirectional communication between nerves and immune cells and investigate the molecular mechanisms in their direct communication through soluble mediators such as neuropeptides.

3.1. Cell Cultures 1. Keep dishes at −20°C (see Note 5). and Cocultures 2. Fill the bottom of a dish with the diluted matrigel solution 3.1.1. Isolation of Nerve kept on ice, using a cold Pasteur pipette. Cells 3. Swirl the dish in a hood to evenly coat the bottom.

Preparation of Matrigel- 4. Take off excess solution and add to the next dish. The film of Coated Dishes matrigel should be very thin. 10 Neuro-immune interactions 175

5. Leave the dishes in hood without the lid for at least 1 h. 6. Store the coated dishes at −20°C (if not used immediately).

Isolation of SCG 1. Add several microliters of HBSS in a 15-mL centrifuge tube, ~5 drops of HBSS in a 60-mm culture dish, and one drop of HBSS in a 35 mm dish. 2. Take a tube of 0.25% trypsin II solution out from refrigerator. 3. Prepare several newborn (0–2 day old) mice. 4. Decapitate a mouse using fine scissors. 5. Pin down the head by two 26 G needles (neck is up-side) and immediately rinse the section of neck with a small amount of HBSS in the tube by a Pasteur pipette to prevent blood clots. 6. Remove one SCG, which is located at bifurcation of carotid artery, with fine forceps under a stereomicroscope. Keep in the 60-mm dish with HBSS. Remove the other SCG from the same mouse and also keep in HBSS in the dish. 7. When all SCG have been dissected, remove nonnervous tissue, such as arteries and fat, using fine forceps under the stereomicroscope. 8. Once each SCG is cleaned, place in the 35-mm dish with one drop of HBSS.

Isolation of Dorsal 1. Place approximately five drops of HBSS in a 60-mm culture Root Ganglia dish and one drop of HBSS in a 35-mm dish. 2. Take a tube of 0.25% trypsin II solution out from refrigerator. 3. Prepare several newborn (0–2 day old) mice. 4. Decapitate a mouse using fine scissors. 5. Cut the spinal cord taken from a mouse longitudinally at dorsal site using fine scissors. 6. Remove dorsal root ganglia (DRG) and transfer to the 60-mm dish with HBSS using fine forceps under a stereomicroscope. 7. When all DRG have been dissected, remove nonnervous tissue using fine forceps under the stereomicroscope. As each ganglion is cleaned, place it in the 35-mm dish with one drop of HBSS.

3.1.2. Establishment 1. Transfer all cleaned ganglia in the tube containing the 0.25% of Cultures of Nerve trypsin II solution using a Pasteur pipette and incubate at 37°C and Immune Cells for 1 h in a CO2 incubator. Swirl the tube every 10 min to and Cocultures prevent the pieces of ganglia from sticking together.

Cultures of Ganglia 2. Remove the tube from the incubator and leave standing upright in hood to allow the pieces of ganglia to collect at the bottom. When ganglia have settled down, remove the supernatant. 3. Add 2 mL of F-12 medium to the tube and leave it standing for several minutes until ganglia settle down. 176 T. Furuno and M. Nakanishi

4. Remove the supernatant and add again 2 mL of F-12 medium. 5. Gently triturate the suspension using a Pasteur pipette. Suck up and expel ~30 times to completely dissociate the ganglia (see Note 6). 6. Adjust volume with F-12 medium (see Note 7) and then add the Ara-C solution at 2 mM as final concentration to prevent proliferation of nonnervous cells such as fibroblasts. 7. Place approximately 200 mL of the ganglia suspension in the middle of matrigel-coated dishes.

8. After incubating for several hours or overnight in a CO2 incu- bator to make the neurons attach to dishes, remove the super- natant and add 2 mL of fresh F-12 medium. 9. Replace with fresh medium every 2 days at most.

Bone Marrow-Derived 1. Add approximately 10 mL of DMEM to two 100-mm dishes Mast Cells (Falcon). 2. Remove the thighbones from 8 to 12-week-old mice and keep in DMEM in one of the dishes. 3. When dissection is complete, remove muscle and fat around thighbones using scissors. 4. Cut cleaned thighbones at upper and lower sites by scissors, and then flush inside with DMEM medium in another dish several times using a 27G needle connected with a 1-mL syringe. Collect the flushed medium including bone marrow cells with a 15-mL centrifuge tube. Carry this operation out at both sites of the thighbones. 5. When bone marrow cells are collected from all thighbones in tubes, leave tubes standing upright for a few minutes to let impurities settle down. 6. Move the cell suspension to fresh centrifugation tubes using a Pasteur pipette and centrifuge for 10 min at 400 × g. 7. After the supernatant is removed, resuspend the cells in 10 mL of fresh DMEM and measure density. 8. Culture cells at a density of 2 × 105 cells/mL in DMEM sup- plemented with IL-3 (10 ng/mL) and SCF (2 ng/mL) in

100 mm dishes in a CO2 incubator. 9. Replace with fresh medium every 7 days.

Maintenance of Cultured 1. Culture a mast cell line, rat basophilic leukemia (RBL) cell, and

Cell Lines of Mast Cells a Th1 cell line, S37 cell, in F-12 medium in a CO2 incubator. and T Cells 2. To maintain adherent RBL cells, scrape by a rubber policeman when they become confluent. 3. To maintain nonadherent S37 cells, dilute every a few days. 10 Neuro-immune interactions 177

Coculture of Nerve 1. Remove the medium from dishes culturing nerve cells (see with Immune Cells Subheading “Cultures of Ganglia”). It is better to use the nerve cell dishes for <3 days after starting culture. 2. Add 2 mL of immune cell suspension to the nerve cell-cultured dishes. 3. The immune cells become attached to nerve cell neurites for several hours as shown in Fig. 1. Maintain the coculture for 1–3 days to measure Ca2+ movement (see Note 8).

3.2. Calcium 1. Add Fluo 3-AM to the coculture dish at 1 mM as a final con- Measurements by centration (see Note 9). Confocal Microscopy 2. Incubate the dish for 30 min in a CO2 incubator at 37°C. 3. Gently wash the dish twice with warmed HEPES buffer (at 37°C). 4. Add 1.8 mL of HEPES buffer to the dish. 5. Place the dish on the heated stage (at 37°C) of a confocal laser fluorescence microscope (LSM-510: Zeiss). 6. Capture a time sequence image (~60 images) of Fluo 3 fluores- cence every few seconds, as shown in Fig. 2 (see Note 10). 7. Add 0.2 mL of stimulant solution to the dish after taking the fifth fluorescence image (see Note 11).

Fig. 1. A differential interference contrast image in coculture dishes of nerve cells with bone marrow-derived mast cells (BMMCs). BMMCs were added to the matrigel-coated plastic dish where nerve cells from SCG were cultured. After incubation for 3 h, the cocul- ture dish was washed twice to remove unattached floating BMMCs. Remaining BMMCs (black arrows) are attached to SCG neurites (white arrows). 178 T. Furuno and M. Nakanishi

Fig. 2. Calcium mobilization images of DRG neurites and RBLs in coculture dishes. (a) Fluo 3 fluorescence images after capsaicin stimulation (3 mM). Addition of capsaicin (3 mM) to DRG cultures resulted in neurite activation, but did not evoke an increase in fluorescence when added directly to RBLs in the absence of DRG neurites. In coculture dishes, capsaicin- induced neurite activation was invariably followed by RBL activation, as indicated by increased fluorescence. An arrow shows an activated RBL by specific neurite stimulation. b( ) Fluo 3 fluorescence images after the addition of BC4, an anti- body against high affinity IgE receptors (FceRI) (×1,000 dilution). Addition of BC4 resulted in RBL activation, but did not directly evoke an increase in fluorescence of DRG neurites. In coculture dishes, BC4-induced RBL activation was invariably followed by neurite activation, as indicated by increased fluorescence. Arrows show an activated DRG neurite by specific RBL stimulation.

4. Notes

1. We usually prepare 50 mL of trypsin solution and aliquot it in 25 tubes.

l 2. F-12 nutrient mixture, NaHCO3, -glutamine, and glucose are dissolved in 1,000 mL of water. Unless stated otherwise, all solutions should be prepared in water that has a resistivity of 18.2 MW-cm. This standard is simply referred to as “water” in this text. The F-12 solution is filtrated in medium bottles, which have been sterilized by autoclave, using a bottle top filter in hood. Following this, NGF and FCS are added in F-12 medium. 3. Siliconized tubes should be sterilized by autoclave beforehand. 10 Neuro-immune interactions 179

4. In our protocol, ×10 HEPES solution (pH 7.2) including

NaCl and KCl and ×100 MgCl2 and CaCl2 solution are prepared. To obtain 100 mL of HEPES buffer, 10 mL of HEPES

solution (×10), 1 mL of MgCl2 and CaCl2 solution (×100), glucose (100 mg), BSA (100 mg), and sulfinpyrazone (10 mg) are added with water to a total volume of 100 mL. 5. Glass bottom dishes and m-dishes are suited to observe nerves and immune cells in high resolution with oil-immersion objec- tive lens because their bottoms are very thin. 6. If nerve cells are not completely separated from ganglia, it is better to cut each ganglion in half using a scalpel after removing nonnervous cells in a 35-mm dish next time. 7. The nerve cells from one ganglion are ordinarily settled to one to two dishes in our methods. 8. Nerve cells are attached to matrigel-coated dishes within 2 h. The extension of neurites from nerve cells is observed within 4 h. The neurite network is seen after 2 days, but it is easily detached from dishes when being cultured for a longer period. 9. Many kinds of fluorescent indicators for Ca2+ are commercially available. One has to choose an indicator to meet his/her own experimental needs based on several features such as optical property (excitation and fluorescence wavelength) and disso- ciation constant for Ca2+. 10. Fluo 3 is excited by an argon laser (488 nm), and its fluores- cence is detected through a long pass filter (>505 nm). 11. We generally use scorpion venom and IgE receptor cross-linker (antigen and antibody) as a specific stimulator of nerves and mast cells, respectively.

References

1. Nakanishi, M., and Furuno, T. (2008) (1987) Intestinal mucosal mast cells in normal Molecular basis of neuroimmune interaction in and nematode-infected rat intestines are in inti- an in vitro coculture approach. Cell. Mol. mate contact with peptidergic nerves. Proc. Immunol. 5:249–259. Natl. Acad. Sci. USA 84:2975–2979. 2. Kraneveld, A.D., Rijnierse, A., Nijkamp, F.P., 5. Blennerhassett, M.G., and Bienenstock, J. and Garssen, J. (2008) Neuro-immune interac- (1990) Apparent innervation of rat basophilic tions in inflammatory bowel disease and irrita- leukaemia (RBL-2H3) cells by sympathetic ble bowel syndrome: future therapeutic targets. neurons in vitro. Neurosci. Lett. 120:50–54. Eur. J. Pharmacol. 585:361–374. 6. Blennerhassett, M.G., Tomioka, and M., 3. Felten, D.L., Felten, S.Y., Bellinger, D.L., Bienenstock, J. (1991) Formation of contacts Carlson, S.L., Ackerman, K.D., Madden, K.S., between mast cells and sympathetic neurons Olschowki, J.A., and Livnat, S. (1987) in vitro. Cell Tissue Res. 265:121–128. Noradrenergic sympathetic neural interactions 7. Suzuki, R., Furuno, T., McKay, D.M., Wolvers, with the immune system: structure and func- D., Teshima, R., Nakanishi, M., and Bienenstock, J. tion. Immunol. Rev. 100:225–260. (1999) Direct neurite-mast cell (RBL) commu- 4. Stead, R.H., Tomioka, M., Quinonez ,G., nication in vitro occurs via the neuropeptide Simon, G.T., Felten, S.Y., and Bienenstock, J. substance P. J. Immunol. 163:2410–2415. 180 T. Furuno and M. Nakanishi

8. Ohshiro, H., Suzuki, R., Furuno, T., and mast cells and neurites. Biol. Pharm. Bull. Nakanishi, M. (2000) Atomic force microscopy 27:1891–1894. to study direct neurite-mast cell (RBL) com- 13. Furuno, T., Ito, A., Koma, Y., Watabe, K., munication in vitro. Immunol. Lett. 74: Yokozaki, H., Bienenstock, J., Nakanishi, M., 211–214. and Kitamura, Y. (2005) The spermatogenic Ig 9. Mori, N., Suzuki, R., Furuno, T., McKay, superfamily/synaptic cell adhesion molecule D.M., Wada, M., Teshima, R., Bienenstock, J., mast-cell adhesion molecule promotes interac- and Nakanishi, M. (2002) Neurite-mast cell tion with nerves. J. Immunol. 174: (RBL) communication: RBL membrane ruf- 6934–6942. fling is restricted to the specific site of contact 14. Obata, K., Furuno, T., Nakanishi, M., and with the activated neurite. Am. J. Physiol. Cell Togari, A. (2007) Direct neurite-osteoblastic Physiol. 283:C1738-C1744. cell communication, as demonstrated by use of 10. Furuno, T., Ma, D., van der Kleij, H.M.P., an in vitro co-culture system. FEBS Lett. Nakanishi, M., and Bienenstock, J. (2004) 581:5917–5922. Bone marrow-derived mast cells in mice respond 15. Koma, Y., Furuno, T., Hagiyama, M., Hamaguchi, in co-culture to scorpion venom activation of K., Nakanishi, M., Hirota, S., Yokozaki, H., and superior cervical ganglion neurites according to Ito, A. (2008) CADM1 is a novel pancreatic islet level of expression of NK-1 receptors. Neurosci. cell adhesion molecule involved in interaction with Lett. 372:185–189. nerves and in hormone secretion. Gastroenterology 11. Suzuki, R., Furuno, T., Okamoto, K., Teshima, 134:1544–1554. R., and Nakanishi, M. (2007) ATP plays a role 16. Romano-Silva, M.A., Ribeiro-Santos, R., in neurite stimulation with activated mast cells. Gomez, M.V., Moraes-Santos, T., and Brammer, J. Neuroimmunol. 192:49–56. M.J. (1994) Tityustoxin-mediated Na+ influx is 12. Suzuki, A., Suzuki, R., Furuno, T., Teshima, more efficient than KCl depolarisation in pro- R., and Nakanishi, M. (2004) N-cadherin plays moting Ca2+-dependent glutamate release from a role in the synapse-like structures between synaptosomes. Neurosci. Lett. 169:90–92. Chapter 11

Electrophysiology

Zhi-Qing David Xu

Abstract

Electrophysiological recording techniques have been widely applied to study the functional role of neuropeptides. The present chapter focuses on the very often used techniques including whole-cell patch-clamp recording, sharp electrode intracellular recording, and extracellular field potential recording in slice preparation as well as in vivo animal recording.

Key words: Electrophysiology, Patch clamp, Sharp electrode recording, Intracellular recording, Extracellular recording, Slice preparation, In vivo studies, Neuropeptides

1. Introduction

Over the decades, our understanding of the involvement of peptides in numerous neuronal functions, such as neurotransmission, learn- ing and memory, pain and feeding behaviors, has been greatly advanced using various techniques including electrophysiology (1, 2). Electrophysiology techniques involve placing electrodes into various preparations of biological tissue and enable to study the electrical properties of neurons by measuring voltage change or elec- tric current on a wide variety of scales, from single ion channel proteins to the electrical activity of neurons (3). Thus, electrophys- iological recording techniques have been widely applied to study the functional role of neurotransmitters, including neuropeptides. In fact, neuropeptides have a wide diversity of direct or modula- tory effects on the electrical responses of target cells, in addition to trophic effects (1, 2). So far, there are various electrophysiological methods fundamentally different from each other in the level of

Adalberto Merighi (ed.), Neuropeptides: Methods and Protocols, Methods in Molecular Biology, vol. 789, DOI 10.1007/978-1-61779-310-3_11, © Springer Science+Business Media, LLC 2011 181 182 Z.-Q.D. Xu

analysis, from subcellular (patch-clamping single-ion channels) to behavioral approaches (neuronal recordings in awake animals). This chapter focuses on those that have been very often used in the field of neuropeptide research.

2. Materials

2.1. In Vitro Recording 1. Rats (200–300 g) or mice. with Slice 2. Artificial cerebrospinal fluid (ACSF): 124 mM NaCl, 2.5 mM Preparations KCl, 1.3 mM MgSO4, 1.24 mM NaH2PO4, 2.4 mM CaCl2,

25 mM NaHCO3, 10 mM glucose, pH 7.35–7.45.

3. 95% O2 and 5% CO2 mixture. 4. Halothane. 5. Small animal guillotine. 6. Vibrating microtome (e.g., Leica VT1000, Leica Microsystems GmbH Wetzlar, Germany). 7. Recording chamber for electrophysiology (RC-26 Recording Chamber, Warner Instruments, Hamden, CT).

2.1.1. Whole-Cell 1. Borosilicate tubes. Patch-Clamp Recording 2. Vertical pipette puller (e.g., PP-830, Narishige, Tokyo, Japan). 3. Internal solution: 135 nM K-gluconate, 20 mM KCl, 2 mM MgATP, 10 mM HEPES, 0.2 mM EGTA, 5 mM phosphocre- atine, pH 7.3; osmolarity, 300 mOsm. 4. Upright microscope fitted with a 40× water-immersion objec- tive, differential interference contrast, and infrared filter. 5. Manipulator (e.g., PCS5000-PCS5400, EXFO Burleigh, npi electronic GmbH, Tamm, Germany). 6. Patch-clamp amplifier (EPC9-2 or Axopatch 200B, Molecular Devices, Sunnyvale, CA). 7. Digitizer (Axon Digidata 1440A, Molecular Devices). 8. Data analysis software (e.g., Mini Analysis, Synaptosoft, Decatur, GA).

2.1.2. Sharp Electrode 1. Brown/Flaming puller (Sutter Instruments, Novato, CA). Intracellular Recording 2. 2 M potassium chloride (DC resistances between 35 and 70 M:). 3. 2 M potassium acetate (resistance 60–90 M:). 4. Stereomicroscope. 11 Electrophysiology 183

5. Manipulator (e.g., M3301 Manipulator, World Precision Instruments Ltd, Stevenage, UK). 6. Amplifier (Axoclamp 2B, Molecular Devices).

2.1.3. Extracellular Field 1. Bipolar stimulating electrodes (TM or TST Tungsten Electrode, Potential Recording World Precision Instruments). 2. Amplifier (e.g., EPC9-2, HEKA Instruments Inc., Bellmore, NY). 3. Stimulator (e.g., Pulsemaster A300, World Precision Instruments).

2.2. Neuropeptide and 1. Calibrated micropipettes (250 nL per div. – tip o.d. = 10 Pm). Drug Applications 2. Picospritzer (General Valve Corporation, Fairfield, NJ). 3. Three-way valve (for bath applications).

2.3. Identification of 1. Biocytin (available from Tocris Biosciences, Bristol, UK). Recorded Neurons 2. Bacitracin (Sigma Chemicals, St. Lous, MO). 3. Sucrose. 4. Cryostat. 5. 4% paraformaldehyde in PBS containing 0.2% picric acid. 6. Avidin conjugated to fluorescein isothiocyanate (FITC – avail- able from Vector Laboratories, Burlingame, CA) 0.1 mg/mL in phosphate-buffered saline (PBS). 7. Triton X-100. 8. Sodium azide. 9. Lissamine rhodamine (LRSC)-labeled secondary antibody (Jackson Immuno Laboratories, Suffolk, UK). 10. Mounting medium: glycerol–PBS solution (10:1) containing 0.1% para-phenylenediamine. 11. Fluorescence microscope (e.g., Nikon Microphot-FX , Tokyo, Japan) equipped with epifluorescence and proper filter combi- nations for FITC (filter cube B-1E; excitation at 480 and 10 nm with a band-pass emission filter passing 520–550 nm) or LRSC (filter cube G-1B; excitation at 546 and 5 nm with a barrier filter at 590 nm).

2.4. In Vivo Animal 1. Rats (250–400 g). Recording 2. Chloral hydrate. 3. Stereotaxic instrument. 4. Heating pad. 5. Skull drill. 6. Hamilton syringe. 184 Z.-Q.D. Xu

7. Polyethylene tubing. 8. Single-barrel glass micropipettes. 9. Brown/Flaming puller (Sutter Instruments). 10. Filling solution: 2 M NaCl containing 2% pontamine sky blue. 11. Oscilloscope. 12. Audio monitor. 13. Micro 1041 and spike 2 software (Cambridge Electronic Design, Cambridge, UK).

2.5. Data Analysis 1. AxoGraph software (Molecular Devices). 2. Axo-Tape software (Molecular Devices). 3. pClamp software (Molecular Devices) or Irgo software (HEKA).

3. Methods

3.1. In Vitro Recording 1. Anesthetize a rat or mouse under halothane and rapidly sacrifice with Slice it with a small animal guillotine. Remove the brain or spinal Preparations cord to isolate the interested region in ice-cold ACSF bubbled

with a mixture of 95% O2 and 5% CO2. 2. Cut slices (250–350 Pm thick) on a vibratome in oxygenated ice-cold ACSF (see Note 1) and maintain at room temperature in a submerged chamber filled with gassed ACSF. Allow slices to equilibrate for at least 1 h before transferring into the record- ing chamber. 3. Transfer slices to a recording chamber continuously perfused (at a speed of 2 mL/min) with oxygen-enriched ACSF at 35–37°C or room temperature. Neuropeptides and drug solutions are introduced at given concentrations through a multibarrel delivery system or the perfu- sion system (Fig. 1) and the changes in membrane potential or membrane current are recorded in the various models as described below.

3.1.1. Whole-Cell 1. Pull patch pipettes (3–7 M:), from borosilicate glass tubes in Patch-Clamp Recording two stages on a vertical pipette puller. 2. Fill with internal solution. 3. Visualize neurons of interest with a 40× water-immersion objective, differential interference contrast, and infrared filter. 4. Approache a cell of interest with the electrode, establish a gigaohm seal, and then rupture the cell membrane to obtain a whole-cell recording (see Note 2). 11 Electrophysiology 185

Fig. 1. Schematic drawing of a setup for in vitro whole-cell/intracellular recording with brain slice preparation.

5. Filter signals at 2 kHz and digitize at 5 kHz. Store data on disk for subsequent analysis by the use of the Igor software. 6. For EPSCs recording: To monitor evoked EPSCs, place the stimulating electrode laterally to the recorded neurons at least 125 Pm away. mEPSCs are recorded under voltage clamp at −60 mV in the presence of tetrodotoxin (TTX; 1 PM) and bicuculline (30 PM).

3.1.2. Sharp Electrode 1. Pull sharp microelectrodes with a Brown/Flaming puller. Intracellular Recording 2. Fill microelectrodes with 2 M potassium chloride (DC resis- tances between 35 and 70 M:), or with 2 M potassium acetate (resistance 60–90 M:). 3. Visualize the recording region by using a stereomicroscope and approach it with the electrode driven by a manipulator. 4. Make conventional intracellular recordings by using the bridge balance or discontinuous single-electrode voltage-clamp mode on an amplifier (see Notes 3 and 4). 5. Routinely hold neurons near their resting membrane potential at −60 mV (holding potential).

3.1.3. Extracellular Field 1. Position bipolar stimulating electrodes and deliver stimuli of Potential Recording square-wave pulses (100 Ps; 3–10 V) at a fixed intensity with frequencies of 0.1 Hz. 2. Record extracellular field potentials with glass microelectrodes filled with KCl (2–4 M:). 3. Amplify DC-coupled signals using an EPC9-2 amplifier. Low-pass filter the resulting signal at 5 kHz. 4. Make baseline recordings for a minimum of 20 min to ensure stationary of responses before experiments are initiated. 186 Z.-Q.D. Xu

5. Elicit paired-pulse facilitation of fEPSPs after paired stimulation with different interpulse intervals (25, 50, 100, and 200 ms). 6. Induce frequency facilitation of fEPSPs by switching stimula- tion frequency from 0.1 to 1 Hz for 20 stimuli.

3.2. Neuropeptide and 1. Dissolve neuropeptides in ACSF as stock solutions (10–3 M, calcu- Drug Applications lated on the basis of purity of the peptide) and store at −70°C in aliquot vials. Dilute neuropeptides and drugs at working concen- tration in warmed, carboxygenated ACSF, just prior to use. 2. For micropipette applications, position the tip of a calibrated micropipette containing various active compounds 100–200 Pm above the slice with a micromanipulator. 3. Apply nitrogen pulses of constant duration and pressure to the end of the pipette via a Picospritzer (see Note 5). 4. For bath application, neuropeptides and drugs are adminis- tered by turning a three-way valve that switches from ACSF to the test solution, for sufficient time to reach a steady-state response (about 2 min or longer) (Fig. 1). 5. On acquisition of a stable recording, apply neuropeptides and other drugs via the bath or micropipette, and record the changes in membrane potential or membrane current.

3.3. Identification 1. At the termination of recording, recorded neurons are filled of Recorded Neurons with biocytin (see Note 6). 2. Fix slices containing biocytin-filled neurons with 4% paraform- aldehyde containing 0.2% picric acid (see Note 7). 3. Cryoprotect slices in increasing concentrations of sucrose (see Note 7) and cut sections with a cryostat. 4. Incubate sections with FITC-conjugated avidin in diluted PBS. 5. For double labeling, incubate sections overnight at 4°C with an antibody against the target of interest diluted at optimal titre in PBS (pH 7.4) containing 0.3% Triton X-100, 0.2% bacitracin and 0.01% sodium azide. 6. Rinse sections in PBS and incubate for 1 h in LRSC-labeled secondary antibody diluted at optimal titre in PBS. 7. Rinse sections in PBS and coverslip them in the mounting medium. Observe in the fluorescence microscope.

3.4. In Vivo Animal 1. Anesthetize male Sprague–Dawley rats weighing 250–400 g Recording with chloral hydrate (400 mg/kg, i.p.) and mount on a stereo- taxic instrument. 2. Maintain body temperature at 37–38°C with a small heating pad. 11 Electrophysiology 187

3. Cannulate a lateral tail vein for supplemental anesthetic and drug administration. 4. Expose the skull and drill two small burr holes over the inter- ested region according to the stereotaxic atlas coordinates. 5. Pull single-barrel glass micropipettes on a Brown/Flaming puller and fill with 2 M NaCl and 2% pontamine sky blue. 6. Position the micropipette to the interested region according to the stereotaxic atlas. 7. Monitor signals online with an oscilloscope and an audio monitor. 8. Record waveform and firing rate with the micro 1041 and spike 2 software. 9. After a stable firing rate (baseline) is established, infuse 1.5 PL of neuropeptides or drugs in the cerebral ventriculi manually through a Hamilton syringe within 1 min. 10. Other drugs can also be administered i.v. through the cannu- lated lateral tail vein. 11. At the end of experiment, mark the recording locus by ionto- phoretic delivery of pontamine sky blue (−15 PA, 20 min) (see Note 8). 12. Verify histologically the injection and recording sites.

3.5. Data Analysis 1. Store all data on a computer for online and off-line analysis with AxoGraph, Axo-Tape, and pClamp software or Irgo software. 2. Work only on all data from preparations that showed significant recovery upon washout. 3. Perform statistical comparisons using Student’s t-test and consider statistical differences significant at P < 0.05.

4. Notes

1. (a) Time is of the essence when preparing brain slices, so make sure your tools and solutions are ready before decapitating the animals.

(b) Prior to beginning, make sure that the dissection glass- ware (small Petri dish, small chamber, large Petri dish, and vibratome chamber) is chilling.

(c) Tissues that are too small to be glued directly to the stage can be first embedded in 2% agar dissolved in ACSF.

(d) The texture of the tissue, the speed and angle of the blade, and the amplitude of the vibration are critical for the quality of the slice. 188 Z.-Q.D. Xu

(e) Use a small paintbrush to guide the brain slice off the blade; do not let the slice curl up. 2. After whole-cell access is achieved, the series resistance is par- tially compensated by the amplifier and continuously checked during the recordings and not allowed to vary more than 25% during the course of the experiment. Both input resis- tance and series resistance are also monitored throughout the experiments. Only those recordings with a stable series resistance (d20 M:) and input resistance are accepted. 3. The bridge has to be frequently checked to ensure that it was balanced throughout an experiment. 4. For the discontinuous single-electrode voltage-clamp (switching frequencies, 5–6 kHz, duty cycle 30%), the head stage output needs to be continuously monitored to ensure adequate elec- trode settling time. 5. The exact volume injected is determined directly by the movement of the fluid meniscus in the pipette. The injection pipette is allowed to contact the bath solution only during the application of drugs, thus reducing the dilution of the pipette solution with bathing ACSF. 6. Biocytin is added to sharp electrodes (1%) and to patch pipettes (0.3–0.5%) as an intracellular marker. The biocytin diffuses into the recorded cells by dialysis during patch recordings, or in the case of sharp electrode recordings is iontophoresed intracellularly at the end of experiments by passing negative current pulses (0.2–0.6 nA, 250 ms, 1 Hz) for 5–30 min. 7. Protocol for fixed, cryoprotected slices: (a) Place slices containing biocytin-filled neurons in a fixative containing 4% paraformaldehyde and 0.2% picric acid at 4°C overnight (16 h maximum). (b) Replace solution with PBS at 4°C X 3, 5–10 min each, then with 10% sucrose in PBS and leave at 4°C for 24 h. 8. After each experiment, the recording site is marked by the ejection of pontamine sky blue dye from the recording elec- trode with a −15 PA current for 20 min. Animals are then anesthetized deeply and perfused transcardially with 4% para- formaldehyde. Brains are removed and stored in a similar solution containing 20% sucrose. The brains are cut into 50-Pm-thick frozen sections, mounted on subbed slides, and counterstained with neutral red and the recording sites are verified by light microscopy. 11 Electrophysiology 189

Acknowledgments

The author would like to thank Professor Tomas Hökfelt for his advice and encouragement. This work was supported by Swedish Research Council (2008:3106), SIDA (348-2006-6688), National Basic Research Program of China (973 Program) (2007CB512307; 2010CB912003), National Natural Sciences Foundation of China (30870815), Natural Science Foundation of Beijing (Key Project 09G0013), and PHR20100510.

References

1. Nässel, D.R. (2009) Neuropeptide signaling in brain--progress during the last six years. near and far: how localized and timed is the Neuropeptides 39, 269–275 action of neuropeptides in brain circuits? Invert 3. Blanton, M.G., Lo T.J. and Kriegstein, A.R. Neurosci. 9, 57–75 (1989). Whole cell recording from neurons in 2. Xu Z.Q., Zheng, K., and Hökfelt, T. (2005) slices of reptilian and mammalian cerebral Electrophysiological studies on galanin effects cortex. J. Neurosci. Meth. 30, 203–210

Chapter 12

Localization of Neuropeptides by Radioimmunoassay

Fred Nyberg and Mathias Hallberg

Abstract

The past half-century has seen an enormous development in the area of biomedical science. This includes also research related to neuroactive peptides. These compounds have been the subject for extensive studies in many cases resulting in knowledge opening for new therapeutic strategies for the management of various neurological disorders. In this research the radioimmunoassay has represented an invaluable tool. This method, first introduced for the assessment of serum and plasma levels of various hormones, meant a transition from bioassay to a more sensitive and precise technique for protein and peptide quantification in samples of clinical relevance. It accounts for an approach which became one of the most widely used methods in routine and research at many clinical and basic laboratories. This relatively simple technique also became a worthwhile instrument in most studies exploring the field of neuropeptides. In this chapter, various approaches to probe neuroactive peptides in tissues and biological fluids are reviewed. It is by no means exhaustive and does cover all aspects of radioimmunoassay, but it serves to give the readers an idea about some essential principles of the practical use of this technique for probing peptides in the above- mentioned compartments.

Key words: Analysis, Antibodies, Body fluids, Cerebrospinal fluid, Neuropeptides, Plasma, Radioimmunoassay, Sample preparation, Serum

1. Introduction

Radioimmunoassay (RIA) was developed nearly 50 years ago and has been used in basic and clinical laboratories for almost as long. Its value as an analytical tool within a broad area of biomedical science has been documented worldwide. In 1977, the Nobel Prize for Physiology or Medicine was awarded for the development of tool. The recipient of this Prize, Rosalyn Yalow, was awarded for the development of radioimmunoassays of peptide hormones and reported the usefulness of this method first for the assessment of ACTH levels in human plasma in 1964 (1). Subsequent to this, the

Adalberto Merighi (ed.), Neuropeptides: Methods and Protocols, Methods in Molecular Biology, vol. 789, DOI 10.1007/978-1-61779-310-3_12, © Springer Science+Business Media, LLC 2011 191 192 F. Nyberg and M. Hallberg

RIA technique was proven to be extremely useful in the measurement of a number of peptide or protein hormones. Several different RIAs were developed and the technique revolutionized the area of clinical endocrinology. For instance, the possibility for rapid screening of circulating levels of insulin, thyrotropin, growth hor- mone, and (2–4) brought about a remarkable diagnostic tool for a number of endocrine disorders. During 1970s, at the time for the beginning of the discovery of a number of peptides acting on the central nervous system (CNS), the so-called neuropeptides, the RIA became an invaluable tool for studies performed in order to understand the function and mechanisms of action of these compounds. The RIA was used to study the role of neuropeptides in relation to various behaviors using experimental animal models, but also to confirm their presence in humans by probing their levels in various body fluids (5–7). Today, the RIA procedure represents the most commonly used instrument for assessing neuropeptide levels in experimental animals as well as in samples of human origin. And yet, although the method has clearly recognized advantages over earlier methods and the length of time that it has been in use, it is still connected with several drawbacks to consider. This chapter briefly outlines the theory behind the RIA technique but it also includes the major procedures involved in the protocol and discusses advantages as well as drawbacks with the procedures. The theory behind RIA is based on the specific interaction between a given antigen and its antibody. Thus, in the case of neu- ropeptides, the essential principle involves the binding of an isotope-labeled peptide to its specific antibody and competition of this binding by the unlabeled peptide in standard buffer solu- tions or by authentic peptides in samples of biological origin. A standard curve is established by plotting the ratio of bound (B) to free (F) labeled peptide (B/F) as a function of the total concen- tration of the unlabeled peptide present in solution at known concentrations. The level of the peptide in the sample being analyzed is subsequently determined from the B/F ratio recorded for the actual sample with reference to the standard curve. Thus, unknown samples are read off against the peptide standards by their ability to block the binding of the radioactive peptide tracer to a small fixed quantity of antibody. For RIA procedures, it is essential that the antibodies selected for use exhibit high structural specificity and energy of reaction to provide assays of high sensitivity. In this context, it is obvious that the schedule and route of immunization is of particular importance. For instance, a schedule including intradermal injec- tions of low doses antigen has been reported to give highly specific antisera in a few weeks (8). In the procedure used for many years in our laboratory, booster doses at multiple sites were injected in rabbits with 3 or 4 weeks intervals (9, 10) generating useful 12 RIA 193 antisera within 4 months. However, it is important to realize that most peptides of molecular weight below 4,000 are connected with a weak immunogenic capacity. Peptides of low molecular weight can be rendered immunogenic by coupling to a carrier protein. In our laboratory, we have conjugated the antigen peptide to thyroglobulin (9, 11, 12). The specificity of antisera to low molecular peptides highly depends on the site of attachment of the peptide antigen to the carrier protein, i.e., the peptide should be linked to the protein in a way that the structurally specific sites of the molecule remained exposed. In general, the N-terminal part of the peptide is attached, leaving its C-terminal proportion free for exposure to the immunoglobulins. A crucial aspect related to all type of immunoassays is the spec- ificity of the antibodies. It seems obvious that the specificity of the immune reaction is not absolute. Antigens from different species may differ in their amino-acid sequence but still they cross-react between their antisera. Also, the antigenic site may represents imprints from amino acids, which are common to reside within proteins and peptides not functionally or structurally related to each other. Certain pairs or triplets of residues are frequently occurring in many peptides and proteins. This may create a prob- lem particularly for short peptides. For instance, the sequence Phe-Arg occurs at the C-terminal of both Met-enkephalin- Arg-Phe and -7, two opioid active peptides of differ- ent origin. There is a risk that these peptides may cross-react with their antibodies. Also, within the tachykinin family of neuropep- tides, high degree of cross-reactivity between various peptides and their antibodies has been described (13, 14). All RIA procedures require an effective method for the prepa- ration of a proper radioligand. A proper radiotracer is essential for the ease, precision, rapidity, reliability, and sensitivity of the assay. However, difficulties associated with the tracer ligand are a common problem with the RIA technique. Therefore, this issue needs particular attention. In some cases, the ligand can be labeled by replacing a nonradioactive isotope in the native molecule with radioactive nuclide of the same element. In these cases, the isotope may be 14C or 3H. Labeling of peptides with these isotopes requires de novo synthesis of suitable precursor compounds that in a labeling procedure can be inserted in the molecule. A drawback is that this is a time-consuming and expensive procedure. An alternative to this is a procedure in which a foreign radioactive atom is used, i.e., an atom not present in the native structure of the peptide to be analyzed. For neuropeptide tracers, the commonly used element for foreign labeling is iodine as this atom can easily be substituted by simple chemical methods. A frequently used method for iodina- tion includes the chloramine-T (N-chloro-p-toluenesulphonamide) as oxidizing agent (15, 16). This approach requires a tyrosine residue included in the peptide sequence. The 125I radio-isotope of iodine 194 F. Nyberg and M. Hallberg

is oxidized in the presence of the neuropeptide to be labeled. This oxidation is believed to result in a cationic iodine (I+), which is incorporated into the phenolic group of tyrosine. In peptides lacking tyrosines, a suitable residue (e.g., ) can be substituted by a tyrosine or the structure can be extended with this residue at any of their terminals. For instance, the tachykinin substance P can be labeled with 125I after substitution of a phe- nylalanine at position 8 with a tyrosine. Polypeptides and proteins are often labeled with 125I utilizing enzymatic iodination. In this case, the protein/peptide to be labeled is mixed with lactoperoxidase, hydrogen peroxide, and radio-iodine. To overcome the difficulty with cross-reactivity between different peptides and also with other material which may interfere in the assay, it is suitable to include a preseparation procedure prior to RIA. Separation of peptides can be achieved by several techniques, such as ion exchange chromatography or reverse-phase chroma- tography on disposable silica gel cartridges. In addition, presepa- ration of cross-reacting molecules extends the application of RIA to the measurement of individual peptides in a single sample. Also, in the analysis of series of samples, it is preferable to have access to procedures that allow for a rapid and effective separation. Minicolumns for ion exchange chromatography, which give com- plete separation of short peptides differing in charge, have been used in our laboratory over the years (10, 17, 18). We as well as many other research groups have also used preseparation on reverse-phase cartridge for recovery of peptides in a variety of biological samples prior to RIA (e.g., (9, 19)). To confirm the structure of the measured RIA activity and in the case of sufficient material, the RIA can be combined with HPLC (19) or electro- phoresis and, in some cases, with mass spectrometry (20–22). In the following, this article describes procedures used in our laboratory for quantitative analysis of neuropeptides in samples originating from CSF, plasma, as well as various CNS tissues. In the RIA, it is also necessary to have access to an efficient procedure for a physical separation of the antibody-bound and free-labeled peptide (radioactive tracer) before the proportion of bound tracer can be measured. To separate antibody-bound peptide tracer from free tracer, chemical precipitation can be applied, i.e., by the addition of ethanol, polyethylene glycol (PEG), or ammonium sulfate. Unbound tracer can also be adsorbed onto dextran-coated charcoal. However, all these approaches require careful optimization and their applications are limited mainly to assays of low molecular weight structures. A more general method uses the immunological precipitation of the bound fraction (double- antibody procedures), i.e., the use of a first or second antibody in an insoluble form (solid-phase methods). In methods to probe neuropeptides of comparatively low molecular size (less than 4,000 Da), both the charcoal adsorption and the double-antibody procedures are in common use (see below). 12 RIA 195

2. Materials (see Note 1)

2.1. Generation 1. 100 mM Phosphate buffer, pH 7.5. of Antisera 2. 25% Glutaraldehyde in water. 3. 0.9% Sodium chloride. 4. 0.2 M Potassium phosphate, pH 7.5. 5. Thyroglobulin (Sigma-Aldrich, Sweden AB, Stockholm). 6. Dialysis membrane (Sigma-Aldrich, Sweden AB, Stockholm). 7. Freund’s complete adjuvant (Sigma-Aldrich, Sweden AB, Stockholm). 8. Rabbits.

2.2. Peptide Labeling 1. Peptide(s) of interest. 2. Na125I (Amersham Biosciences, Uppsala, Sweden). 3. 0.2 M Sodium phosphate buffer, pH 7.4. 4. Chloramine-T (Sigma-Aldrich, Sweden AB, Stockholm). 5. Buffer A for HPLC: 15% acetonitrile (ACN) in 0.04% trifluo- roacetic acid (TFA). 6. Buffer B for HPLC: 40% ACN in 0.04% TFA. 7. Reverse-phase HPLC column (e.g., Hichrom C-18, 20 × 4 cm, Hichrom, Theale, UK).

2.3. Sample 1. Homogenizer (Branson, Danbury, CONN). Preparation 2. General laboratory centrifuge. 3. 1 M Acetic acid or methanol/0.1 M HCl (1:1). 4. SepPak cartridges (Waters, Milford, MA). 5. SP-Sephadex C-25 (Amersham Pharmacia, Uppsala, Sweden). 6. Plastic columns (Sigma-Aldrich, Sweden AB, Stockholm). 7. Speed Vac centrifuge (Savant, Hicksville, NY). 8. Buffer I: 0018 M pyridine/0.1 M formic acid, pH 3.2. 9. Buffer II: 0.1 M pyridine/0.1 M formic acid, pH 4.4. 10. Buffer III: 0.35 M pyridine/0.35 M formic acid, pH 4.4. 11. Buffer IV: 0.8 M pyridine/0.8 M formic acid, pH 4.4. 12. Buffer V: 1.6 M pyridine/1.6 M formic acid, pH 4.4. 13. Buffer VI: 4% acetic acid in distilled water or 0.04% TFA in distilled water. 14. Buffer VII: 4% acetic acid in 96% methanol or 0.04% TFA in methanol. 196 F. Nyberg and M. Hallberg

2.4. Radioimmu- 1. Diluent buffer for samples: Methanol/0.1 M HCl (1:1). noassay 2. Buffer A: 0.05 M sodium phosphate, 0.15 M NaCl, 0.02% sodium azide. 3. Assay buffer: 0.1% gelatin, 0.1% bovine serum albumin (BSA), 0.1% Triton X-100 in Buffer A. 4. Dilution buffer for the RIA: 0.05 M sodium phosphate, pH 7.4, 0.1% gelatin, 0.1% BSA, 0.82% NaCl, 0.93% EDTA (see Note 2). 5. Charcoal suspension: 750 mg active charcoal, 75 mg dextran T 70 dissolved in 200 mL 50 mM sodium phosphate buffer, pH 7.4 (see Note 3). 6. Microcentrifuge (e.g., Beckman Microfuge, Beckman Coulter, Brea, CA). 7. Gamma counter (e.g., 1470 Wizard, Wallac, Turku, Finland).

3. Methods (see Note 4)

3.1. Generation 1. Dissolve 5 mg thyroglobulin with 1 mg of the actual peptide in of Antisera 300 ML 100 mM sodium phosphate buffer (pH 7.5) and cool to 0°C. 3.1.1. Conjugation of Peptides to Carrier Proteins 2. Dilute (1:100 v/v) glutaraldehyde in 25% aqueous solution with cold (0°C) distilled water. 3. Add 180 ML dropwise to the reaction mixture. 4. Stir for 30 min at 0°C and subsequently for 24 h at room temperature. 5. Subject the mixture to extensive dialysis against 0.9% NaCl before the formed peptide–thyroglobulin conjugate can be injected into rabbits.

3.1.2. Immunization 1. Emulsify 500 ML of the peptide–thyroglobulin conjugate with an equal volume of Freund’s complete adjuvant. The emulsion will be used for immunization in rabbits. 2. Injected animals intracutaneously on their backs at multiple sites. Use booster doses in volumes of 250 ML, corresponding to 50–100 Mg, for injections at intervals of 3–4 weeks. After 3–4 booster injections (i.e., 3–4 months), useful antisera can be collected and stored at −20°C. The obtained antisera are characterized regarding titers and cross-reaction with similar and related molecules.

3.2. Peptide Labeling 1. Mix the peptide (1–5 Mg) dissolved in 40 ML 0.2 M sodium phosphate buffer, pH 7.4, with 0.5 mCi Na125I (see Note 5). 12 RIA 197

2. Start the reaction by adding 10 ML of the chloramine-T solution (0.2 mg/mL). 3. After 40–60 s, terminate the reaction by adding 100 ML 15% acetonitrile (ACN). 4. Use a reversed-phase HPLC column (e.g., Hichrom C-18, 20 × 4 cm) for purification of the labeled peptide. 5. Elute the HPLC column is eluted with a gradient of ACN (15–45%) containing 0.04% TFA.

3.3. Sample Prepa- 1. Add 1 M acetic acid (1 mL per 10 mg CNS tissue) to samples, ration (see Note 6) heat to 95°C for 5 min, and cool on ice.

3.3.1. Tissue Extraction 2. Homogenize and heat for another 5 min. 3. Cool on ice and centrifuge at 9,000 × g for 20 min. Collect for further processing on minicolumns for ion exchange or reverse- phase chromatography.

3.3.2. CSF Samples 1. Thaw frozen CSF samples (2 mL). 2. For separation on minicolumns with ion exchanger, dilute samples five times with Buffer I. 3. Before reverse-phase separation on SepPak cartridges, acidify samples by the addition of 100 ML 1 M HCl per mL CSF and diluted (1:5) with water containing 0.04% TFA.

3.3.3. Plasma Samples 1. Thaw frozen plasma samples on ice and thereafter centrifuge at 4°C for 10 min at 3,000 × g. 2. Collect the supernatants (2 mL) and subsequently dilute (1:5) with Buffer I and take for separation on ion exchange chromatography. 3. In the case of reverse-phase separation on SepPak cartridges, acidify plasma samples by the addition of 100 ML 1 M HCl per mL plasma, and thereafter dilute (1:5) with water containing 0.04% TFA.

3.3.4. Sample Preparation 1. Pour SP-Sephadex C-25 in Buffer I into small plastic columns (packed gel volume = 1 mL) (see Note 7). Ion Exchange Chromatography 2. Wash columns with 20 mL of Buffer I before adding samples. 3. Apply the diluted samples in Buffer I on the columns in volumes of 5 mL. 4. Prior to elution wash samples with addition 10 mL of Buffer I. 5. Add to each eluate 16 mL of Buffer I. 6. Elute samples stepwise as follows: 4 mL of Buffer II, 4 mL of Buffer III, 4 mL of Buffer IV, and finally by 4 mL of Buffer V. 7. Evaporate eluates in a Speed Vac centrifuge and then analyze by RIA. 198 F. Nyberg and M. Hallberg

Reverse-Phase Separation 1. Precondition the reverse-phase cartridges by successive washes on C-18 Cartridges with 10 mL Buffer VI and 10 mL Buffer VII. (SepPak) 2. Apply samples and wash cartridges with 4 mL of Buffer VI. 3. Elute in increasing concentrations of methanol. 4. Evaporate the eluates in the Speed Vac centrifuge.

3.4. Radioimmu- 1. Add a 25 ML aliquot of sample or standard in diluent buffer to noassay (see Note 8) the assay vial. 2. Incubate 100 ML of antisera and 100 ML of iodinated peptide (~4,500 cpm) in triplicate overnight at 4°C. The antisera and the labeled peptide are diluted in assay buffer. 3. Add 200 ML dextran-coated charcoal suspension to separate the bound and free peptides. 4. After 10 min incubation, centrifuge the mixture for 1 min at 9,000 × g in a microcentrifuge. 5. Remove a sample (300 ML) of the supernatant and count the radioactivity for 4 min in a gamma counter. 6. Assess the detection limit (analytical sensitivity) calculated as 3× the standard deviation for replicates of standards with the lowest peptide concentration. 7. Determine also the interassay coefficient of variation calculated

from the IC50 values of the standard curves as well as the intraassay coefficient of variation, calculated from controls in the same concentration range as the samples and evenly dis- tributed within the RIA (see Note 9). 8. Assays using double-antibody procedure are carried out identi- cally except for separation of free and bound radioligand. To separate antibody-bound and free-labeled peptide, use 50 ML of the immunoprecipitate. The immunoprecipitate in this case is an anti-rabbit antiserum commercially available.

4. Notes

1. All buffers and solutions are not stored longer than 14 days and all phosphate buffers should always be stored at 4–5°C. 2. To solve the gelatin, the buffer should be heated to approxi- mately 60°C. Add the BSA when the solution has reached room temperature or lower. 3. Dissolve the dextran and charcoal separately before mixing. The charcoal solution should swell overnight before use. 4. Although the RIA techniques has provided an excellent and long-lasting method for quantitative analysis of neuroactive 12 RIA 199

peptides and peptide hormones over decades and still is in frequent use, it is obvious that the procedure also has its limita- tions and has been connected with problems. Among the most recognized difficulties are problems with interfering compounds and cross-reacting entities, which underline the necessity of sample preseparation. Other difficulties concern problems with the radioactive tracer, low specificity of the antibodies, instability of the peptide to be measured, and of course the fact that most neuropeptides are present at very low concentrations, in particular in body fluids. 5. A properly labeled tracer of high stability is essential for a reliable RIA. First, the use of radioactive iodine is preferable since this isotope is easily detected by its gamma emission. It increases the sensitivity of the RIA. A drawback is that the half-life time of 125I is around 60 days, which means that for routine assays the peptide tracer needs to be re-labeled approximately every second or third month. Furthermore, in many cases, the actual neuropeptide lacks tyrosine residues and in these cases an ana- logue containing a tyrosine residue with retained affinity for the antibodies needs to be synthesized. Also, in the labeling procedure, some tyrosines may be mono-iodinated, whereas others are di-iodinated. To obtain a homogenous tracer, it is therefore necessary to combine the labeling procedure with HPLC separation. Finally, the introduction of an iodine atom in the aromatic ring of tyrosine will certainly affect the size of the tracer and may in worst case affect the binding to the antibody. However, this can be avoided by using antibodies directed to the nontyrosine containing part of the peptide. 6. Depending of tissue or body fluid, a number of peptide, proteins, and nonpeptides impurities may appear in tissue extracts and body fluids at considerable high amounts. If these impurities represent peptides/proteins containing amino acid sequences similar to the compound to be measured, it certainly may inter- act with the binding to the antibodies and give rise to false responses in the assay. Moreover, peptides to be measured may also attach to proteins by nonspecific binding. To avoid or minimize cross-reaction and possible interactions with proteins and other contaminating structures, it is important to include effective extractions and preseparation procedures as those described. 7. It is well known that many neuropeptides due to their basic nature are easily adsorbed to the walls of tubes and tubings. To minimize these effects, the tubes or tubing of material resis- tant toward these kinds of interactions should be chosen. For example, polyethylene and polypropylene should be preferred before polystyrene or glass. It is also favorable to choose tubes that have been precoated with some material that reduces 200 F. Nyberg and M. Hallberg

adsorption to the walls or to add some protein, such as serum albumin, which can prevent the peptides to be measured from adsorption. 8. Instability of peptide or peptide tracer is often a major source of error in RIA in addition to methodological errors. During the sampling procedure, it is important to keep in mind that failure to prevent degradation in plasma and tissues or during incubation in the RIA vials may severely affect the responses. 9. In order to clarify the chemical nature of the measured peptide in the RIA, it is essential to have access to techniques that reli- able confirm the structure of the recorded immunoreactivity. Comparing the behavior of the RIA active material with the authentic peptide by means of HPLC (e.g., (6, 19, 23)) or electrophoresis (24, 25) may at least partly help in this regard. A more precise identification can be achieved by HPLC com- bined with mass spectrometry (20–22). However, this perfor- mance can be carried out under the condition that sufficient amount of RIA active material is available.

Acknowledgment

This study was supported by The Swedish Medical Research Council (Grant 9459).

References

1. Yalow, R. S., Glick, S. M., Roth, J., and Berson, in spinal cord and brain following traumatic S. A. (1964) Radioimmunoassay of human injury to the spinal cord: influence of p-chlo- plasma ACTH, J. Clin. Endocrinol. Metab. 24, rophenylalanine. An experimental study in 1219–1225. the rat using radioimmunoassay technique, 2. Soeldner, J. S., and Slone, D. (1965) Critical Neuropharmacology 32, 711–717. variables in the radioimmunoassay of serum 7. Lindh, C., Liu, Z., Lyrenas, S., Ordeberg, G., insulin using the double antibody technic, and Nyberg, F. (1997) Elevated cerebrospinal Diabetes 14, 771–779. fluid substance P-like immunoreactivity in 3. Odell, W. D., Wilber, J. F., and Paul, W. E. patients with painful osteoarthritis, but not (1965) Radioimmunoassay of thyrotropin in in patients with rhizopatic pain from a herni- human serum, J. Clin. Endocrinol. Metab. 25, ated lumbar disc, Scand. J. Rheumatol. 26, 1179–1188. 468–472. 4. Midgley, A. R. (1967) Radioimmunoassay for 8. Vaitukaitis, J., Robbins, J. B., Nieschlag, E., human follicle-stimulating hormone, J. Clin. and Ross, G. T. (1971) A method for pro- Endocrinol. Metab. 27, 295–299. ducing specific antisera with small doses of 5. Zhou, Q., Liu, Z., Ray, A., Huang, W., Karlsson, immunogen, J. Clin. Endocrinol. Metab. 33, K., and Nyberg, F. (1998) Alteration in the 988–991. brain content of substance P (1–7) during 9. Glamsta, E. L., Morkrid, L., Lantz, I., and withdrawal in -dependent rats, Nyberg, F. (1993) Concomitant increase in blood Neuropharmacology 37, 1545–1552. plasma levels of immunoreactive hemorphin-7 6. Sharma, H. S., Nyberg, F., Thornwall, M., and and beta-endorphin following long distance Olsson, Y. (1993) Met-enkephalin-Arg6-Phe7 running, Regul. Pept. 49, 9–18. 12 RIA 201

10. Bergstrom, L., Christensson, I., Folkesson, R., 19. Nyberg, F., Lieberman, H., Lindstrom, L. H., Stenstrom, B., and Terenius, L. (1983) An ion Lyrenas, S., Koch, G., and Terenius, L. (1989) exchange chromatography and radioimmuno- Immunoreactive beta--8 in cere- assay procedure for measuring opioid peptides brospinal fluid from pregnant and lactating and substance P, Life Sci. 33, 1613–1619. women: correlation with plasma levels, J. Clin. 11. Christensson-Nylander, I., Nyberg, F., Endocrinol. Metab. 68, 283–289. Ragnarsson, U., and Terenius, L. (1985) A 20. Renlund, S., Erlandsson, I., Hellman, U., general procedure for analysis of Silberring, J., Wernstedt, C., Lindstrom, L., B derived opioid peptides, Regul. Pept. 11, and Nyberg, F. (1993) Micropurification and 65–76. amino acid sequence of beta-casomorphin-8 in 12. Von Schenck, H. (1977) Production and char- milk from a woman with postpartum psychosis, acterization of an antiserum against pancreatic Peptides 14, 1125–1132. glucagon, Clin. Chim. Acta 80, 455–463. 21. Eriksson, U., Andren, P., Silberring, J., Nyberg, 13. Sharma, H. S., Nyberg, F., Olsson, Y., and Dey, F., and Wiesel, F. A. (1994) Characterization of P. K. (1990) Alteration of substance P after neurotensin-like immunoreactivity in human trauma to the spinal cord: an experimental study cerebrospinal fluid by high-performance liquid in the rat, Neuroscience 38, 205–212. chromatography combined with mass spec- 14. Sakurada, T., Le Greves, P., Stewart, J., and trometry, Biol. Mass Spectrom. 23, 225–229. Terenius, L. (1985) Measurement of substance 22. Eriksson, U., Andren, P. E., Caprioli, R. M., P metabolites in rat CNS, J. Neurochem. 44, and Nyberg, F. (1996) Reversed-phase high- 718–722. performance liquid chromatography combined 15. Hunter, W. M., and Greenwood, F. C. (1962) with tandem mass spectrometry in studies of a Preparation of iodine-131 labelled human substance P-converting enzyme from human growth hormone of high specific activity, cerebrospinal fluid, J. Chromatogr. A 743, Nature 194, 495–496. 213–220. 16. Bolton, A. E., and Hunter, W. M. (1973) The 23. Sharma, H. S., Nyberg, F., and Olsson, Y. labelling of proteins to high specific radioactivi- (1992) Dynorphin A content in the rat brain ties by conjugation to a 125I-containing acylat- and spinal cord after a localized trauma to the ing agent, Biochem. J. 133, 529–539. spinal cord and its modification with p-chloro- 17. Hallberg, M., Johansson, P., Kindlundh, A. M., phenylalanine. An experimental study using and Nyberg, F. (2000) Anabolic-androgenic radioimmunoassay technique, Neurosci. Res. steroids affect the content of substance P and 14, 195–203. substance P(1–7) in the rat brain, Peptides 21, 24. Nyberg, F., Wahlstrom, A., Sjolund, B., and 845–852. Terenius, L. (1983) Characterization of elec- 18. Johansson, P., Hallberg, M., Kindlundh, A., trophoretically separable in human and Nyberg, F. (2000) The effect on opioid CSF, Brain Res. 259, 267–274. peptides in the rat brain, after chronic treat- 25. Nyberg, F., le Greves, P., and Terenius, L. ment with the anabolic androgenic steroid, (1985) Identification of substance P precursor nandrolone decanoate, Brain Res. Bull. 51, forms in human brain tissue, Proc. Natl. Acad. 413–418. Sci. USA 82, 3921–3924.

Chapter 13

Reversed-Phase HPLC and Hyphenated Analytical Strategies for Peptidomics

Anne-Marie Hesse, Sega Ndiaye, and Joelle Vinh

Abstract

Peptide study and analysis widely involve liquid chromatography. Among the different strategies available, reversed-phase liquid chromatography (RP-HPLC) is one of the methods of choice to separate species in a nontargeted approach. The compounds are sorted according to their hydrophobicity, even though the experimental order of elution could change according to the nature of the mobile phase and the stationary phase. In our work, we have developed protocols to resolve hundred of peptidic species. To overcome the limitations of peak capacity of RP-HPLC alone, it has been coupled downstream to tandem mass spectrometry using two different ionization modes. To overcome the limitations of peak capacity of RP-HPLC MS/MS, it has been coupled upstream to strong cation exchange liquid chromatography. Multidimensional analysis allows for a deeper description of a sample because the limit of detection is often due to a lack of dynamic range of the detection itself rather than due to a lack of sensitivity. In this chapter, different protocols are presented. They should be considered as examples that could be used as starting point for new protocols optimization. Even if RP-HPLC is a universal peptide separation method, it should be optimized according to the specific characteristics of the peptide(s) of interest.

Key words: Reversed-phase liquid chromatography, Peptidomics, Mass spectrometry, ESI, MALDI, Peak capacity

1. Introduction

The study of peptides in biological fluids by separation techniques has been widely used for decades. Reversed-phase high-pressure liquid chromatography (RP-HPLC) is a method of choice for peptide characterization, because of its direct interface with mass spec- trometry. It counts among several analytical techniques for the determination of neurotransmitters such as capillary electrophoresis, enzyme assays, sensors, and mass spectrometry (for a review see ref. 1). First dealing on the field of proteomics, this technique is

Adalberto Merighi (ed.), Neuropeptides: Methods and Protocols, Methods in Molecular Biology, vol. 789, DOI 10.1007/978-1-61779-310-3_13, © Springer Science+Business Media, LLC 2011 203 204 A.-M. Hesse et al.

also of high value for the separation and detection of endogenous peptides (2). The development of miniaturized approaches such as micro and nano-HPLC opened the way toward direct biological applications where sample amounts are limited such as the detection and characterization of bioactive peptides. These applications have been reviewed extensively. Saz and Marina have reviewed the works published since 2001 on the micro/nano-HPLC analysis of bioac- tive and biomarker peptides (3). Boonen et al. also have presented an overview of the strategies involving mass spectrometry for neu- ropeptide discovery and analysis (4, 5). Briefly, the separation is based on the hydrophobic properties of the compounds. The peptides are injected in aqueous buffer at a controlled pH in a chromatographic column packed with a hydrophobic stationary phase. The most widely used stationary phase for peptides is the octadecyl carbon chain (C18) bonded silica in association with a mobile phase containing a mixture of acidic aqueous buffer and polar organic solvents. Other phases have been evaluated for diverse applications, working on different retention mechanisms (6, 7) or on the nature of the stationary phase, studying the influence of silica vs. polymeric beads (8), or looking for peak capacity optimization (9). The elution of the peptides is sequentially obtained in isocratic mode by percolation of the mobile phase or with a gradient of the organic solvent concentration. The most frequent organic solvents are acetonitrile or methanol. The organic solvent should be misci- ble with water. The pH of the separation is an important parame- ter, since it controls the apparent charge of the peptides and can influence the strength of the interactions of the peptides with the stationary phase. A good separation resolution can be reached only if the injection volumes can be considered as negligible in compari- son with the column volume itself. Biological samples are often available at low concentrations in large volumes equivalent to several times the column volume and thus cannot be injected in the column as they are. Moreover, bio- logical matrices contain high concentrations of salts that could interact with the analytical elution buffer. Sample cleanup has been studied and online configurations have been proposed (10, 11). A classical strategy to eliminate those two hindrances is the imple- mentation of an upstream preconcentration and desalting step. Because very low amounts of material are targeted, the separation does not accept any external contamination that would hide the signal coming from the species of interest. With this aim, we have developed an online strategy for the efficient removal of plastic component from the buffer (12). Finally, a classic protocol for sample fractionation using strong cation exchange (SCX) chroma- tography is presented as a pseudoorthogonal approach to RP-HPLC when highly complex samples are studied. The detection for such low amounts of sample requires high sensitivity and specificity: mass spectrometry is a method of choice 13 RP HPLC for Peptidomics 205

to detect eluted peptides. Two kinds of interfaces are presented, either associated to electrospray ionization (ESI) or matrix- assisted laser desorption/ionization (MALDI). To validate our experimental setup, the separation of a six-standard proteins tryp- tic digest is given as a reference at the end of the chapter. To minimize sample cross-contamination, we use a total flush method. Examples of such protocols are detailed in the follow- ing. However, one should keep in mind that those are only exam- ples that are only given as a starting point. Each sample requires its own optimization according to the characteristics of the molecular species of interest and of the surrounding biological matrix.

2. Materials

2.1. Monodimensional As examples, three nano-HPLC hardware configurations are given RP-HPLC Hardware (see Note 1). Configuration 1. Famos-Switchos-UltiMate (LC Packings, The Netherlands) or U3000 Dionex (Dionex, Sunnyvale, CA) or 1200 series Agilent HPLC (Agilent Technologies, Santa Clara, CA). 2. Nano-RP-HPLC column: Acclaim C18 PepMap100 (3 Mm, 100 Å, 75 Mm internal diameter (i.d.), 150 mm length – Dionex) or C18 Zorbax 300SB (3 Mm, 300 Å, 75 Mm i.d., 15 cm length – Agilent Technologies) (see Note 2). 3. Capillary SCX HPLC column: BioBasic SCX (300 Å, 5 Mm, 320 Mm i.d. 150 mm length – Thermo Fisher Scientific, Waltham, MA). 4. Regular precolumns for preconcentration and desalting: C18 PepMap100 (5 Mm, 100 Å, 300 Mm i.d., 5 mm length – Dionex) or C18 Zorbax300 SB (5 Mm, 300 Å, 300 Mm i.d., 5 mm length – Agilent Technologies). 5. Large precolumns for buffer cleanup: C18 PepMap100 (5 Mm, 100 Å, 1 mm i.d., 15 mm length – Dionex).

2.2. Chemicals 1. MilliQ Water (Millipore, Billerica, MA – see Note 3). 2. Acetonitrile (ACN) (available from Fisher Scientific (Acetonitrile Optima® LC/MS packaged under nitrogen, 0.2 Mm filtered) or from Mallinckrodt Baker, Phillipsburg, NJ (Ultra Gradient HPLC Grade Baker HPLC analyzed, packaged under nitro- gen, 0.2 Mm filtered)). 3. Isopropanol (IPA) (Fisher Scientific (2-Propanol Optima® LC/MS packaged under nitrogen, 0.2 Mm filtered)). 4. Methanol (MeOH) (VWR Prolabo, West Chester, PA (HiPerSolv Chromanorm for HPLC – Isocratic grade)). 206 A.-M. Hesse et al.

5. Formic acid (FA) (puriss. p.a. eluent additive for LC–MS, Fluka Analytical, Sigma Aldrich, St. Louis, MO) (see Note 4). 6. Trifluoroacetic acid (TFA – protein sequencing grade, protein sequencer reagent) (available from Applied Biosystems, Foster City, CA). 7. 25% Ammonia solution (Merck, Darmstadt, Germany). 8. A-Cyanohydroxycinnamic acid (CHCA – available from LaserBiolabs, Sophia-Antipolis, France). 9. Ammonium citrate (citric acid diammonium salt, 98% capillary GC – available from Sigma-Aldrich). 10. Tryptic digest consisting of six proteins with molecular weight from 11 to 135 kDa (cytochrome C, lysozyme, alcohol dehy- drogenase, bovine serum albumin, serotransferrin, and B-galac- tosidase) (available from Dionex).

2.3. Buffers and 1. Sample buffer: Sample is reconstituted in Solvent A or in 1–2% Solvents (see Note 5) aqueous TFA/ACN 98:2 (v/v).

2. Solvent A: H2O/ACN/FA, 98:2:0.1 (v/v/v).

3. Solvent B: ACN/H2O/FA, 90:10:0.1 (v/v/v). 4. Flush buffer: ACN/IPA/MeOH 1:1:1 (v/v/v). 5. MALDI matrix solution: 5 mg/mL CHCA in 6:4 ACN/0.1%

TFA in H2O v/v, with 10 mM ammonium citrate (see Note 6).

6. Buffer Ac: H2O/ACN/FA, 95:5:0.1 (v/v/v).

7. Buffer Bc: 100 mM ammonium formate in H2O buffer directly prepared with formic acid and ammonia solution 25%. pH is adjusted to 2.5 with TFA. 5% ACN is added to give the final buffer Bc.

2.4. Interface with ESI 1. Fused silica tubing (i.d. 20 Mm). Hardware 2. Nanoelectrostray emitter (SilicaTip™ picotip emitter) with dis- Configuration tal conductive coating, 360 Mm outer diameter (o.d.), 20 Mm i.d., 10 ± 1 Mm tip i.d. (New Objective, Woburn, MA). 3. Electric contact for spray with a voltage 1.2–1.5 kV, for 10–20 nA ion current.

2.5. Interface with 1. Automat for MALDI sample preparation: Probot (Dionex). MALDI Hardware 2. Stainless steel MALDI target compatible with the mass spec- Configuration trometer available.

2.6. Bidimensional 1. U3000 Dionex in dual configuration (see Note 7). HPLC Hardware 2. First dimension column: Capillary SCX HPLC (see Configuration Subheading 2.1, item 3). 3. Second dimension column: Nano-RP-HPLC (see Subheading 2.1, item 2). 13 RP HPLC for Peptidomics 207

3. Methods

3.1. Sample The need to detect very low quantities of analytes from limited Enrichment and amount of diluted biological samples is one of the reasons of sam- Desalting ple enrichment to achieve more sensitivity. Moreover, direct punc- tual injection of biological samples is generally not possible. Indeed, the typical nanocolumn volume is 0.5 ML (see Subheading 2). Injection volume should be less than 5 nL to be considered as punctual. To set the experiment above the detection limit, typical injection volume is 5–10 ML. This is the reason why sample enrich- ment is always performed for biological salts removal and peptide preconcentration. This procedure is identical for 1D- or 2D-liquid chromatography (LC) modes. 1. Trap peptides onto a regular precolumn. 2. Desalt and concentrate with a 15 ML/min flow of solvent A between 5 and 15 min according to the hydrophilic properties of the peptides and to the salt concentration in sample (see Fig. 1).

3.2. Monodimensional 1. Switch the valve V1 and elute the enriched and desalted sample RP-HPLC (1D-LC) (see from the regular precolumn toward the RP nanocolumn (see Notes 8 and 9) Fig. 1). 2. Separate peptides according to their hydrophobicity with an ACN gradient (see Notes 10 and 11).

3.3. RP-HPLC ESI MS/ 1. Use a fused silica capillary with a conductive distal coating at MS Coupling an approximate distance of 2 mm with 1.4 kV potential in front of a grounded counter electrode at the entrance of the mass spectrometer to directly connect the RP-HPLC effluent to the nano-ESI probe (see Note 12).

3.4. RP-HPLC MALDI 1. Use the Probot as LC-MALDI deposition device (Fig. 2) to MS/MS Coupling collect and spot the RP-HPLC effluent on the stainless steel MALDI plate. 2. Begin collection 15 min after gradient start (the first part of the chromatogram usually corresponds to preconcentration, desalting and transfer from the trap column to the analytical column). 3. Mix continuously the matrix solution at 436 nL/min with the eluted sample using an external coaxial capillary added at the exit of the column. 4. Collect one fraction giving one MALDI spot every 10 s. For each run, a total of 240 spots are collected for 40 min. 5. Dry the MALDI samples under atmospheric pressure at 20°C. 6. Blown out any residual possible dust just before the introduction of the MALDI target in the mass spectrometer. 208 A.-M. Hesse et al. ab Load Inject

Sample Loop 20μL Loop 20μL

Purification Purification cartridge cartridge Waste Waste Loading Pump Loading Pump Sample loading cartridge regeneration 100% solvent A 100% solvent B

Trap column Trap column RP-HPLC RP-HPLC column column MicroPump MicroPump RP sample elution RP column equilibration Solvent B gradient 100% solvent A MS detector MS detector

sample desalting, column equilibration sample elution, cartridge regeneration

Fig. 1. Schematic representation of a classical 1D-LC system with a purification cartridge added to remove polymers. (a) Peptides are loaded into the sample loop using a syringe (load sample position), they are injected with 100% solvent A from the loading pump (inject position), they are retained and desalted on the regular trap column. In the mean time, con- taminants are trapped on the purification large precolumn (purification cartridge). (b) Valve V1 switches, the trap column goes to the micropump fluidics pathway, the micropump delivers a positive gradient of solvent B (acetonitrile gradient) to elute peptides from the trap column for them to be separated into the analytical column. In the meantime, the purification cartridge is regenerated with 100% solvent B delivered by the loading pump in parallel of sample elution and RP gradient.

3.5. Bi-dimensional For very complex samples, such as tissues extracts or whole cells HPLC (2D-LC) (see lysates, the total number of peptides widely exceeds the peak capac- Notes 8 and 9) ity of conventional RP-HPLC columns. Co-elution of tens of pep- tides decreases the percentage of peptides that can be further characterized and sequenced. This limits the sample coverage. In this case, 2D-LC offers an additional separation dimension that is useful to overcome this bottleneck. Even if strong cation exchange (SCX) and RP are not totally orthogonal, SCX-LC followed by RP-LC is the default coupling in proteomics and different protocols can be used (see Note 13). An online configuration is presented here. 1. Fractionate peptides on the capillary SCX column. For peptide fractionation, the gradient profile consists of a multilinear gra- dient from 0 to 100 mM ammonium formate (see Table 1). Micropump flow rate changes over separation time. 13 RP HPLC for Peptidomics 209

Fig. 2. Schematics of the RP-HPLC MALDI interface. The LC setup is analogous to the one detailed in Fig. 1. The effluent of the column is added a continuous flow of MALDI matrix solution at a flow-rate ratio sample–matrix 1:2 (v/v) roughly. As presented in the scheme, the matrix solution was added via a tee into an external metallic needle coaxial with the fused silica capillary containing the nano-LC effluent. The two solutions are mixed at the end of the metallic needle just at the contact with the MALDI target. In the picture, the collection frequency was set to 10 s/spot, the MALDI matrix solution was A-cyano-4-hydroxycinnamic acid at 5 mg/mL in 60% ACN, ammonium citrate 10 mM, at 436 nL/min. Up to 15 LC separa- tions can be collected on the same plate.

2. Trap eluted peptides alternatively on the regular precolumn 1 or on the precolumn 2 (Trap Col 1 and 2, in Fig. 3). Valve V2 is switched every 71 min. 3. After the trapping and desalting step, switch valve V3 and elute the trapped peptides toward the analytical column for a RP-HPLC separation (see Note 14). 4. During last RP gradient, equilibrate the SCX column 100% buffer Ac for 1 h.

3.6. Online Buffer Continuous contamination with impurities can be highly detri- Purification and Total mental to the quality of the LC profiles and to the efficiency of Flush detection by mass spectrometry, as observed in our hands (12). It decreases sensitivity of detection and/or the contaminant signals overlap with compounds of interest. This observation has also been made in other laboratories with other equipments. Several peaks showing the well-known PEG MS pattern with m/z values 44.026 units apart are detected, covering approximately 4 min of retention time in every LC run. These compounds are eluted in the same elution window as peptides. It is difficult, if not impossible, to conclude about the source of contamination: solvent or liquid pathway bleeding or common contamination due to containers. 210 A.-M. Hesse et al.

Table 1 Salt gradient characteristics for online SCX–RP-HPLC separation

% of Buffer Bc

Time (min) Start End Flow rate (ML/min)

From 0 to 17 0 0 4 From 17 to 230 0 3 4 From 230 to 301 3 5 3 From 301 to 372 5 7.1 2 From 372 to 443 7.1 8.8 2 From 443 to 514 8.8 10.9 2 From 514 to 585 10.9 17.3 1 From 585 to 656 17.3 24 1 From 656 to 727 24 46 1 From 727 to 798 46 100 1.5 From 798 to 869 100 100 4 From 869 to 929 0 0 0.5 From 929 to 934 0 0 4 Composition of buffer Bc is detailed in Subheading 2. RP-HPLC gradient is given in the Subheading 3. A new RP gradient is started every 71 min for a total SCX gradient time of 934 min

1. To remove contamination in 1D-LC, insert a large precolumn in LC fluidics pathway between the micropump outlet and the injection valve (Fig. 1). 2. Regenerate the purification cartridge by independent flushing at 40 ML/min with 100% solvent B in parallel during the ana- lytical gradient period. 3. At the end of the run, equilibrate the cartridge with solvent A for 15 min. A similar approach is used in our 2D-LC system: 1. Insert a first large precolumn after loading pump 2 and a sec- ond identical precolumn after micropump 1 (Fig. 4). 2. Flush the first cartridge at 40 ML/min with 100% solvent B in parallel of each RP gradient. 3. Flush the second precolumn with solvent B after the last SCX fraction (see Note 15). 13 RP HPLC for Peptidomics 211

Injection valve

MicroPump 1 Salt gradient SCX column

Purification Collection valve cartridge

Trap Trap Waste Loop 20μL column 1 column 2 External valve Sample Loading Loading Pump 1 Pump 2 Waste Purification cartridge

Position A Elution valve Position B RP-HPLC column MicroPump 2 Acetonitrile gradient MS detector

Fig. 3. Schematics of the online 2D-LC configuration. Injection valve: in position A, sample is loaded; in position B, sample is injected on the SCX column. External valve: in position A, the micropump 1 delivers a salt gradient to separate peptides on the SCX column; in position B, the cartridge 2 is regenerated after the last SCX fraction at a high flow rate of solvent B using loading pump 1. Collection valve V2: in position A, peptides are collected on the trap column 1 during desalting and sample elution from trap column 2; in position B, it is the opposite. Elution valve V3: in position A, the loading pump 2 is used for desalting, the RP column is equilibrated; in position B, the micropump 2 delivers the acetonitrile gradient to sepa- rate compounds on the RP column, the loading pump 2 delivers solvent B at high flow rate to regenerate the purification cartridge 1.

4. Notes

1. All nano-HPLC hardware configurations are equipped with a loading pump (two solvent channels, isocratic mode for flow rates between 5 and 50 ML/min are required), a nanopump (three solvent channels, isocratic and gradient modes for flow rates between 50 and 350 nL/min are required), a refrigerated autosampler (set at 4°C) for injection volumes between 1 and 20 ML, a solvent organizer with online desalting, and a column oven (stable temperature at 20°C is used). 2. The stationary phases presented in this work are the one we use, but it does not exclude other phases. One should be aware that different C18 silica phases can give different LC profiles with different elution orders. According to the peptide(s) of interest, the C18 phase of choice will change. 3. Unless stated otherwise, all solutions should be prepared in water that has a resistivity of 18.2 M7 cm and total organic 212 A.-M. Hesse et al.

RT: 15.00 - 54.97 NL: 2.63E6 61 59 TIC F: FTMS + p 28.5 535. 100 NSI Full ms [470.00- 32.23 95 695.34 2000.00] MS G110622_0989_ 28.26 24.89 653.36 90 722.82 e_slm

85

80

75 35.82 582.32 9 32

70 4 30.2 547.3

65 39.63 484.74

60 35.97 30.68 474.23 549.26 55 4 50 26.20 728.84 36.55

45 668.84 33.64 626.34 37.46 Relative Abundance Relative 679.29 41.35 23.29

40 682.35 512.25 26.87 570.74 35

30 40.01 724.40

25 39.33 20 699.33 8.93 7.59 2.28 1.05 22 51 52 15 997 21.85 503.24 41.62 10 21.14 536.17 1132.99 19.31 18.52 519.14 519.14 44.32 823.50 5

0 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 Time (min)

Fig. 4. LC chromatogram from the tryptic digest of 20 fmol of six standard proteins mixture (see Subheading 2). Presented here is a chromatogram detected with the total ion current recorded in nano-ESI interface: more than 400 species have been detected, selected and 100 distinct peptides have been successfully sequenced online by mass spectrometry. The profile illustrates the lack of peak capacity even for a simple peptidic mixture, with many peaks overlapping, for a MHWH of 30 s, which is a good average value for gradient separation in RP-HPLC. This profile was obtained using the RP-HPLC U3000 setup (see Subheading 2 and 3), using a Pepmap Acclaim column coupled to a nano-ESI LTQ-FT mass spectrometer (Thermo Fisher Scientific). The signal was filtered to select only the total ion current from the Fourier Transform Mass Spectrometry full survey scan on 470–2,000 Da mass range.

content of less than five parts per billion. This standard is

referred to as “H2O” in the text. 4. Instead of FA, TFA can be used in mobile phases when an LC-MALDI setup is chosen. It will enhance peptides resolu- tion. In the case of a LC-ESI setup, FA is preferred because TFA is known to suppress the ESI signals of analytes due to ion pair formation. 5. LC buffers should be at least weekly prepared if not daily. The bottles should be changed and washed carefully each time. The

last step should be a thorough rinse with MilliQ H2O. 6. The MALDI matrix solution should be prepared extempora- neously and stored in dark. 7. For 2D-LC experiments, two loading pumps are needed (two solvent channels, isocratic mode for flow rates between 5 and 50 ML/min are required) a micropump (three solvent chan- nels, isocratic and gradient modes for flow rate between 0.5 and 4 ML/min are required), a nanopump (three solvent 13 RP HPLC for Peptidomics 213

channels, isocratic and gradient modes for flow rates between 50 and 350 nL/min are required), a refrigerated autosampler (set at 4°C) for injection volumes between 1 and 20 ML, a sol- vent organizer, and a column oven (stable temperature at 20°C is used). 8. LC setups should be regularly checked for performances in terms of peptides retention times and intensities. Pressure pro- file should be stable. We used a 20-fmol mix of six proteins tryptic digest for sample recovery control. The proteins and the associated proteolytic peptides that are detected and iden- tified on a routine basis are described in Table 2 and are our reference for validation. 9. If the LC system should be kept on standby for a while, do not stop the mobile phase flow rate completely in the RP-HPLC nanocolumn. RP column and trap columns are kept in ACN– H2O, 50:50 (v/v), and SCX column in water. 10. For the Dionex column, the flow rate is 220 nL/min. The default gradient profile consists in a linear gradient from 0 to 50% solvent B in 35 min, then in an isocratic step of 10 min 100% solvent B, and finally 20 min of 100% solvent A as the final equilibration step. 11. For the Agilent column the flow rate is 300 nL/min. The lin- ear gradient starts from 1 to 13% solvent B in 3 min, then from 13 to 44% solvent B in 42 min, 15 min 100% solvent B, and finally 15 min 100% solvent A. 12. The dead volume between the exit of the analytical column and the nano-ESI probe should be as low as possible. We selected a connection in fused silica with 20 Mm i.d. as short as possible to maintain the column in the column oven regulated at 20°C. The connections between the exit of the column, the fused silica capillary, and the nano-ESI needle are realized with pieces of Teflon tubing (10 mm length max), since there is negligible pressure drop between these three parts. The volt- age is applied at the outer surface of the coated nano-ESI nee- dle with no contact with the sample itself. 13. This protocol can be adapted for different peptidic samples. For example, very complex mixtures may require increasing the separation time by lowering the gradient slope. Starting from endogenous peptidic extract, it is likely that all species will not be resolved with overlapping peaks. Therefore, the coupling of the LC with mass spectrometry is highly informa- tive since it allows the detection of different coeluted species. 14. Peptides samples could be very easily lost by nonspecific adsorp- tion in the plastic vials. This phenomenon increases with stor- age time and temperature. It is also more pronounced for very low concentrations. Therefore, it is advisory to store the sample 214 A.-M. Hesse et al. M (ppm) Sequence Peptide Modification(s) Peptide D Theorical MW (Th) r0 t / r t (min) r t 22.22 1.0223.17 1249.620824.64 1.06 1534.747426.4 1.13 −0.31 1444.625726.53 1.2126.76 1.21 1480.4915 −1.22 FKDLGEEHFK 1422.485427.13 1.23 −0.26 1140.465728.49 1.24 LKEccDKPLLEK 1902.8534 0.1128.93 1.30 YIcDNQDTISSK −0.34 1504.581429.03 1.32 −0.33 ETYGDMADccEK 1108.497629.23 1.33 ETYGDMADcCEK −0.28 2 CaM (C) 1439.810830.25 1.34 ccTESLVNR −0.3 CaM (C) 1305.716930.41 1.39 NEcFLSHKDDSPDLPK −0.33 2 CaM (C) 1639.936830.63 1.39 CaM (C) −0.73 EYEATLEEccAK 1930.7511 CaM (C) 30.82 1.40 EAcFAVEGPK 0.57 1749.673231 1.41 RHPEYAVSVLLR −0.65 2 CaM (C) 1752.615331.79 HLVDEPQNLIK 0.33 1.42 2 CaM (C) 32.73 1.46 KVPQVSTPTLVEVSR 1555.6357 0.06 1142.7139 1.50 CaM (C) ccAADDKEAcFAVEGPK 0.37 1577.7507 YNGVFQEccQAEDK 3 CaM (C) −0.85 −0.41 EccHGDLLEcADDR −0.62 2 CaM (C) DDPHAcYSTVFDK KQTALVELLK 2 CaM (C) LKPDPNTLcDEFK CaM (C) CaM (C) , MW = 69,248 Da) Bos Taurus Table 2 Table Identification of 20 fmol a tryptic digest of six standard proteins mixture in RP−HPLC Protein ALBU_BOVINE (Serum albumin, 13 RP HPLC for Peptidomics 215 (continued) AVAVVK M (ppm) Sequence Peptide Modification(s) Peptide D Theorical MW (Th) r0 t / r t (min) r 33.5434.69 1.54 1283.710434.87 1.59 1511.841535.72 1.60 1881.904235.85 1.64 −0.23 1163.629435.86 1.64 −0.88 1002.582338.51 1.64 HPEYAVSVLLR −0.53 1420.678138.6 1.76 VPQVSTPTLVEVSR −1.15 1014.619540.38 1.77 RPcFSALTPDETYVPK −0.7540.96 1.85 1415.6851 LVNELTEFAK 0.23 1479.794844.49 1.88 CaM (C) LVVSTQTALA 0.09 1888.9261 2.04 SLHTLFGDELcK −1.8 1399.6912 −0.49 QTALVELLK −0.4 TVmENFVAFVDK LGEYGFQNALIVR −1.04 CaM (C) HPYFYAPELLYYANK TVMENFVAFVDK Ox (M) t 23.45 1.0726.04 1347.599926.32 1.19 1483.684027.72 1.21 1167.572127.98 1.27 0.02 1768.858628 1.28 −0.42 1216.6030 WcTISTHEANK28.49 0.5 1.2828.55 1.30 KNYELLcGDNTR −1.54 1311.6540 1757.859528.77 1.31 −0.08 KENFEVLcK 1604.806130.56 1.32 HSTVFDNLPNPEDRK CaM (C) 1594.7389 1.40 CaM (C) LLEAcTFHKP −0.65 0.04 1122.5791 −0.42 DKPDNFQLFQSPHGK ELPDPQESIQR 0.33 CaM (C) DNPQTHYY 0.05 CaM (C) KTYDSYLGDDYVR DLLFRDDTK ) MW = 77,703 Da

, Bos Taurus Protein TRFE_BOVINE (Serotransferrin, 216 A.-M. Hesse et al. M (ppm) Sequence Peptide Modification(s) Peptide D Theorical MW (Th) r0 t / r t (min) r 25.42 1.16 1299.62365 0.55 ELNYGPHQWR 21.84 1.00 1507.69578 −0.04 YSQQQLMETSHR 46.48 2.13 1550.8020 −1.08 TAGWNIPMGLLYSK 42.31 1.94 1363.6931 0.33 cGLVPVLAENYK CaM (C) 42.17 1.93 1566.7979 −0.42 TAGWNIPmGLLYSK Ox (M) 41.9 1.92 1830.8573 −0.35 GEADAMSLDGGYLYIAGK 40.04 1.83 1996.8269 0.25 SVTDcTSNFcLFQSNSK 2 CaM (C) 39.24 1.80 1397.6433 −0.57 cLMEGAGDVAFVK CaM (C) 38.44 1.76 1645.6932 −1.21 FDEFFSAGcAPGSPR CaM (C) 35.0836.48 1.6138.37 1.67 1996.7841 1.76 1336.6804 1846.8515 0 −1 −0.72 cAcSNHEPYFGYSGAFK ILESGPFVScVK GEADAmSLDGGYLYIAGK 2 CaM (C) Ox (M) CaM (C) 34.39 1.57 1413.6374 −1.07 cLmEGAGDVAFVK CaM (C ); Ox (M) 34.06 1.56 1466.6421 −0.91 TYDSYLGDDYVR 32.15 1.47 1355.5892 −0.3 NYELLcGDNTR CaM (C) 32.13 1.47 1039.4764 −0.13 ENFEVLcK CaM (C) 32.13 1.47 1389.6756 −0.16 TSDANINWNNLK 30.6731.55 1.40 1.44 1464.7774 1640.7653 0.41 −0.69 ILESGPFVScVKK HSTVFDNLPNPEDR CaM (C) t , MW = 116,388 Da) Escherichia coli BGAL_ECOHS (Beta-Galactosidase, Table 2 Table (continued) Protein 13 RP HPLC for Peptidomics 217 (continued) M (ppm) Sequence Peptide Modification(s) Peptide D Theorical MW (Th) r0 t / r t (min) r 25 1.1425.7 1136.574428.41 1.18 1.30 1402.7357 1071.6407 0.75 −0.18 GVIFYESHGK −0.16 ANGTTVLVGmPAGAK EKDIVGAVLK Ox (M) 45.96 2.10 1446.79679 −1.22 TMITDSLAVVLQR 42.39 1.94 1742.89577 −0.14 LSGQTIEVTSEYLFR 42.12 1.93 1462.79343 −0.02 TmITDSLAVVLQR Ox (M) 40.2240.85 1.84 1787.7301 1.87 1757.85979 −1.65 −0.48 WSDGSYLEDQDMWR VNWLGLGPQENYPDR 36.96 1.69 1472.75126 −0.95 IGLNcQLAQVAER CaM (C) 34.68 1.59 1083.52103 −0.74 GDFQFNISR 33.38 1.53 1428.68549 −0.84 DWENPGVTQLNR 33.34 1.53 1621.75355 −1.58 AVLEAEVQmcGELR Ox (M), CaM (C ) 33 1.51 1425.66226 0.18 YHYQLVWcQK CaM (C) 32.89 1.51 1099.55255 −0.53 IDPNAWVER 32.42 1.48 989.4867 −0.68 WLPAMSER 32.57 1.49 1341.66896 0.28 VDEDQPFPAVPK 30.58 1.40 1252.65805 −0.45 LAAHPPFASWR 29.95 1.37 1265.61671 −0.59 HQQQFFQFR 29.36 1.34 1457.72243 −0.49 APLDNDIGVSEATR 26.8428.41 1.23 1584.78701 1.30 954.43485 −0.5 −0.15 RDWENPGVTQLNR LTAAcFDR CaM (C) t Saccharomyces MW = 36,800 Da) = MW Deshydrogenase 1, cerevisiae, ADH1_YEAST (Alcohol Protein 218 A.-M. Hesse et al. M (ppm) Sequence Peptide Modification(s) Peptide 0.76 ANGTTVLVGMPAGAK D

Theorical MW (Th) r0 t / r t (min) r 33.54 1.54 1045.54217 −0.38 GTDVQAWIR 31.79 1.46 1334.65191 −0.22 cKGTDVQAWIR CaM (C) 24.92 1.14 1428.64926 −0.71 FESNFNTQATNR 39.52 1.81 964.53419 −0.85 EDLIAYLK 31.42 1.44 1386.7400 34.48 1.58 1306.69913 −0.82 GEREDLIAYLK 33.0533.55 1.51 1013.598334.38 1.54 1251.668637.35 1.57 1618.843039.88 1.71 −0.72 1357.575540.7 1.83 −0.54 1447.803342.64 1.86 ANELLINVK −0.36 1.95 SISIVGSYVGNR −0.58 968.4832 1312.6781 VLGIDGGEGKEELFR −0.73 ccSDVFNQVVK −0.44 VVGLSTLPEIYEK −0.22 EALDFFAR SIGGEVFIDFTK 2 CaM (C) 33.74 1.54 1092.62942 −0.51 EDLIAYLKK 32.02 1.47 1168.62131 −0.78 TGPNLHGLFGR 30.02 1.37 1434.79502 −0.11 KGEREDLIAYLK t 26.16 1.20 1456.67011 −0.17 TGQAPGFSYTDANK 22.27 1.02 1584.76492 −0.25 KTGQAPGFSYTDANK ) MW = 11,696 Da gallus, MW = 16, 228 Da) = gallus, MW Bos_Taurus, LYSC_CHICK (Lysozyme C, Gallus Table 2 Table (continued) Protein CYC_BOVINE (Cytochrome C, 13 RP HPLC for Peptidomics 219 is the experimental R t om the elucidated structure of the peptide. The . Chromatographic characteristics are given: 1 is measured in our setup on a routine basis. According to tandem mass 0 t M (ppm) Sequence Peptide Modification(s) Peptide D ) and the schematics are given in Fig. 3 Theorical MW (Th) and 2 r0 t / r t (min) r 41.34 1.89 1675.79986 −0.7 IVSDGNGMNAWVAWR 38.99 1.79 1326.61325 −1.13 GYSLGNWVcAAK CaM (C) 37.46 1.72 1691.79451 −0.85 IVSDGNGmNAWVAWR Ox (M) 36.08 1.65 1753.83343 −1.01 NTDGSTDYGILQINSR t is calculated using the first identified peptide as a reference (ESKPPDSSKDEC(CaM)M(Ox)VK from bovine serrotransferrin, where ` k Protein retention time, the relative retention factor CaM stands for a carboxymethyl on Cys and Ox an oxidized Met) because no experimental spectrometry analysis, sequences and observed peptide modifications are summarized, and the theoretical masses are calculated fr protein associated to each proteolytic peptide is given in the first column The setup used a PepMap Acclaim column (see Subheadings 220 A.-M. Hesse et al.

as concentrated as possible and to analyze the sample immediately after its preparation. The surface of the storage vial seems to have a great influence (13). Use vial with low protein adsorp- tion properties. 15. In the case of persistent contamination, the whole system should be flushed for 4–6 h, and all the valves should be switched every 30–45 min. As described in Subheading 2, we selected a mixture of solvent with strong elution power. However, the use of isopropanol increases the pressure drop during the transition between water–acetonitrile and metha- nol–acetonitrile–isopropanol, so the transition between the two solvent systems should be done at lower flow rates during the equilibration steps in both directions.

Acknowledgments

The authors would like to thank Emmanuelle Demey and Iman Haddad for technical assistance. The 2D-LC work was part of A.M. Hesse’s PhD work, which was cofinanced by the CNRS (Centre National de la Recherche Scientifique) and by L’Oréal. The nano-LC-MALDI work was part of S. Ndiaye’s PhD work, which was financed by UPMC Paris 6. The hardware configuration was financed by the town of Paris (ESPCI ParisTech) for the dual LC configuration and by the Réseau National des Genopoles (RNG) for the monodimensional LC configurations.

References

1. Perry, M., Li, Q., and Kennedy, R.T. (2009) tissues. In: Soloviev, M. (ed.), Peptidomics, Review of recent advances in analytical tech- Meth. Mol. Biol. 615 Humana Press, Springer niques for the determination of neurotransmit- Science. ters. Analytica Chimica Acta 653, 1–22. 6. Pesek, J.J., Matyska, M.T., and Pindi Venkat, J. 2. Fricker, L.D., Lim, J., Pan, H., and Che, F.Y. (2008) Evaluation of protein, peptide, and (2006) Peptidomics: identification and quanti- amino acid retention on C5 hydride-based sta- fication of endogenous peptides in neuroendo- tionary phases. J. Sep. Sci. 31, 2560–2566. crine tissues. Mass. Spec. Rev. 25, 327–344. 7. Wujcik, C.E., Tweed, J., and Kadar, E.P. (2010) 3. Saz, J.M., and Marina, M.L. (2008) Application Application of hydrophilic interaction chroma- of micro- and nano-HPLC to the determina- tography retention coefficients for predicting tion and characterization of bioactive and bio- peptide elution with TFA and methanesulfonic marker peptides. J. Sep. Sci. 31, 446–458. acid ion-pairing reagents. J. Sep. Sci. 33, 4. Boonen, K., Landuyt, B., Baggerman G., et al 826–833. (2008) Peptidomics: The integrated approach 8. Li, Y., and Lee, M.L. (2009) Biocompatible of MS, hyphenated techniques and bioinfor- polymeric monoliths for protein and peptide matics for neuropeptide analysis. J. Sep. Sci. 31, separations. J. Sep. Sci. 32, 3369–3378. 427–445. 9. Donato, P., Dugo, P., Cacciola, F., et al (2009) 5. Boonen, K., Husson, S.J., Landuyt, B., et al High peak capacity separation of peptides (2010) Identification and relative quantifica- through the serial connection of LC shell- tion of neuropeptides from the endocrine packed columns. J. Sep. Sci. 32, 1129–1136. 13 RP HPLC for Peptidomics 221

10. Machtejevas, E., Marko-Varga, G., Lindberg,C. 12. Hesse, A.M., Marcelo, P., Rossier, J. et al et al (2009) Profiling of endogenous peptides (2008) Simple and universal tool to remove by multidimensional liquid chromatography: on-line impurities in mono- or two-dimen- On-line automated sample cleanup for bio- sional liquid chromatography-mass spectrom- marker discovery in human urine, J. Sep. Sci. etry analysis. J. Chromatogr. A 1189, 32, 2223–2232. 175–182. 11. Storme, M.L., t’Kindt, R.S., and Van Bocxlaer, 13. Kraut, A., Marcellin, M., Adrait, A. et al. J.F. (2009) Improved analyte detectability of (2009) Peptide storage: are you getting the proteins and peptide lysates by means of multi- best return on your investment? Defining ple large-volume injection in LC-MS. J. Sep. optimal storage conditions for proteomics Sci. 32, 2346–2352. samples. J. Proteome Res. 8, 3778–3785.

Chapter 14

Neuropeptidomics: Mass Spectrometry-Based Qualitative and Quantitative Analysis

Ping Yin, Xiaowen Hou, Elena V. Romanova, and Jonathan V. Sweedler

Abstract

Neuropeptidomics refers to a global characterization approach for the investigation of neuropeptides, often under specific physiological conditions. Neuropeptides comprise a complex set of signaling molecules that are involved in regulatory functions and behavioral control in the nervous system. Neuropeptidomics is inherently challenging because neuropeptides are spatially, temporally, and chemically heterogeneous, making them difficult to predict in silico from genomic information. Mature neuropeptides are produced from intricate enzymatic processing of precursor proteins/prohormones via a range of posttranslational modifications, resulting in multiple final peptide products from each prohormone gene. Although there are several methods for targeted peptide studies, mass spectrometry (MS), with its qualitative and quantitative capabilities, is ideally suited to the task. MS provides fast, sensitive, accurate, and high- throughput peptidomic analysis of neuropeptides without requiring prior knowledge of the peptide sequences. Aided by liquid chromatography (LC) separations and bioinformatics, MS is quickly becoming a leading technique in neuropeptidomics. This chapter describes several LC-MS analytical methods to identify, characterize, and quantify neuropeptides while emphasizing the sample preparation steps so integral to experimental success.

Key words: Sample preparation, Liquid chromatography, Mass spectrometry, Peptide identification, Stable isotope labeling, Quantitation

1. Introduction

Acting as cell to cell signaling molecules, neuropeptides participate in many regulatory and behavioral functions, such as feeding, sleep- ing, and learning. Neuropeptidomics is the comprehensive analysis of neuropeptides in cells and tissues in the nervous system. Because of the variations inherent to neuropeptide prohormone processing, the

Adalberto Merighi (ed.), Neuropeptides: Methods and Protocols, Methods in Molecular Biology, vol. 789, DOI 10.1007/978-1-61779-310-3_14, © Springer Science+Business Media, LLC 2011 223 224 P. Yin et al.

presence of posttranslational modifications (PTMs), and the intrinsic low relative abundance of neuropeptides, neuropeptidomics method- ologies generally demand high sensitivity, great selectivity, and a wide dynamic range of detection (1–6). Assisted by the develop- ment of enhanced sample preparation techniques and high-perfor- mance liquid chromatography (HPLC), mass spectrometry (MS) has proven to be particularly useful in neuropeptidomics, primarily due to its capability to unambiguously characterize peptides in com- plex biological samples without requiring prior knowledge of the peptide sequences (6, 7). Since its early success in characterizing thyrotropin-releasing hormone (8), MS has been used to character- ize hundreds of putative signaling peptides in a range of animals (9–17). In addition, several MS-based measurement approaches have been developed that enable relative quantitation of peptide levels in biological samples, including correlating peptide-level changes to specific traits, conditions, and even behaviors (18–20). By implementing the appropriate sample preparation protocols and LC-MS platforms, MS-based neuropeptidomics analysis can provide a wealth of information about the cells/tissues under investigation: identification and characterization (21, 22), distribu- tion and location (23, 24), temporal and spatial release dynamics (25), and relative level changes (18–20, 26–28). Specifically, this chapter describes generalized MS-based approaches to achieve peptide identification and relative quantitation while allowing flexibility in sample type and size. The protocol steps describe the mechanical disruption of cells or tissues by homogenization, extrac- tion of peptides with organic solvents and/or acids, desalting and preconcentration, and LC fractionation followed by peptide measurement, sequencing, identification (Figs. 1a and 2), and quan- titation (Figs. 1b and 3). Scrupulous sampling, optimized LC separation, and appro- priate MS detection are essential to successful neuropeptidomics analysis. We emphasize sampling because the detection of neuro- peptides in complex biological matrices can be compromised by the presence of salts, lipids, and numerous protein degradation products. Protocols specific to LC-MS characterization are also described here. Although there are a number of LC-MS platforms available, we describe capillary LC coupled to electrospray ionization (ESI)-ion trap (IT) MS for peptide measurement, sequencing, and identification. ESI-IT MS is commonly used for peptide identifi- cation due to its high sensitivity (29). With the advent of effective labeling reagents (26, 27, 30), quantitative analysis of peptide levels became possible. Using stable isotopic labeling, neuropeptides from experimental and control samples are chemically modified with the addition of structur- ally identical functional groups that incorporate different isotopes of the same element. Thus, after labeling, neuropeptides from the control and sample treatments are distinguished by mass (Fig. 1b), 14 Qualitative and Quantitative Neuropeptidomics 225

Fig. 1. Workflow of sample processing (a) prior to LC-MS data analysis in a typical MS-based neuropeptidomics approach for peptide identification, and (b) sample processing and succinic anhydride labeling prior to LC-MS data analysis for MS-based neuropeptide quantitation.

and the relative change in peak heights between these conditions are obtained for each peptide pair. Common isotopic reagents

target the N-terminus and the E-amino group of Lys (e.g., d0- or

d6-acetic anhydride, d0-or d4-succinic anhydride (SA), and d0- or

d9-trimethylammonium butyrate) (26, 27), or the C-terminus

(e.g., d0- or d3-methanol) (30). We elaborate on a labeling proto- col that uses the commercially available reagent succinic anhy- dride for peptide quantitation because it produces stable labeling products with measurable mass differences between light and heavy forms (although other reagents are available) (7, 26–28). Nano-scale ultra performance liquid chromatography (UPLC) cou- pled to ESI quadrupole time-of-flight (Q-TOF) MS is used to mea- sure the relative peptide levels. This approach has been selected because the high resolution and speed of ESI-Q-TOF enhances the ability to differentiate the labeled peptides (29) in complex biological samples. 226 P. Yin et al.

LC Relative intensity 20 30 40 50 60 70 Time [min]

2+ +MS 2+ +MS 769.88 719.02 * * Relative intensity Relative intensity

600 650 700 750 800 850 900 950 m/z 600 650 700 750 800 850 900 950 m/z Search against in-house prohormone database +MS2 (769.88) No hit

Manual de novo sequencing

+MS2 (719.02) T S V I/L G K I/L F V T b ion Relative intensity V F I/L K G I/L V S y ion 200 400 600 800 1000 1200 1400 m/z Search against in-house prohormone database Relative intensity PLDSVYGTHGMSGFA The identified peptide is from 200 400 600 800 1000 1200 m/z Pedal peptide-1 precursor MNVAKIPIFLLLVLCGSALCEETNDVTADSDSEEVEAAKRAPEVAKR PLDSVYGTHGMSGFAKRPLDSVYGTHGMSGFAKRPFDSISQGEGL Manual de novo sequence tag (TSVLGKIFVT) SGFAKRPLDSVYGTHGLSGFAKRPLDSVYGTHGMSGFAKRPLDSV matches to auto de novo sequence tag (TSVLGKIFVT). YGTHGMSGFAKRPLDSVYGTHGMSGFAKRPLDSVYGTHGMSGFA KRPLDSVYGTHGMSGFAKRPLDSVYGTHGMSGFAKRPLDSVYGTH Add to in-house Search against EST and whole- GMSGFAKRPLDSVYGTHGMSGFAKRPLDSVYGTHGMSGFAKRPL prohormone database genome shotgun databases DSVYGTHGMSGFAKRPLDSVYGTHGMSGFAKRPLDSVYGTHGMS GFAKRPLDSVYGTHGMSGFAKRPLDSVYGTHGMSGFAKRPLDSVY Newly identified prohormone: Atrial gland peptide B/califin B precursor GTHGMSGFAKRSVDSDEEEAETQAEE MKANTMFIILCLTLSTLCVSSQFTSVLGKIFVTNRAVKSSSYEKYPFDLSK EDGAQPYFMTPRLRFYPIGKRAAGGMEQSEGQNPETKSHSWRERSVL TPSLLSLGESLESGISKRISINQDLKAITDMLLTEQIQARQRCLAALRQRLL DLGKRDSDVSLFNGDLLPNGRCS

Fig. 2. Typical LC-ESI-IT MS results using the animal model Aplysia californica. Left: a peptide was identified from the existing prohormone database via MS/MS. Right: De novo sequencing of unassigned MS/MS spectra facilitate the refine- ment of the existing prohormone database. The sequences in italics indicate the signal peptide of the prohormone.

2. Materials

2.1. Peptide Extraction 1. HPLC-grade acetone (available from Fisher Scientific, Pittsburgh, PA). 2. Deionized water from a Milli-Q filtration system (Millipore, Bedford, MA). 3. HCl (12 M) (available from Fisher Scientific). 4. Acidified acetone (acetone–water–hydrochloric acid (40:6:1), v/v/v). 14 Qualitative and Quantitative Neuropeptidomics 227

Fig. 3. LC-ESI-Q-TOF MS spectra of bradykinin standards labeled with isotopic succinic anhydride tags (molecular weight, 1059.55; masses after labeling 1159.62(H)/1163.66(D); charge state +2). (a) Forward labeling is within 4% of the expected ratio, 2:1. (b) Reverse labeling is within 18% of the expected ratio, 1:2.

5. Homogenizer: pellet pestle cordless motor and pellet pestles with microtubes (Kimble Chase, Vineland, NJ). 6. Centrifuge 5804R with rotor, FA-45-30-11 (9.5 cm radius) with a maximum rotation speed of 20,800 × g (Eppendorf, Hauppauge, NY). 7. Savant SpeedVac Vacuum Concentrator (Thermo Scientific, Waltham, MA). 8. LC-MS-grade acetonitrile (ACN) with 0.1% formic acid (FA) and 0.01% trifluoroacetic acid (TFA) (Sigma-Aldrich, St. Louis, MO).

9. LC-MS-grade water (H2O) with 0.1% FA and 0.01% TFA (Sigma-Aldrich).

10. Reconstitution solution: ACN–H2O (2:98, v/v) with 0.1% FA and 0.01% TFA.

2.2. Peptide Desalting 1. Microcon centrifugal filter unit YM-10 membrane, NMWCO and Preconcentration 10 kDa (Millipore). 2. Deionized water (e.g., from a Milli-Q filtration system – Millipore). 3. C18 spin columns (Pierce, Rockford, IL) or Sep-Pak C18 car- tridges (Waters, Milford, MA).

4. Activating solution: ACN–H2O (50:50, v/v) with 0.1% FA and 0.01% TFA.

5. Equilibrating and desalting solution: ACN–H2O (5:95, v/v) with 0.1% FA and 0.01% TFA.

6. Eluting solution: ACN–H2O (70:30, v/v) with 0.1% FA and 0.01% TFA. 228 P. Yin et al.

2.3. Peptide 1. HPLC system: Micromass™ CapLC system (Micromass, Fractionation and Manchester, UK) with manual injector (Valco Instruments Co. Identification Inc., Houston, TX) and OPTI-PAK® Trap Column 0.5 ML C18 (Optimize Technologies, Inc., Oregon City, OR). 2. HPLC column: LC Packings™ 300 Mm i.d. × 15 cm, C18 PepMap100, 3 Mm particle size, 100 Å pores (LC Packings, San Francisco, CA).

3. Solvent A: ACN–H2O (5:95, v/v) with 0.1% FA and 0.01% TFA as mobile phase.

4. Solvent B: ACN–H2O (95:5, v/v) with 0.1% FA and 0.01% TFA as mobile phase.

5. Solvent C: ACN–H2O (2:98, v/v) with 0.1% FA and 0.01% TFA as loading solvent. 6. ESI-IT MS system: HCTultra PTM Discovery System™ (Bruker Daltonics, Billerica, MA). 7. Software: Bruker Daltonics Hystar to integrate the HPLC and the ESI-IT MS, and to establish the HPLC method. 8. Software: Bruker Daltonics EsquireControl to set up the ESI-IT MS acquisition method and control the ESI-IT MS. 9. Software: Bruker Daltonics DataAnalysis to generate the ESI-IT MS and tandem MS (MS/MS) spectra for peptide identification. 10. Online search engine: Mascot (http:\\www.matrixscience.com, Matrix Science, London, UK) to search the MS/MS spectra against peptide sequences in the selected database. 11. Software: Peaks Studio (Bioinformatics Solutions Inc., Waterloo, ON, Canada) as an alternative algorithm for data- base searching and automatic de novo sequencing.

2.4. Peptide 1. Peptide extraction materials (see Subheading 2.1). Quantitation with 2. Solution of 1 M NaOH in deionized water. Succinic Anhydride 3. Solution of 1 M phosphate buffer in deionized water (pH 9.5). Labeling 4. Labeling reagents: 2 M H4-succinic anhydride (SA-H) in

dimethyl sulfoxide (DMSO), and 2 M D4-succinic anhydride (SA-D) in DMSO (Sigma-Aldrich). 5. pH paper. 6. Solution of 2.5 M glycine in deionized water. 7. Solution of 2 M hydroxylamine in DMSO. 8. Sample cleanup and preconcentration materials (see Subheading 2.2). 9. UPLC system: nanoAcquity UPLC with autosampler (Waters). 10. UPLC column: nanoAcquity UPLC Atlantis dC18 Column, 75 Mm i.d. × 15 cm, 3 Mm particle size, 100 Å pores (Waters). 14 Qualitative and Quantitative Neuropeptidomics 229

11. Solvent A: H2O with 0.1% FA and 0.01% TFA as mobile phase. 12. Solvent B: ACN with 0.1% FA and 0.01% TFA as mobile phase. 13. ESI-Q-TOF MS system: API Premier US™ (Waters). 14. Software: MassLynx (Waters) to set up both UPLC separation and MS acquisition methods, control the UPLC-ESI-Q-TOF MS system, and generate MS spectra.

3. Methods

MS-based qualitative and quantitative analyses for neuropeptidomics include several steps. The first involves peptide extraction from a pool of many cells or a tissue region, with the goal of having a sample large enough to ensure the peptides are detectable. However, larger samples do not always ensure success. In some cases, the pep- tides are highly concentrated and localized into a small region within the tissue. Another important factor in sample preparation is the reduc- tion of peptides attributable to degradation of high-abundance proteins. To prevent postmortem protein degradation, extraction procedures have been developed to deactivate the protein proteases. Protease activity can be reduced by using a strong, acidified organic solution for extraction (21), by microwaving the samples immedi- ately after the dissection (28), or by sacrificing the animals with focused microwave irradiation (3). Here, we describe a peptide extraction protocol using fresh tissues placed in an acidified acetone solution. Larger proteins in the extracts are filtered out based on molecular size. The filtrates are further desalted and preconcen- trated with a reversed solid-phase column before being analyzed by LC-ESI-IT MS and MS/MS. Software and search engines containing either public precursor protein databases or in-house prohormone databases are used specifically for neuropeptide/hormone identifi- cation. The advantage of using a tailored prohormone database is that more targeted results are obtained, especially if likely prohor- mone PTMs are included in the library and thus can be predicted using expert systems such as NeuroPred (31). For quantitative analysis, we detail the peptide labeling reaction with succinic anhydride and introduce UPLC-ESI-Q-TOF MS analysis for characterizing relative peptide-level changes.

3.1. Peptide Extraction 1. Homogenize the biological samples (e.g., isolated cells or tissues from the nervous system) in acidified acetone (see Notes 1 and 2). 2. Centrifuge the homogenate at 14,000–20,000 × g at ambient temperature in a 1.5-mL microcentrifuge tube for at least 5 min or until all the solid debris is precipitated. 230 P. Yin et al.

3. Transfer the supernatant into a different microcentrifuge tube. 4. Vacuum-dry the supernatant in a SpeedVac concentrator.

5. Reconstitute the samples in 20–40 ML of ACN–H2O (2:98, v/v) with 0.1% FA and 0.01% TFA and vortex well. 6. Centrifuge the reconstituted samples at 14,000–20,000 × g at ambient temperature for at least 5 min or until the solid debris is precipitated and save the supernatant.

3.2. Peptide Desalting 1. Filter the supernatant through a Microcon YM-10 cut-off filter and Preconcentration to remove proteins larger than 10 kDa from the extract (see Note 3). 2. Desalt the samples with C18 spin columns or Sep-Pak C18 cartridges following the manufacturer’s instructions for the corresponding products (see Note 4). 3. Vacuum-dry the elution solution from the desalting step in a SpeedVac concentrator.

4. Reconstitute the samples in 20–25 ML of ACN–H2O (2:98, v/v) containing 0.1% FA and 0.01% TFA, vortex well, and centrifuge. 5. Place the samples in a 4°C refrigerator for short-term storage or in a −20°C freezer for long-term storage.

3.3. Peptide 1. Thaw the frozen samples at ambient temperature if necessary. Fractionation 2. Vortex the samples for 5 min. and Identification 3. Centrifuge the samples at 14,000–20,000 × g at ambient temperature for 5 min. 4. Prepare the LC-MS system by first washing the injection loop a few times with an organic solution (e.g., ACN or MeOH), followed by deionized water. 5. Set up the HPLC method: set the isocratic flow of solvent C at a flow rate of 8 ML/min for 5 min to load the sample onto the C18 trap column, and the separation gradient of the mobile phases at a flow rate of 2.5 ML/min, starting from 2 to 80% of solvent B in 40–80 min (determined by the sample complexity), staying at 80% of solvent B for 5–10 min to elute any hydro- phobic compounds, then ramping back to 2% of B, and main- taining at 2% of B for 10–15 min to equilibrate the column (see Note 5). If UV detection is available prior to MS, the wave- length is generally set up at 214 nm for peptide detection. 6. Equilibrate the HPLC system with a blank run (no sample injection) to make sure that the column and lines are clean. 7. Inject the samples at a volume not greater than the HPLC injection loop size into the HPLC system with a syringe. 14 Qualitative and Quantitative Neuropeptidomics 231

8. Separate the samples using a reversed-phase column with the established HPLC method. 9. The Hystar software triggers the ESI-IT MS acquisition at the same time as the HPLC separation. Data-dependent MS/MS acquisition is activated once the peptide ion peaks in the MS scan reach a certain threshold, as indicated in the MS acquisition method in the EsquireControl. The MS acquisition method also includes the selection of 2–3 precursor ions with highest intensities for MS/MS acquisitions within 1 min, and dynamic exclusion of already fragmented precursor ions after 2–3 iterations within 1 min. The MS scan is generally in the range of 300– 2,000 m/z, and MS/MS is typically performed in the range of 50–2,000 m/z. The preferred mass-to-charge ratio (m/z) and charge state (z) can also be defined. 10. Use the Bruker Daltonics DataAnalysis software to display the MS and MS/MS spectra, and to find compounds for identifi- cation by selection and deconvolution of the MS/MS spectra. A typical LC-MS result is shown in Fig. 2. The processed MS/ MS data can be further converted to a universal format (e.g., mgf directly through DataAnalysis, or mzMXL found at http://ionsource.com/functional_reviews/CompassXport/ CompassXport.htm). 11. Import the converted data files to the selected search engine (e.g., Mascot or PEAKS Studio). 12. Set the search parameters, such as mass tolerance (see Note 6) and PTMs (see Note 7), in the search engine for both database searching and automatic de novo sequencing, if available. 13. Search against public or in-house databases and initiate auto- matic de novo sequencing at the same time, if available (Fig. 2). 14. Manually check all of the positive search results on peptide identities for correct peak assignments, reasonable cleavage sites, and PTMs (see Notes 6–8). 15. For MS/MS data that do not result in positive sequence assign- ments via database search or automatic de novo sequencing, conduct manual de novo sequencing to generate sequence tags by matching the space between two fragment peaks to an

amino acid with or without neural losses (e.g., −18 Da for H2O

loss and −17 Da for NH3 loss). The sequence tags can be used to search against genome databases with the assistance of bio- informatics (9, 32), as shown in Fig. 2.

3.4. Peptide 1. Peptide extraction is conducted for all the samples (see Quantitation with Subheading 3.1 and Note 9). Succinic Anhydride 2. Adjust the pH of the control and the experimental sample Labeling solutions to ~9 using 1 M phosphate buffer and 1 M NaOH. 232 P. Yin et al.

3. Add equal amounts of the labeling reagents (2 M SA-H and SA-D) to the both the control and the experimental samples (see Note 10). 4. Vortex the mixed reactants and centrifuge. 5. Incubate the reactants at ambient temperature for 10 min. 6. Adjust the pH of the sample solutions to ~9 with the 1 M NaOH solution. 7. Repeat steps 3–6 at least five times for each sample. 8. Quench extra SA in the samples with the 2.5 M glycine solu- tion for 40 min at room temperature before combining the control and the experimental samples (see Note 11). 9. Use a Microcon YM-10 cut-off filter to remove proteins larger than 10 kDa from the combined sample solutions (see Note 3). 10. Adjust the pH of the combined sample solutions to ~9 using the 1 M NaOH solution. 11. Add 2 M hydroxylamine solution into the combined sample solutions (see Note 11). 12. Incubate the solutions at ambient temperature for 30 min. 13. Desalt and preconcentrate the labeled samples with C18 spin columns or Sep-Pak C18 cartridges (see Subheading 3.2, steps 2–5). 14. Analyze the samples with UPLC-ESI-Q-TOF. The UPLC method is similar to the HPLC method described above in Subheading 3.3, step 5 with autosampler; here, inject the samples using solvent A at a flow rate of 400 nL/min to load the samples directly onto the nano-column, and a mobile phase set to a flow rate of 400 nL/min to separate the samples. MS scans are acquired in the range of 300–2,000 m/z. 15. Use MassLynx to display the MS spectra; the original peptide mass is determined by converting the experimentally mea- sured mass of the labeled peptide into that expected from an unlabeled peptide (see Eq. 1). The peak assignment is per- formed by matching the calculated original peptide mass with the known peptide mass previously identified under Subheading 3.3 and using peptide elution orders to confirm the assignment.

1 MmzzL= [( / )HD ×u 100.030  ( mz / ) 2 (1) uzL u104.054]  1.008 z

M is the calculated original peptide mass; (m/z)H and (m/z)D are the monoisotopic mass-to-charge ratios of the light and heavy form peptides, respectively; z is the ion charge; L is the number of labeled tags. 14 Qualitative and Quantitative Neuropeptidomics 233

16. Generate extracted ion chromatograms for the MS spectra of both the light and the heavy forms of the labeled peptides using MassLynx, and sum the intensities of the mass spectra over the entire elution time for the paired peptides, respec- tively, for quantitation. The relative intensities or areas of paired peaks in the MS scan reflect the relative amounts of peptides in the control and the experimental samples. With SA-H/SA-D as labeling reagents (Fig. 3), light and heavy isotope-labeled peak pairs are separated by 4 Da for singly charged ions and 2 Da for doubly charged ions. 17. Perform statistical analysis (e.g., Student’s t-test) to identify significant differences between the control and experimental samples and evaluate data consistency between technical/ biological replicates.

4. Notes

1. Alternative extraction solutions that work well for peptides

include acidified methanol – methanol–H2O–HAc = 90:9:1,

v/v/v (9), methanol–H2O–HAc = 90:1:9, v/v/v (33), or 10 mM HCl (26–28). To improve the efficiency of peptide extraction, thorough homogenization of biological samples and the combination of supernatants from a multistage approach using the same or different extraction solutions can be employed (15, 16). 2. It is recommended that a minimum volume of extraction solu- tion is used to enable complete homogenization without over- diluting the samples. For example, Hatcher et al. (21) used 40 ML of acidified acetone to homogenize SCN punches in rats, and Che et al. (28) suggested 100 ML of 10 mM HCl per pituitary be used for peptide extraction in mice. 3. The cut-off filter should be rinsed with deionized water or other buffers before use, as stated in the manufacturer’s instruc- tions, to prevent discharge of polymers into the samples. In addition, the filter needs to be washed multiple times during sample filtration, followed by combining the individual filtrate fractions together to improve sample recovery. 4. Desalting and preconcentration with C18 spin columns or Sep-Pak C18 cartridges requires activation and equilibration of the sorbent, loading the samples multiple times to improve sample recovery, washing away the salts, and eluting out the peptides. Attention should be paid to prevent drying of the packing materials in the spin columns and/or cartridges. 5. Two-dimensional LC may be needed for complex samples, in which different separation principles (e.g., ion exchange or 234 P. Yin et al.

reversed phase), different mobile phases (e.g., ACN–water or methanol–water), and different ion pairing reagents (FA or acetic acid) can be used (9, 33, 34). 6. Mass tolerance is determined by both the MS platform and calibration selected. It is generally suggested to set the mass tolerance at 0.5 Da for both ESI-IT MS and MS/MS. 7. Common modifications include C-terminal amidation, N-terminal pyroglutamate formation from glutamine or glutamic acid, and disulfide bonding. Other modifications such as meth- ionine oxidation, N-terminal acetylation, tyrosine sulfation, and phosphorylation of tyrosine, threonine, and serine can be considered, as they are sometimes encountered with neuro- peptides. 8. Neuropeptide precursor processing involves the basic site cleavages of the prohormones, although many basic sites are not cleaved (31). Therefore, putatively identified neuropeptides that form through the cleavages of the prohormone at KR, RR, RK, KK, or RXnR (n = 0, 2, 4, or 6), or other predicted cleavages, are more likely to be correct assignments (1, 4, 6, 33). In addition, at least three consecutive amino-acid matches are required for a positive identification. 9. At least three biological replicates are required to obtain statistics for quantitative experiments. Generally, simultaneous forward (Sample A with SA-H and Sample B with SA-D) and reverse labeling (Sample B with SA-H and Sample A with SA-D) are recommended. It is essential to simultaneously extract all the samples using the same extraction approach and minimize the sample loss for more consistent results. 10. Usually, 1–2 ML of labeling reagent is added at a time. The desired amount of labeling reagent for complete reactions is ~500 times more than the amount of peptides in the samples (27). 11. The total moles of glycine or hydroxylamine used here should equal that of added SA in the samples.

Acknowledgments

This material is based upon work supported by the National Science Foundation (NSF) under Award No. CHE-0526692, by Award No. NS031609 from the National Institute of Neurological Disorders and Stroke (NINDS) and Award No. DA018310 from the National Institute On Drug Abuse (NIDA). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NSF, NIDA, NINDS, or National Institutes of Health. 14 Qualitative and Quantitative Neuropeptidomics 235

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26. Che, F.Y., and Fricker, L.D. (2002) Quantitation 31. Southey, B.R., Amare, A., Zimmerman, T.A. of neuropeptides in Cpe(fat)/Cpe(fat) mice et al (2006) NeuroPred: a tool to predict cleavage using differential isotopic tags and mass spec- sites in neuropeptide precursors and provide trometry. Anal. Chem. 74:3190–3198. the masses of the resulting peptides. Nucleic 27. Che, F.Y., and Fricker, L.D. (2005) Quantitative Acids Res. 34:W267–272. peptidomics of mouse pituitary: comparison of 32. Amare, A., Hummon, A.B., Southey, B.R. et al different stable isotopic tags. J. Mass. Spectrom. (2006) Bridging neuropeptidomics and genom- 40:238–249. ics with bioinformatics: Prediction of mamma- 28. Che, F.Y., Lim, J., Pan, H. et al (2005) lian neuropeptide prohormone processing. Quantitative neuropeptidomics of microwave- J. Proteome Res. 5:1162–1167. irradiated mouse brain and pituitary. Mol. Cell. 33. Dowell, J.A., Heyden, W.V., and Li, L. Proteomics 4:1391–1405. (2006) Rat neuropeptidomics by LC-MS/MS 29. Kinter, M., and Sherman, N.E. (2000) Protein and MALDI-FTMS: enhanced dissection sequencing and identification using tandem and extraction techniques coupled with mass spectrometry, John Wiley & Sons, Inc., 2D RP-RP HPLC. J. Proteome Res. New York. 5:3368–3375. 30. Goodlett, D.R., Keller, A., Watts, J.D. et al 34. Holm, A., Storbråten, E., Mihailova, A. et al (2001) Differential stable isotope labeling of (2005) Combined solid-phase extraction and peptides for quantitation and de novo sequence 2D LC–MS for characterization of the neuro- derivation. Rapid Commun. Mass Spectrom. peptides in rat-brain tissue. Anal. Bioanal. 15:1214–1221. Chem. 382:751–759. Chapter 15

Suppression Subtractive Hybridization

Mohamed T. Ghorbel and David Murphy

Abstract

Comparing two RNA populations that differ from the effects of a single independent variable, such as a drug treatment or a specific genetic defect, can establish differences in the abundance of specific transcripts that vary in a population dependent manner. There are different methods for identifying differentially expressed genes. These methods include microarray, Serial Analysis of Gene Expression (SAGE), and quantitative Reverse-Transcriptase Polymerase Chain Reaction (qRT-PCR). Herein, the protocol describes an easy and cost-effective alternative that does not require prior knowledge of the transcriptomes under examination. It is specifically relevant when low levels of RNA starting material are available. This protocol describes the use of Switching Mechanism At RNA Termini Polymerase Chain Reaction (SMART-PCR) to amplify cDNA from small amounts of RNA. The amplified cDNA populations under comparison are then subjected to Suppression Subtractive Hybridization (SSH-PCR). SSH-PCR is a technique that cou- ples subtractive hybridization with suppression PCR to selectively amplify fragments of differentially expressed genes. The resulting products are cDNA populations enriched for significantly overrepresented transcripts in either of the two input RNAs. These cDNA populations can then be cloned to generate subtracted cDNA library. Microarrays made with clones from the subtracted forward and reverse cDNA libraries are then screened for differentially expressed genes using targets generated from tester and driver total RNAs.

Key words: Differential expression, cDNA amplification, SMART-PCR, Subtractive hybridization, Suppressive PCR, Subtracted cDNA

1. Introduction

Changes in gene expression are associated with a wide range of biological and pathological processes. To gain a better understanding of the molecular and cellular mechanisms underlying disease pro- gression or biological development, the identification of differen- tially expressed genes has been crucial. In recent years, different methods including Suppression Subtractive Hybridization (SSH)

Adalberto Merighi (ed.), Neuropeptides: Methods and Protocols, Methods in Molecular Biology, vol. 789, DOI 10.1007/978-1-61779-310-3_15, © Springer Science+Business Media, LLC 2011 237 238 M.T. Ghorbel and D. Murphy

(1) and gene microarrays (2) have been developed to help identify differentially expressed genes. Although commercial gene microarrays have greatly acceler- ated the identification of differentially expressed genes, such microarrays suffer by being limited to known gene sequences to serve as templates for probe design. Consequently, it is still neces- sary to use additional methods to identify novel genes. Subtractive hybridization is an appealing method for enriching differentially expressed genes. Bautz and Reilly used first this method to purify phage T4 mRNA in the mid-1960s (3). The need for a large quan- tity of mRNA to drive hybridization to completion as well as the difficulty in cloning the small amount of cDNA remaining after hybridization made pure subtractive method to be of limited use. Duguid and Dinauer greatly improved this method by adapting generic linkers to cDNA (4), allowing the selective PCR amplifica- tion of tester cDNA between hybridization cycles. Diatchenko et al. further improved the method by introducing the SSH in which differentially expressed genes could be normalized and enriched over 1,000-fold in single round of hybridization (1). The commercialization of a PCR-Select cDNA Subtraction kit by Clontech (Contech Laboratories, Palo Alto, CA) has led to its increasing popularity in research laboratories. A succession of molecular events occurs during PCR-Select cDNA subtraction (Fig. 1). First, cDNA is synthesized from RNA generated from the two types of tissues or cells being compared. The tester and driver cDNAs are digested with RsaI, yielding blunt ends. The tester cDNA is then divided into two parts, and each is ligated with a dif- ferent cDNA adaptor. Because the ends of the adaptor do not con- tain a phosphate group, only one strand of each adaptor attaches to the 5c ends of the cDNA. The two adaptors have portions of iden- tical sequence to allow annealing of the PCR primer once the recessed ends have been filled in. Two hybridizations are then performed. In the first, an excess of driver is added to each sample of tester. The samples are then heat denatured and allowed to anneal, generating a, b, c, and d types of molecules in each sample (Fig. 1). The concentration of high- and low-abundance sequences is equalized among the type a molecules because reannealing is faster for the more abundant molecules due to the second-order kinetics of hybridization. At the same time, type a molecules are significantly enriched for differen- tially expressed sequences, while cDNAs that are not differentially expressed from type c molecules hybridize with the driver. During the second hybridization, the two primary hybridiza- tion samples are mixed together without denaturing. Now, only the remaining equalized and subtracted ss tester cDNAs can reas- sociate and form new type e hybrids. These new hybrids are ds 15 Subtractive Hybridization 239

Tester cDNA Tester cDNA + Driver cDNA + Adaptor 1 (in excess) Adaptor 2R

First hybridization

a a

b b

c c

d d

Second hybridization

a, b, c, d Mix samples. Add fresh denatured driver. Anneal + e

Fill in the ends

a a

b b

c c

d

e

Add primers Amplify by PCR

a, d No amplification b b’ No amplification c Linear amplification

5’ 3’ e %XPONENTIALAMPLIFICATION 3’ 5’

Fig. 1. Schematic diagram of PCR-Select cDNA subtraction. 240 M.T. Ghorbel and D. Murphy

tester molecules with different ends corresponding to the sequences of Adaptors 1 and 2R. Fresh denatured driver cDNA is added (again without denaturing the subtraction mix) to further enrich fraction e for differentially expressed sequences. After filling in the ends by DNA polymerase, the type e molecules (the differentially expressed tester sequences) have different annealing sites for the nested primers on their 5c and 3c ends. The whole population of molecules is then subjected to PCR to amplify the differentially expressed sequences. During this PCR, type a and d molecules cannot be amplified because they are miss- ing primer annealing sites. Because of the suppression PCR effect (Fig. 2), most type b molecules form a pan-like structure that pre- vents their exponential amplification. Type c molecules have only one primer annealing site and amplify linearly. Only type e mole- cules – the equalized, differentially expressed sequences with two different adaptors – amplify exponentially. The suppression occurs when complementary sequences are present on each end of a sin- gle-stranded cDNA. During each primer annealing step, the hybridization kinetics strongly favor (over annealing of the shorter primers) the formation of a pan-like secondary structure that pre- vents primer annealing (Fig. 2). Occasionally, when a primer

ITR ITR 5’ 3’ 3’ 5’

Melting

Cooling Next PCR Cycle Next Self-annealing PCR Cycle

Primer annealing No primer binding: “pan-like” structure suppresses PCR

Elongation DNA synthesis

Fig. 2. Suppression PCR. 15 Subtractive Hybridization 241

anneals and is extended, the newly synthesized strand will also have the inverted terminal repeats and form another pan-like structure. Thus during PCR, nonspecific amplification is efficiently sup- pressed, and specific amplification of cDNA molecules with differ- ent adaptors at both ends can proceed normally. Next, a secondary PCR amplification is carried out using nested primers to further reduce any background PCR products and enrich for differentially expressed sequences. The resulting cDNAs can be directly inserted into a T/A cloning vector. Alternatively, site-specific cloning can be performed using the NotI (also SmaI, XmaI) site on Adaptor I and the EagI site on Adaptor 2R. Blunt- end cloning requires use of the RsaI site at the adaptor/cDNA junction. This cloning allows identification of differentially expressed RNAs by sequence and/or hybridization analysis. Additionally, the PCR mixture can be used as a hybridization probe to screen DNA libraries. After the subtracted cDNA library has been obtained, it is important to confirm that individual clones indeed represent dif- ferentially expressed genes. One way to accomplish this is by microarray screening of the SSH-subtracted cDNA libraries (5). To maximize the sensitivity of the differential screening, two sub- tractions are performed: the original intended subtraction (forward subtraction), and a reverse subtraction in which tester serves as the driver and the driver as tester. The forward and reverse subtracted libraries are spotted onto nylon membrane. Radiolabeled targets generated from tester and driver total RNAs are then hybridized to the subtracted libraries’ microarray. The spot signal intensities are then captured and examined to identify the differentially expressed genes (5).

2. Materials

All materials should be stored at −20°C unless otherwise stated. Working stocks should be kept on ice unless otherwise stated.

2.1. Preparation and 1. MMLV Reverse Transcriptase, RNase H Minus (e.g., Amplification of cDNA PowerScript from Clontech Laboratories, Palo Alto, CA or Superscript II from Invitrogen, Carlsbad, CA). 2. 5× first-strand buffer (supplied with RT). 3. 3c cDNA synthesis primer (3c SMART CDS Primer IIA, 12 PM).

5c-AAGCAGTGGTATCAACGCAGAGTACT(30)VN-3c (N = G, A, T, C. V = G, A or C) Rsa I 4. 5c cDNA synthesis primer (SMART IIA Oligonucleotide, 12 PM, store at −80°C). 242 M.T. Ghorbel and D. Murphy

5c-AAGCAGTGGTATCAACGCAGAGTACGCGGG-3c (see Note 1) Rsa I 5. 5c PCR primer IIA (12 PM) 5c-AAGCAGTGGTATCAACGCAGAGT-3c (see also item 1 in Subheading 2.5). 6. Thermo-stable DNA Polymerase Mix (e.g., Clontech Advantage 2 PCR kit) (see Note 2). 7. 10× PCR buffer (supplied with the polymerase). 8. 1× TNE buffer: 10 mM Tris–HCl (pH 8), 10 mM NaCl, 0.1 mM EDTA. Store at room temperature. 9. CHROMA SPIN 1000 purification columns (Clontech). Store at room temperature.

2.2. Restriction Digest 1. Rsa I (10 Units/PL). of cDNA 2. 10× Rsa I buffer (supplied with the enzyme, e.g., from New England Biolabs, Ipswich, MA). 3. Silica matrix purification system and associated buffers (e.g., NucleoTrap from Clontech or Qiaex II from Qiagen, Valencia, CA or equivalent). Store components according manufactur- er’s specification.

2.3. Preparation 1. T4 DNA ligase (400 U/PL) (containing 3 mM ATP) available of Tester cDNAs from Clontech or Invitrogen. 2. 5× T4 DNA Ligase buffer (supplied with the ligase). 3. Adapter 1: (10 PM) 5c-CTAATACGACTCACTATAGGGCTCGAGCGGCC G CCCGGGCAGGT-3c 3c-GGCCCGTCCA-5c 4. Adapter 2R: (10 PM) 5c-CTAATACGACTCACTATAGGGCAGCGTGGTCG C GGCCGAGGT-3c 3c-GCCGGCTCCA-5c Underlined (regular and bold) bases match the PCR primer sequence. Bold underlined bases match the nested (secondary) PCR primer sequences (see items 2 and 3 in Subheading 2.5).

2.4. Subtractive 1. 4× Hybridization Buffer: 50 mM HEPES (pH 8.3), 0.5 M Hybridizations NaCl, 0.02 mM EDTA (pH 8.0), 10% (w/v) PEG 8000. 2. Dilution buffer: 20 mM HEPES (pH 6.6), 20 mM NaCl, 0.2 mM EDTA (pH 8.0). 15 Subtractive Hybridization 243

2.5. Primary and 1. PCR primer 1 (10 PM): 5c-CTAATACGACTCACTAT Secondary PCRs and A GGGC-3c. Subtracted cDNA 2. Nested PCR primer 1 (10 PM): 5c-TCGAGCGGCCGC Library Construction C CGGGCAGGT-3c. 3. Nested PCR primer 2R (10 PM): 5c-AGCGTGGTCGCG G CCGAGGT-3c. 4. 5c G3PDH primer (10 PM): 5c-ACCACAGTCCATGCCA T CAC-3c. 5. 3c G3PDH primer (10 PM): 5c-TCCACCACCCTGTTGC T GTA-3c. These primers will amplify human, mouse, and rat G3PDH.

2.6. Microarray 1. Nytran + supercharge nylon membranes (Schleicher and Schuel Analysis of the cDNA BioScience, Dassel, Germany). Libraries Generated 2. Krylon repositional adhesive (Schleicher and Schuel by SSH BioScience). 3. Affymetrix 417 arrayer (Affymetrix, Santa Clara, CA). 4. Stratalinker 2400 (BD Biosciences Clontech). 5. ImageQuant software (Amersham Biosciences, Little Chalfont, UK). 6. Superscript II RT (Invitrogen). 7. 10- to 20-mer poly(dT) primer (Invitrogen). 8. Microhyb (Invitrogen). 9. ImageQuant software (Amersham Biosciences). 10. STORM phosphorimager (Amersham Biosciences). 11. GeneSpring software version 7.0 (Agilent Technologies, Stockport, UK). 12. ABI BigDye terminator cycle sequencing kit (Applied biosystems). 13. Universal M13 forward primer (Applied biosystems). 14. ABI 377XL Prism DNA Sequencer (Applied biosystems). 15. Gene Jockey software (Biosoft).

2.7. General Reagents General reagents should be stored and used at room temperature unless otherwise stated. 1. 10 mM dNTP’s (10 mM each, dGTP, dATP, dTTP, and dCTP). Store at −20°C and keep working stocks on ice. 2. Dithiothreitol (DTT), 20 mM. 3. 20× EDTA–glycogen Mix: 0.2 M EDTA, 1 mg/mL glycogen. 4. Phenol–Chloroform–Isoamyl alcohol (25:24:1) (see Note 3): Store at 4°C and keep working stock at room temperature. 244 M.T. Ghorbel and D. Murphy

5. n-Butanol. 6. 4 M ammonium acetate (pH 7.0). 7. Absolute Ethanol. Store at −20°C. 8. 80% EtOH (v/v, 80 parts absolute ethanol and 20 parts sterile, deionized water). 9. TE buffer: 10 mM Tris (pH 7.8), 1 mM EDTA. 10. 0.5 M EDTA (pH 8.0).

11. Sterile, deionized H2O. 12. Agarose gel electrophoresis system. 13. Molecular weight markers.

3. Methods

3.1. Preparation and 1. For each RNA sample, combine the following components in Amplification of cDNA a nuclease-free 0.5-mL microfuge tube: 1–3-PL RNA sample (0.05–1 Pg of total RNA – see Note 4), 1 PL 3c cDNA synthe- 3.1.1. First-Strand cDNA sis primer (12 PM), 1 PL 5c cDNA synthesis primer (12 PM), Synthesis × PL nuclease-free H2O to 5 PL total volume. 2. Mix contents and spin briefly. 3. Incubate at 70°C in the thermal cycler for 2 min. 4. Spin the tube briefly to collect reaction mix at the bottom of the tube. 5. For each sample, prepare and add the following RT Mix (5 PL): 2 PL 5× RT buffer, 1 PL DTT (20 mM), 1 PL dNTP Mix (10 mM of each dNTP), and 1 PL MMLV RNase H minus Reverse Transcriptase. 6. Gently vortex and spin briefly (10 PL total volume). 7. Incubate the tubes at 42°C for 1 h in an air incubator (see Note 5). 8. Add 40 PL of TE and heat-denature at 70°C for 10 min. 9. The samples (50 PL of first-strand cDNA) can be stored at −20°C for up to 3 months.

3.1.2. cDNA Amplification Overcycling of the cDNA amplification reactions can lead to distor- by SMART PCR tion of the relative representation of cDNAs in the amplified prepa- rations. Therefore, it is necessary to determine the optimal cycle number for each sample that generates sufficient amplified cDNA without saturating the PCR reaction. Three identical PCR reactions are set up for each sample – one optimization reaction and two pre- parative reactions. All reactions are subjected to 15 cycles of PCR, after which the two preparative tubes are temporarily transferred to 4°C. An aliquot is removed from the third (optimization) tube, 15 Subtractive Hybridization 245 which is then subjected to further cycles of PCR. Additional aliquots are removed from the optimization tubes after every three cycles. These aliquots, collected at 15, 18, 21, and 24 cycles, are then analyzed by electrophoresis. The two preparative tubes held at 4°C are then returned to the thermal cycler and subjected to the remainder of the optimal cycle number. The optimal cycle number is defined as one less than the number of cycles required to achieve the PCR saturation point. The amount of first-strand cDNA input used for amplification is dependent on the amount of RNA used in the first-strand synthesis reaction. Use the suggested volumes of first-strand cDNA indicated in Table 1. 1. Dispense the appropriate volume of each cDNA (from Table 1)

into a clean PCR tube and adjust to 10 PL with H2O. 2. Prepare enough of the following PCR mix for three PCR reac- tions for each cDNA sample. Per reaction: 74 PL deionized

H2O, 10 PL 10× PCR buffer, 2 PL dNTP Mix (10 mM of each dNTP), 2 PL cDNA PCR primer (12 pmol/PL), 2 PL 50× Advantage 2 Polymerase Mix, 90 PL total volume. 3. Mix well by vortexing and spin briefly to collect the mix at the bottom of the tube. 4. Program your thermal cycler as follows and preheat it to 95°C. 95°C 1 min × cycles 95°C, 15 s 65°C, 30 s 68°C, 6 min 5. Add 90 PL of the PCR Mix to each 10 PL cDNA sample, mix well, and centrifuge briefly. 6. Place all tubes (three identical reactions for each sample) in the preheated thermal cycler.

Table 1 Starting material for PCR reactions

RNA input in first-strand Volume first-strand cDNA synthesis reaction used in PCR reactions

50–100 ng 10 PL 100–250 ng 4 PL 250–500 ng 2 PL 500–1,000 ng 1 PL 246 M.T. Ghorbel and D. Murphy

7. Perform 15 cycles of PCR on all samples. 8. At the end of the 15th cycle, remove the two preparative reaction tubes of each sample and place at 4°C. 9. Remove a 15-PL aliquot from each optimization reaction, place it in a fresh tube, and store at 4°C (see Note 6). 10. Return the optimization reaction tubes to the thermal cycler and continue the cycling program. 11. At the end of 18th cycle, remove another 15-PL aliquot from each optimization reaction tube and place at 4°C. 12. At the end of the 21st cycle, remove another 15-PL aliquot from each optimization reaction tube and place at 4°C. 13. At the end of the 24th cycle, remove another 15-PL aliquot from each optimization reaction tube and place at 4°C. 14. Run the15-PL aliquots of each PCR reaction cycled for 15, 18, 21 and 24 cycles on a 1.2% (w/v) agarose electrophoresis gel using a 1-kb ladder as a marker. 15. Determine the optimal number of cycles for cDNA amplifica- tion, corresponding to one cycle less than the saturation point of the PCR reaction. 16. Retrieve the preparative PCR reaction tubes from 4°C storage and subject these to the remaining number of cycles required to reach the optimal cycle number for each sample. 17. Add 2 PL of 0.5 M EDTA to each tube to terminate the reaction. 18. Combine the contents of both preparative tubes for each amplified cDNA. 19. Confirm that the PCR reactions were successful by running 10–15 PL on a 1.2% (w/v) agarose gel electrophoresis. You should now have optimally amplified, double-stranded cDNA from the two mRNA populations being compared. These samples will now be purified and digested prior to linker ligation and subtractive hybridization.

3.1.3. Purification 1. Set aside a 5-PL aliquot of each sample at −20°C as an “input” of Amplified cDNA control (for step 19 in Subheading 3.1.3, below). 2. Add an equal volume (~190 PL) of phenol–chloroform– isoamyl alcohol (25:24:1) and vortex well to extract the amplified cDNA products. 3. Centrifuge at 14,000–200,000 × g for 10 min. 4. Transfer the upper aqueous phase to a clean 1.5-mL tube. 5. Add two to three volumes (700 PL) of n-butanol and vortex well to extract the aqueous phase and concentrate the cDNAs. 15 Subtractive Hybridization 247

6. Centrifuge at 14,000–20,000 × g for 1 min. Extract the aqueous phase. 7. Discard the UPPER organic phase. The cDNA remains in the LOWER aqueous phase and should be approximately 50 PL. Transfer the aqueous phase to a new 1.5-mL tube. 8. Prepare a CHROMA SPIN 1000 purification column for each sample by inverting the column several times to resuspend the column matrix. 9. Remove the top cap then bottom cap and let the buffer to drain through by gravity flow. 10. Equilibrate the column with 1.5 mL room-temperature 1× TNE buffer. 11. Discard the collected buffer and proceed with purification. 12. Apply fully and slowly the cDNA sample to the center of the gel bed’s flat surface and allow it to enter the column. 13. Apply 25 PL of 1× TNE buffer and allow the buffer to drain out of the column. 14. Apply 150 PL 1× TNE buffer and allow the buffer to drain out of the column. 15. Transfer column to a clean 1.5-mL microfuge tube. 16. Apply 320 PL 1× TNE buffer and collect the entire eluate as your purified cDNA fraction. 17. Transfer column to a clean 1.5-mL microfuge tube. 18. Apply 75 PL 1× TNE buffer and collect the eluate. This frac- tion may contain additional cDNA. 19. On a 1.2% (w/v) agarose gel, analyze the 5 PL “input” control (from step 1 in Subheading 3.1.3) and a 10-PL aliquot of the two eluted column fractions. Use a 1-kb ladder as a marker. 20. The first (320 PL) fraction should contain the majority of recovered cDNA. If the second eluate (75 PL) contains a sig- nificant amount of cDNA, combine it with the first (320 PL) eluate ONLY if the first fraction contains less than approxi- mately 30% of the total input.

3.2. Restriction Digest The purified cDNAs will now be digested with Rsa I. This gener- of cDNA ates blunt ends for linker ligation and produces smaller fragments better suited to subtractive hybridization. 1. Set aside a 10-PL aliquot of each purified ds cDNA sample at −20°C as an “uncut” control (for details see step 4, below). 2. To the purified cDNA, add an appropriate volume of 10× Rsa I buffer to bring the mixture to 1× Rsa I buffer. 248 M.T. Ghorbel and D. Murphy

3. Add ten units of Rsa I, mix well, and incubate at 37°C for 3 h (see Note 7). 4. On a 1.2% (w/v) agarose gel, analyze the 10 PL “uncut” controls and 10 PL of the digestion products. 5. Add 8 PL 0.5 M EDTA (pH 8.0) to terminate the reaction. 6. Purify the digestion products using a silica matrix-based PCR purification system (see item 12 in Subheading 2.2) according to the manufacturer’s recommendations (see Note 8). 7. Transfer the eluted, purified cDNA from the final pellet of silica matrix to a fresh tube (see Note 9).

8. Add 1/2 volume of 4 M NH4OAc to the final elution volume containing the silica purified Rsa I digested cDNAs and mix. 9. Add 2.5 volumes of EtOH to each sample and mix to precipi- tate the digested cDNA. 10. Centrifuge at full speed (20,000 × g) for 20 min at room tem- perature and discard the supernatants. 11. Rinse the pellets with 500 PL 80% (vol/vol) EtOH, centrifuge at full speed (20,000 × g) for 10 min at room temperature, and discard the supernatants. 12. Air-dry the pellets at room temperature for 10–15 min. Do not use a speed vac. 13. Resuspend the pellets in 6.7 PL of distilled water. 14. Use 1.2 PL to determine the concentration of the purified digested cDNAs by absorbance at 260 nm. You should recover between 2 and 6 Pg Rsa I digested cDNA after purification. If the concentration is >300 ng/PL, dilute to 300 ng/PL. 15. Store the remaining 5.5 PL of each Rsa I digested and purified cDNA at −20°C.

3.3. Preparation Label the prepared cDNAs as number 1- and number 2-. An ali- of Tester cDNAs quot of each digested cDNA preparation, 1- and 2-, will be ligated to two separate adaptors; 1 and 2R, making a total of four separate 3.3.1. Ligation of Adaptors ligation reactions. The ligation reactions are labeled 1-1, 1-2R and 2-1, 2-2R. Two additional ligation reactions, labeled 1-c and 2-c, will be prepared by mixing aliquots of reactions 1-1 with 1-2R and 2-1 with 2-2R. Reaction products 1-c and 2-c will later serve as unsubtracted controls. 1. Prepare a ligation mix sufficient for four reactions. Per reac-

tion: 3 PL H2O, 2 PL 5× ligation buffer, 1 PL T4 DNA ligase, total 6 PL. 2. Dilute 1 PL of each RsaI-digested and purified cDNA from step 13 in Subheading 3.2 with 5 PL distilled water. 15 Subtractive Hybridization 249

3. Set up the following ligation reactions with the following labels:

Tube 1-1 1-2R 2-1 2-2R

Diluted cDNA 1 2 PL 2 PL Diluted cDNA 2 2 PL 2 PL Adaptor 1 2 PL 2 PL Adaptor 2R 2 PL 2 PL Ligation mix 6 PL 6 PL 6 PL 6 PL Volume (total) 10 PL 10 PL 10 PL 10 PL

4. Mix well and spin briefly. 5. Transfer 2 PL from tube 1-1 and 2 PL from tube 1-2R to a fresh tube labeled 1-c and mix. 6. Transfer 2 PL from tube 2-1 and 2 PL from tube 2-2R to a fresh tube labeled 2-c and mix. 7. Centrifuge all tubes briefly. 8. Incubate all (six) ligation reactions overnight at 16°C. 9. Add 1 PL EDTA–glycogen mix to each tube to stop the liga- tion reaction. 10. Heat all tubes at 72 C for 5 min to inactivate the ligase. 11. Briefly centrifuge all tubes. These are your adaptor-ligated tes- ter cDNAs to be used in step 1 in Subheading 3.3.2. 12. Transfer 1 PL from tube 1-c to a fresh tube containing 1 mL

of distilled H2O. Label this tube “FORWARD unsubtracted 1-c”. 13. Transfer 1 PL from tube 2-c to a fresh tube containing 1 mL

of distilled H2O. Label this tube “REVERSE unsubtracted 2-c”. 14. Store all tubes at −20°C.

3.3.2. Ligation Efficiency It is crucial to determine if the ligation reactions were efficient by Assay using a PCR assay. The two adaptors used in Subheading 3.3.1 share a region of common sequence. PCR reactions using a primer pair comprised of a G3PDH-specific primer and the adaptor-tar- geted PCR primer generate a specific product if the adaptor sequence is present (the “test” reaction). This product is compared to PCR reaction products generated from a primer pair comprised of two G3PDH targeted primers (control reactions). If the efficiency of the test reaction is similar to that of the control, it indicates efficient ligation of the adaptors to the digested cDNAs. 250 M.T. Ghorbel and D. Murphy

1. Dilute 1 PL of each of the four ligation products (1-1, 1-2R,

2-1, and 2-1R) into 200 PL H2O. 2. For each diluted sample, set up two PCR reactions (eight reac- tions in total) as follows:

Reaction 1 (test) 2 (control)

Diluted sample: 1 PL1 PL (1-1, 1-2R, 2-1 or 2-2R) 3c G3PDH primer (10 PM) 1 PL1 PL 5c G3PDH primer (10 PM) 1 PL Adaptor-targeted PCR primer (10 PM) 1 PL Volume (total) 3 PL3 PL

3. Prepare a PCR mix sufficient for eight reactions (mix well by

vortexing). Per reaction: H2O 18.5 PL, 10× PCR buffer 2.5 PL, dNTP mix, 0.5 PL, Polymerase mix 0.5 PL, total vol- ume 22 PL. 4. Add 22 PL of the PCR mix to each reaction tube from step 2. 5. Mix well and then subject all tubes to the following PCR program: 75°C 5 min (this step fills in the ends of the adaptors to produce fully double-stranded fragments) 95°C 2 min

25 cycles (see Note 10): 95°C 30 s 65°C 30 s 68°C 2.5 min

6. Analyze the PCR products alongside a molecular weight marker on a 2% (w/v) agarose gel. For rat or mouse samples, you should observe a ~1.2-kb band in the test reactions. For human samples, you should observe a ~0.75-kb band (see Note 11). For other species, you will need to design specific primers. Successful ligations will result in nearly equal band intensity from both the test and control reactions. If the band intensity of the test reaction products is less than approximately 40% that of the control, you should repeat the procedure starting from Subheading 3.1.2.

3.4. Subtractive Two subtractions will be performed: a FORWARD and a REVERSE Hybridizations subtraction. For each subtraction, the adaptor-ligated products are designated the tester cDNAs and the un-ligated cDNAs (with no adaptors) are designated driver cDNAs. Adaptor-ligated products 1-1 and 1-2R tester cDNAs will be independently hybridized to an 15 Subtractive Hybridization 251

excess of driver cDNA 2 in two primary, FORWARD subtraction hybridizations (one for each adaptor-ligated preparation, 1-1 and 1-2R). Likewise, adaptor-ligated products 2-1 and 2-2R tester cDNAs will be independently hybridized to excess driver cDNA 1 in two primary REVERSE subtraction hybridizations.

3.4.1. First Hybridization 1. Set up the following First Hybridization reactions using the adaptor-ligated tester cDNAs from step 11 in Subheading 3.3.1 and Rsa I digested and purified driver cDNAs from step 15 in Subheading 3.2: FORWARD REVERSE

First hybridization number (1) (2) (3) (4) Rsa I digested driver cDNA 1 1.5 PL 1.5 PL Rsa I digested driver cDNA 2 1.5 PL 1.5 PL Adaptor ligation cDNA tester 1-1 1.5 PL Adaptor-ligated cDNA tester 1-2R 1.5 PL Adaptor ligation cDNA tester 2-1 1.5 PL Adaptor-ligated cDNA tester 2-2R 1.5 PL 4× Hybridization Buffer 1.0 PL 1.0 PL 1.0 PL 1.0 PL Volume (total) 4.0 PL 4.0 PL 4.0 PL 4.0 PL

2. Incubate the hybridization reactions in a thermal cycler at 98°C for 1.5 min. 3. Hybridize the denatured cDNAs in a thermal cycler at 68°C for 8 h. Do not exceed 12 h. 4. Centrifuge briefly to collect the reaction mixes at the bottom of the tube and return to 68°C briefly while preparing the sec- ondary hybridization reaction mix.

3.4.2. Second 1. Prepare fresh denatured drivers for the second hybridization as Hybridization follows: FORWARD REVERSE

Rsa I digested driver cDNA 1 1.0 PL Rsa I digested driver cDNA 2 1.0 PL 4× Hybridization Buffer 1.0 PL 1.0 PL

Sterile H2O 2.0 PL 2.0 PL Volume (total) 4.0 PL 4.0 PL

2. Incubate these driver cDNA solutions at 98°C for 1.5 min in a separate thermal cycler or heat block (see Note 12). 252 M.T. Ghorbel and D. Murphy

3. Put 1 PL of each freshly denatured driver cDNA 1 and 2 into new 0.5-mL tubes and keep at 98°C. The First Hybridization products (1) and (2) will now be mixed with freshly denatured driver cDNA 2 in the FORWARD subtraction. Similarly, the First Hybridization products (3) and (4) will be mixed with freshly denatured driver cDNA 1 in the REVERSE subtraction. Use the following procedure to simultaneously mix the driver with first hybridization samples (1) and (2). This ensures that the two hybridizations samples mix together only in the presence of freshly denatured driver cDNA 2. (a) Set a micropipettor at 15 PL. (b) Gently touch the pipette tip to the first hybridization product (2) and draw the sample into tip. (c) Remove the pipette tip from the tube and draw a small amount of air into the tip, creating a slight air space below the droplet of sample. (d) Carefully touch the pipette tip to the 1 PL of freshly dena- tured driver cDNA 2 and repeat steps b and c. (e) Transfer the entire mixture to the tube containing first hybridization product (1). (f ) Mix by pipetting up and down. (g) Briefly spin if necessary. (h) Incubate at 68°C. This is the FORWARD subtractive hybridization. Label this tube FORWARD subtracted cDNA. 4. Incubate at 68°C overnight. 5. Repeat this process with 1 PL freshly denatured driver cDNA 1 and First Hybridization (3) and (4) and return the mixture to the thermal cycler at 68°C. This is your REVERSE subtrac- tive hybridization. Label this tube REVERSE subtracted cDNA. 6. Incubate the FORWARD and REVERSE subtractive hybrid- ization mixes at 68°C overnight. 7. Add 200 PL of dilution buffer to each subtraction and mix by pipetting. 8. Heat at 68°C for 7 min in a thermal cycler. 9. Store the “FORWARD” and “REVERSE” subtraction mixes at −20°C.

3.5. Primary and The unsubtracted cDNAs prepared in steps 12 and 13 in Secondary PCRs Subheading 3.3.1 are used as controls the next steps of primary and Subtracted cDNA (suppressive) and secondary (nested) PCRs. Library Construction 15 Subtractive Hybridization 253

3.5.1. Primary 1. Prepare enough of the following PCR mix for four reactions. (Suppressive) PCR Per reaction: H2O 19.5 PL, 10× PCR buffer 2.5 PL, dNTP mix (10 mM) 0.5 PL, PCR primer (10 PM) 1.0 PL, 50× DNA Polymerase Mix 0.5 PL, total volume 24.0 PL. 2. Aliquot 1 PL of each of the following into separate PCR reac- tion tubes, labeled P1, P2, P3, and P4 (P = “Primary”). (P1) FORWARD subtracted cDNA (from step 9 in Subheading 3.4.2) (P2) FORWARD unsubtracted 1-c (from step 12 in Subheading 3.3.1) (P3) REVERSE subtracted cDNA (from step 9 in Subheading 3.4.2) (P4) REVERSE unsubtracted 2-c (from step 13 in Subheading 3.3.1) 3. Add 24.0 PL of the PCR mix to each tube (to make 25-PL reaction volumes), mix, and spin briefly to collect contents of each at the bottom of the tube. 4. Program your thermal cycler as follows and subject all reaction mixes to the following program:

1 cycle: 75°C for 5 mina 27 cycles: 94°C 30 s 66°C 30 s 72°C 1.5 min a This step extends the adaptors to create a complementary target for the PCR primer 5. These are your primary (suppressive) PCR products, P1–P4. When the cycling is complete, transfer 8 PL of each reaction product to a fresh, appropriately labeled tube and store at −20°C for later gel analysis.

3.5.2. Secondary 1. Prepare 1:10 dilutions of each primary PCR product P1–P4 (Nested) PCR with water (e.g., 3 PL PCR product and 27 PL water). 2. Aliquot 1 PL of each diluted primary PCR product into sepa- rate PCR reaction tubes labeled S1, S2, S3, and S4 (S = “Secondary”). (S1) FORWARD subtracted cDNA (S2) FORWARD unsubtracted cDNA (S3) REVERSE subtracted cDNA (S4) REVERSE unsubtracted cDNA 3. Prepare enough of the following PCR mix for four reactions.

Per reaction: H2O 18.5 PL, 10× PCR buffer 2.5 PL, dNTP mix (10 mM) 0.5 PL, Nested PCR primer 1 (10 PM) 1.0 PL, 254 M.T. Ghorbel and D. Murphy

Nested PCR primer 2R (10 PM) 1.0 PL, 50× DNA Polymerase Mix 0.5 PL, total volume 24.0 PL. 4. Add 24.0 PL of the PCR mix to each tube (to make 25-PL reaction volumes), mix, and spin briefly to collect contents of each at the bottom of the tube. 5. Subject all reaction mixes to the following PCR program:

12 cycles: 94°C 30 s 68°C 30 s 72°C 1.5 min

6. Transfer 8 PL of each resulting reaction product to an appro- priately labeled tube for gel analysis. 7. The remaining 17 PL of the secondary (nested) PCR products are your final products. They comprise the following: FORWARD subtracted and unsubtracted cDNA; REVERSE subtracted and unsubtracted cDNA. 8. Electrophorese the 8-PL aliquots of each suppressive and nested PCR product, P1–P4 from step 15 in Subheading 3.5.1, and S1–S4 from step 6, respectively, on a 2% (w/v) agarose gel. Both primary and secondary PCR products should appear as a smear, with or without discrete bands. 9. The final products (S1–S4) can now be cloned to make sub- tracted cDNA libraries and/or used as probes to screen sub- tracted cDNA, global cDNA, or genomic DNA libraries.

3.5.3. Assay 1. In fresh tubes, prepare 1:10 dilutions of each secondary PCR for Subtraction Efficiency product S1–S4 with water (e.g., add 3 PL PCR product to 27 PL water as before). 2. Place 1.0 PL of each diluted secondary PCR product in a fresh PCR tube. 3. Prepare enough of the following PCR mix for four reactions.

Per reaction: H2O 22.4 PL, 10× PCR buffer 3.0 PL, dNTP mix (10 mM) 0.6 PL, G3PDH 5c primer (10 PM) 1.2 PL, G3PDH 3c primer (10 PM) 1.2 PL, 50× DNA Polymerase Mix 0.6 PL, total volume 29.0 PL. 4. Add 29.0 PL of the PCR mix to each tube (to make 30-PL reaction volumes), mix, and centrifuge briefly to collect con- tents of each at the bottom of the tube. 5. Program your thermal cycler as follows:

18 cycles: 94°C 30 s 60°C 30 s 68°C 2 min 15 Subtractive Hybridization 255

6. Place the reaction mixes in the thermal cycler and run for 18 cycles. 7. Remove 5 PL from each reaction and place in separate clean tubes (see Note 6). Put the rest of the reaction back into the thermal cycler for five additional cycles. 8. Repeat step 7 twice (i.e., remove 5 PL after 28 and 33 cycles). 9. Examine the 5-PL aliquots collected at 18, 23, 28, and 33 cycles on a 2% (w/v) agarose electrophoresis gel. Efficient subtraction is indicated by depletion of G3PDH sequences from the subtracted cDNAs for both the FORWARD and REVERSE subtractions. Unsubtracted cDNAs should show an increase in G3PDH product accumulation with increasing cycle number, to the point of a plateau.

3.5.4. T/A Cloning Clone 3 PL of the secondary PCR product from the forward and reverse subtractions using a T/A-based cloning system according to the manufacturer’s protocol. For this application, it is important to optimize your cloning efficiency; a low cloning efficiency will result in a high background.

3.5.5. Site-Specific Site-specific or blunt-end cloning will result in removing the adap- or Blunt-End Cloning tor sequences from your cDNAs. Therefore, the inserts can no lon- ger be amplified using the nested primers. 1. For site specific cloning, digest the secondary PCR product at the Eag I or at Eag I and Sma I (Xma I) sites in the adaptor and then ligate into an appropriate vector for site-specific liga- tion (Eag I will cut at Not I sites). 2. For blunt-end cloning, cleave the Rsa I site in the adaptors and then ligate into an appropriate blunt-end vector.

3.6. Microarray The putative differentially expressed clones were arrayed on a nylon Analysis of the cDNA matrix to generate the tester-driver array. Libraries Generated 1. Amplify cDNA inserts using PCR in a 96-well plate format. by SSH 2. Check the PCR products by gel electrophoresis. 3.6.1. Amplification 3. Transfer to 96-well “V” bottom plates, ethanol-precipitate, of Clones and air-dry overnight. 4. Resuspend pellets in 40 PL of 1× Tris-EDTA. 5. Add 0.1 M NaOH to each sample to denature immediately before arraying.

3.6.2. Array Preparation 1. Coat Nytran + supercharge nylon membranes with Krylon repositional adhesive. 2. Cut to the correct size and trim the edges with a razor; wear gloves at all times. 256 M.T. Ghorbel and D. Murphy

3. Place the membrane units into the arrayer, with enough to fill the entire plate. This allowed 42 microscope slide size areas to be printed. 4. Print the cDNA microarray using an Affymetrix 417 arrayer. This machine is capable of arraying 1,152 spots in duplicate, producing 42 identical arrays in 8 h of continuous printing. 5. Spot cDNA using a 300-Pm pin and spot spacing of 665 Pm center to center in 16 identical 12 × 12 grids. 6. Number the membranes individually and UV cross-link twice using Stratalinker 2400 at 120 mJ/cm2. 7. Remove membranes and bake at 70°C for 1–2 h before storing at −20°C in heat-sealed plastic bags (see Note 13).

3.6.3. Target Generation 1. Probe separate microarrays three times using independently and Array Hybridization generated targets from either driver or tester cDNA. 2. Generate radiolabeled target from 3 to 10 Pg of total RNA (from either tester or driver) template. 3. Use Superscript II RT and 10- to 20-mer poly(dT) primer to incorporate [{alpha}-33P]CTP into cDNA. 4. Place arrays DNA-side inward into 50-mL disposable Falcon tubes and wash with 50 mL of 2× SSC. 5. Prehybridize membranes for 4 h at 50°C with 4.0 mL of Microhyb containing 10 PL of 1 mg/mL human Cot 1 DNA and 10 PL of 8 mg/mL poly(dA), both denatured at 95°C for 5 min before use (see Note 14). 6. Denature the labeled target for 5 min at 95°C and then add to the 4.0-mL prehybridization solution and allow hybridizing to the probe at 50°C for 12–18 h. 7. Wash the membranes in 50 mL of 2× SSC-1.0% (w/v) SDS at 50°C, followed by 2 × 15 min washes in 2× SSC-0.1% (w/v) SDS at 50°C. 8. Align moist membranes on a plastic plate, wrap tightly in plastic, and expose to phosphorimager screens for 1–3 days. 9. Capture spot intensities into ImageQuant software by scan- ning screens at 50-Pm resolution on a phosphorimager.

3.6.4. Microarray Data The microarray experiment generated 2,304 volume intensities Analysis corresponding to gene expression levels obtained for 1,152 ESTs spotted in duplicate. 1. Transfer ImageQuant readings from the phosphorimager scans to Microsoft Excel spreadsheets, predesigned to import ImageQuant information, and label correct gene identities. 2. Average duplicate values for each array and then transfer into GeneSpring software version 7.0 for normalization and analysis. 15 Subtractive Hybridization 257

3. Normalize data using the following criteria: any values below 0.01 are set to 0.01. Chip normalization is achieved by divid- ing each measurement by the 50th percentile of all measure- ments in that sample. The percentile is calculated with all raw measurements above 0.01. Gene normalization involves divid- ing each gene measurement by the median of its measurements in all samples. If the median of the raw values is below 0.01, then each measurement for that gene is divided by 0.01 if the numerator is above 0.01; otherwise, the measurement was thrown out. 4. Filter the normalized data to exclude driver vs. tester fold changes of <1.5. 5. Apply a Welsh t-test with a P value cutoff of 0.01.

3.6.5. Sequence Analysis 1. Sequence target clones to an accuracy of 99.5% over a single of Putative Differentially 650–700 bp read using ABI 377XL Prism DNA sequencers Expressed Genes with ABI BigDye terminator, using universal M13 forward primer and reverse primer. 2. Scrutinize sequences with the use of a variety of computational tools to determine their identity. 3. Compare sequences with each other using GeneJockey soft- ware to check for repetitions. 4. (BLAST)n (http://www.ncbi.nlm.nih.gov/BLAST) sequences to determine their identity. 5. Classify clones as “known” or “novel.” 6. Submitted novel sequences to the public database at GenBank (http://www.ncbi.nlm.nih.gov/GenBank/index.html), using the BankIt dbEST database, and obtain accession numbers.

4. Notes

1. This oligonucleotide is available as part of the SMART PCR cDNA synthesis kit from Clontech. Details of their modifica- tions are proprietary. 2. The Taq polymerase used for cDNA amplification should be nuclease deficient, be formulated specifically for amplification of long fragments, and include the Kellog antibody for auto- matic hot start. 3. Phenol–Chloroform–Isoamyl alcohol should be prepared with fresh (<2 weeks) phenol saturated with 50 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA. If phenol appears yellow from oxidation, it should not be used. 258 M.T. Ghorbel and D. Murphy

4. RNA quality is crucial for successful comparisons of two RNA populations. Assess the concentration and purity of the total RNA samples by spectrophotometry (Nanodrop, Wilmington, DE). Then further analyze samples with a sufficiently high yield and purity (an A260:A280 ratio of close to 2) for integ- rity with an Agilent Bioanalyser (Agilents Technologies, Stockport, UK). Use only RNA with RNA Integrity Number of 8–10. 5. The use of prewarmed air incubator (as opposed to a heat block) could minimize evaporation and condensation in the tube, which can significantly reduce cDNA yields. 6. It is crucial that aliquots are removed from each sample while at the 68°C extension step and not at the denaturation step. Denatured samples will not migrate correctly during electrophoresis. 7. Digestion must go to completion. You can remove aliquots (~10 PL) at several time points and analyze them on an agarose gel. If there is no further decrease in molecular weight with time, the digestion is complete. 8. Alternatively, you could extract samples with phenol–chloro- form–isoamyl alcohol (25:24:1) and ethanol precipitated, although this may lead to decreased adaptor ligation efficiency. 9. After transferring to a fresh tube, you can recentrifuge the elu- ate at full speed for 1 min and transfer to another clean tube to eliminate possible carry over of silica matrix. 10. If G3PDH is highly abundant in your cDNA populations, reduce the cycle number to avoid saturation of the PCR reactions. 11. If a band of ~1.2 kb is generated from a human G3PDH PCR product, this indicates incomplete digestion because the human G3PDH contains an Rsa I site. 12. If possible, have a second heat block or thermal cycler near the thermal cycler used for the hybridizations to avoid premature renaturation of the freshly denatured driver cDNAs. 13. As a quality-control measure, inspect arrays by sight. When the printing head makes contact with the membrane, a mark will be visible. If spots are missing on an array, do not use that array. 14. Before prehybridization and after the draining of SSC, gently remove air bubbles. 15 Subtractive Hybridization 259

Acknowledgments

MTG is funded by the Bristol NIHR BRU in Cardiovacular Medicine and the Garfield Weston Foundation.

References

1. Diatchenko, L., Lau, Y. F., Campbell, A. P., 3. Bautz, E. K., and Reilly, E. (1966) Gene- Chenchik, A., Moqadam, F., Huang, B., specific messenger RNA: isolation by the dele- Lukyanov, S., Lukyanov, K., Gurskaya, N., tion method, Science 151, 328–330. Sverdlov, E. D., and Siebert, P. D. (1996) 4. Duguid, J. R., and Dinauer, M. C. (1990) Suppression subtractive hybridization: a Library subtraction of in vitro cDNA libraries to method for generating differentially regulated identify differentially expressed genes in scrapie or tissue-specific cDNA probes and libraries, infection, Nucl. Ac. Res. 18, 2789–2792. Proc. Natl Acad. Sci. USA 93, 6025–6030. 5. Ghorbel, M. T., Sharman, G., Hindmarch, C., 2. Chee, M., Yang, R., Hubbell, E., Berno, A., Becker, K. G., Barrett, T., and Murphy, D. (2006) Huang, X. C., Stern, D., Winkler, J., Lockhart, Microarray screening of suppression subtractive D. J., Morris, M. S., and Fodor, S. P. (1996) hybridization-PCR cDNA libraries identifies novel Accessing genetic information with high-density RNAs regulated by dehydration in the rat supra- DNA arrays, Science 274, 610–614. optic nucleus, Physiol. Genomics 24, 163–172.

Chapter 16

Neuropeptide Microdialysis in Free-Moving Animals

Tetsuya Kushikata and Kazuyoshi Hirota

Abstract

Microdialysis is a technique that collects extracellular fluid through a semipermeable membrane. Various compounds are obtained with this technique in vivo in free-moving animals. Originally, this technique was developed to measure several biogenic amines in rat brain, for example, dopamine from the striatum, acetylcholine from the cerebral cortex, and noradrenaline from the hypothalamus. Recently, the membrane quality and detection limit of many substances have been improved; thus, the microdialysis techniques are widely used to quantify large molecules in the brain. These molecules include neuropeptides and hormones. In this chapter, we describe a principle of the microdialysis technique, how to prepare a microdialysis system, and how to obtain samples from the brain of free-moving animals.

Key words: Microdialysis, Neuropeptides, Free moving, In vivo sampling, Extracellular fluid, Rat

1. Introduction

Microdialysis is a well-established technique that allows collecting various extracellular compounds. These include neuropeptides, neurotransmitters, hormones, and other low-molecular weight substances such as glucose, lactic acids, and nitric oxide. The micro- dialysis technique was introduced at late 1950s (1), and then it had been improved in mid-1980s (2). In the early periods, the technique was mainly used for collecting from brain perfusates different types of neurotransmitters such as catecholamines, acetylcholine, , and amino acids. In more recent years, in parallel with an improving of the technique, other substances have become targets for analysis. A typical microdialysis system consists of several modules: a perfusate driving unit, a microdialysis probe, and an analysis unit. The perfusate driving unit includes a syringe pump that is capable to push a perfusate, typically artificial cerebrospinal

Adalberto Merighi (ed.), Neuropeptides: Methods and Protocols, Methods in Molecular Biology, vol. 789, DOI 10.1007/978-1-61779-310-3_16, © Springer Science+Business Media, LLC 2011 261 262 T. Kushikata and K. Hirota

fluid, at a low flow rate (usually less than 2 ML/min), a Teflon tube in which perfusate is running, collection tubes, and a cage where the animal is to be kept. The microdialysis probe is a vital module of the microdialysis set up. The probe is composed of an inflow and outflow port and a tube with the dialysis membrane. Any substance will move from the extracellular space to the inside of the tube by crossing the dialysis membrane and then it will be pushed through the outflow port into collection tubes. The dialysis membrane is semipermeable to allow for small molecules passing through it. Therefore, samples obtained are blood and microbe free. Usually, the cut-off limit of molecules is less than 20,000 Da. Cut-off of membranes for large molecule such as neuropeptides is 100,000 Da. Several methods can be employed for actual analysis for the substance(s) collected. The method(s) of choice depend(s) on the properties of the substance(s) under study. In general, radioim- munoassay (RIA) or enzyme immunoassay (EIA) is preferred for neuropeptides, while high-performance liquid chromatography (HPLC) with an electrochemical or ultraviolet detector is desired to quantify low molecular weight neurotransmitters (Fig. 1). The set up described in figure is designed for monitoring central ner- vous system (CNS) function, but can be easily modified to monitor peripheral organ function (e.g., liver, skin, heart). The microdialysis technique was originally designed for succes- sive sample collections from a single animal for several hours.

Syringe Pump HPLC with ECD

Sampling Tube HPLC with UV Detector

RIA Inlet Flow Outlet Flow EIA

Dialysis Probe

Fig. 1. Overview of a microdialysis system. Perfusing fluid is delivered with microsyringe pump. An ideal perfusate in vivo brain dialysis should contain exact same composition around the probe insertion region. Therefore, perfusate of in vivo microdi- alysis is usually artificial cerebrospinal fluid (aCSF). A typical composition of the aCSF is following (in mM): NaCl 128, KCl

2.6, CaCl2 1.3, MgCl2 0.9, NaHCO3 20, Na2HPO4 1.3. The aCSF should be sterile. The fluid is running into a microdialysis probe that has a semipermeable membrane, then the fluid is collected with sampling tube. The substance will be quantified by HPLC with ECD or UV detector, or RIA or EIA. 16 Microdialysis 263

Thus, it was possible to reduce the number of animals to use and to easily compare changes in substance(s) concentration before and after any treatment. However, this technique has several limi- tations: as the perfusate flow rate is low (usually less than 2 ML/min), time resolution is in many cases over 10 min. The time resolution is also depending on the amount of sample. Thus, any change that happens in minutes or seconds is difficult to quantify with a microdialysis technique. In such case, alternative methods should be considered such as voltammetry. The ratio between the actual extracellular concentration of substance and concentration of the dialysate is usually less than 40%. It depends on the properties of the substance itself and of the dialysis membrane, environmental temperature, and flow rate of the perfusate. In ordinary condi- tions, peptide concentration of the dialysate is around 5% or less (3). The spatial zone where extracellular substances can be retrieved into the dialysis membrane also depends on the same conditions described above, which may vary within a quite small range. Therefore, the microdialysis probe should be implanted under strict stereotaxic control directly into the desired region where one expects release of the substance occurs so that it can be quantified. The membrane length of the microdialysis is probe usually 2–4 mm. A longer probe can retrieve more substance. One should choose which length is desirable depending on the study purpose and in consideration with the length of the probe and the depth of the brain area of interest. For example, a 4-mm long membrane is suit- able for rat cerebral cortex, whereas a 2-mm long membrane is suitable for the rat hypothalamus. There are three types of microdialysis probes: U shape, hori- zontal, and concentric. U-shape probes have been widely used in early age of microdialysis because of their high recovery rate and simplicity of construction. However, this type of probe tends to damage tissues around the region of insertion compared to the concentric type. Horizontal probes are mainly designed for periph- eral organs, such as the liver. Concentric probes better suit CNS analysis and have recently took over the U-shape probes (Fig. 2). This chapter describes the use of concentric probes for neuro- peptide microdialysis. Although it is possible to manufacture U type and horizontal probes, concentric probes, especially those for neuropeptide research, are particularly complicate to construct. Therefore, we recommend the use of commercial probes. Details can be found at following links: http://www.microdialysis.se/Home http://www.eicom-usa.com/microdialysis http://www.harvardapparatus.com/webapp/wcs/stores/servlet/ haicat1_10001_11051_37263_-1_HAI_Categories_N http://www.instechlabs.com/Infusion/systems/microdialysisrats. php 264 T. Kushikata and K. Hirota

0ERFUSATE

Inflow Outflow Perfusate Dialysate

$IALYSATE

Membrane region

Fig. 2. Substances around the microdialysis probe come across the semipermeable membrane according to a concentra- tion gradient (dialysis). The substance in dialysate is collected for analysis.

2. Materials

2.1. Microdialysis 1. Animals (rats). Probe Implantation 2. Anesthetics: pentobarbital, ketamine with xylazine. 3. Disinfectants: povidone iodine, 70% ethanol. 4. Antibiotics. 5. Brain Map for stereotaxic coordinates (e.g., (7)). 6. Stereotaxic frame with probe holder for guide cannula (David Kopf, Tujunga, CA). 7. Guide cannula for microdialysis probe (available from CMA Microdialysis AB, Soina, Sweden; or Instech, Plymouth Meeting, PA; or Harvard Apparatus, Holliston, MA; or EICOM, Kioto, Japan). 8. Dummy cannula for temporary occupation of the guide cannula (CMA Microdialysis AB, Instech, Harvard Apparatus, or EICOM). 9. Spatula. 10. Dental drill. 11. Dental cement. 12. Anchor screw (stainless). 16 Microdialysis 265

13. Cotton puff. 14. Scalpel. 15. Jeweler screwdriver and forceps. 16. Forceps. 17. Radio knife (optional). 18. Hair shaver.

2.2. In Vivo 1. Artificial cerebrospinal fluid (aCSF): 128 mM NaCl, 2.6 mM

Microdialysis in KCl, 1.3 mM CaCl2, 0.9 mM MgCl2, 20 mM NaHCO3,

Free-Moving Animals 1.3 mM Na2HPO4 (see Note 1). 2. Bovine serum albumin (BSA) (see Note 2). 3. Animals implanted with guide cannula for microdialysis probe (concentric). 4. Liquid Swivel with tether (CMA Microdialysis AB, Instech, Harvard Apparatus, or EICOM). 5. Teflon tubing. 6. Concentric microdialysis probe (CMA Microdialysis AB, Instech, Harvard Apparatus, or EICOM). 7. Low rate (0.2–10 ML/min) infusion pump (e.g., CMA 400 Syringe Pump). 8. Glass gas-tight microsyringe (e.g., Hamilton 500 ML). 9. Collection apparatus, vials in refrigerated fraction collector. Usually, a fraction collector for HPLC is acceptable as the collection apparatus. However, it needs to be placed in refrigerator. 10. Cage for free moving.

3. Methods

3.1. Microdialysis 1. All surgical procedure should be done in aseptic manner with Probe Implantation sterile instruments. Before surgery, make sure all devices are sterile and ready for use. Check if microdialysis probe has neither leak nor broken membrane. Check if the guide cannula chosen is suitable for desired microdialysis. Usually, the guide cannula allows the tip of microdialysis probe to protrude 2–4 mm below. 2. Anesthetize rat: restrain animal gently and give anesthetics intraperitoneally. Sodium pentobarbital is given at 50 mg/kg, or 35 mg/kg ketamine + 5 mg/kg xylazine. 3. Wait for a while until animal loss of lighting (ordinary within 5 min). 266 T. Kushikata and K. Hirota

4. Confirm if animal keeps its breath in regular rhythm (see Note 3). 5. Give antibiotics subcutaneously. 6. Shave the animal hair of head. 7. Mount the animal in the stereotaxic frame. Hold animals gently with your left hand. Be careful to keep it breathing freely (no air obstruction). Seek the animal’s left auditory meatus by lightly pushing ear bar by your right hand. When the ear bar will be placed into the auditory meatus correctly, a pop sound can be heard that means the tympanic membrane is ruptured with the ear bar. Then, keep pushing the ear bar into the meatus, lock the ear bar carefully. 8. Hold animal with your right hand and remind to keep the ear bar in the meatus. Seek the other side auditory meatus (right side) with the second ear bar with your left hand. Place it into the right meatus as same manner of the first ear bar. Lock the second ear bar carefully. If you placed both ear bars correctly, the animal head can only move along the sagittal plane. 9. Gently open the mouth of animal. Insert the upper incisors over the incisor bar. Adjust nose clamp. 10. Make a midline incision of the head skin after disinfection of the skin with povidone iodine or 70% ethanol. Be careful that these reagents do not leak on animal’s eyes. Retract the skin with serrefine forceps. 11. Peel off the fascia and connective tissue of the skull with the forceps. Wipe any blood with a cotton puff moistened with 70% ethanol. If bleeding is hard to arrest, a radio knife may be useful, if available. Another way to obtain hemostasis is applying hydrogen peroxide on the skull surface. However, hydrogen peroxide potentially can damage tissues. Therefore, wipe it away from the skull surface completely. Keeping the skull face dry is very important for proper adherence of dental cement. 12. Identify the bregma. The bregma is the anatomical marker on the skull at which the coronal suture is intersected perpendicu- larly by the sagittal suture. The bregma is an often-used reference point to calculate coordinates of any region where the microdi- alysis probe is placed in. 13. Attach the guide cannula into the stereotaxic holder and contact the tip of the cannula with the bregma. Read the dimensions (anteroposterior, lateral, and vertical). In case of the vertical dimension, if you use 2 mm microdialysis probe, the guide cannula tip should be placed 2 mm above the actual microdi- alysis probe. For example, if one wants to place the microdialysis probe in the posterior hypothalamus, the stereotaxic coordi- nates are: AP: 3.6 mm, L: 0.5 mm, V: 9.0 mm (Paxinos and Watson, 1986). Therefore, the tip of guide cannula should be placed vertically 7 mm below the bregma. 16 Microdialysis 267

14. Move the guide cannula from the bregma to desired point with coordinate calculation. Make any marks with ink to the point. 15. Drill the point with dental drill. Cut the dura with a sterilized needle. Move back the guide cannula and insert it into the brain slowly. Make sure the coordinate is correct. 16. Make two or three other holes in the skull for anchor screw, and then implant the anchor. 17. Apply dental cement on the skull surface to wrap the base of the guide cannula and the anchor screws. Make sure that the skull surface is kept dry. 18. Wait until the cement dries, then remove any skin from the cement to make the wound open. Apply some antibiotics around the wound (e.g., gentamicin powder). 19. Remove the cannula holder gently. Put a dummy cannula into the guide cannula. 20. Place the animal in the warm cage. Prevent hypothermia. Observe if animal breaths normally. Keep animal solely to avoid the implant is damaged by other rats. 21. The animal can be used the day after the surgery for free-moving microdialysis, but if you do not want any interaction of anes- thetics to neuropeptide kinetics, it is better to use the animal 3 or 4 days later.

3.2. In Vivo 1. Prepare the microdialysis probe (see Note 4). Microdialysis in 2. Prior to actual implantation of the microdialysis probe into the Free-Moving Animals rat brain, confirm that the probe works properly: Connect a microsyringe (e.g., Hamilton 1.0 mL) filled with aCSF, swivel, inflow and outflow port of the microdialysis probe with Teflon tubing. 3. Put the probe into a vial securely filled with distilled water. Drive pump at a relatively high rate in comparison to ordinary rate (e.g., 5.0 ML/min). 4. Observe the aCSF spilling out from the outlet flow. Check that there is no leak at the membrane. Then, adjust pump speed at normal rate (e.g., 1.0 ML/min). 5. Prepare a cage for free moving, liquid swivel with tether, hold- ing arm for the swivel and animal restrainer. Commercial-based equipments are highly recommended. 6. Move an animal that has a guide cannula implanted as described in Subheading 3.1 into the cage. 7. Fix it with tether gently. 8. Keep the animal free for a while to make it familiar with the new environment. 268 T. Kushikata and K. Hirota

9. Hold animal gently in one hand (usually left hand). Remove the dummy cannula with your right hand and then insert the microdialysis probe gently. If the animal is calm, the insertion will be done easily, but if the animal is restless, application of a light anesthesia is recommended (see Note 5). 10. After insertion of the probe, keep pump driving at normal flow rate (usually less than 1 ML/min). 11. Check that dialysate spills out from the outflow port (see Note 6). 12. Collect samples in the same way as for actual sampling methods, and then quantify changes in substance concentration in the dialysates. When the concentration becomes constant, the recovery is completed. Record the duration of the interval from probe insertion until a constant concentration of substance is retrieved. This is right recovery duration for the substance. 13. After an adequate recovery period, collect samples with an appropriate sampling device. 14. Collect first several successive samples as control values, and then perform experimental procedures, e.g., drug delivery or beginning of a behavioral test. 15. Compare samples collected following any experimental proce- dures to controls. 16. Quantify substance with appropriate methods. For neuropep- tides, RIA or EIA are popular while HPLC with an electro- chemical detector is good for biogenic amines or traditional neurotransmitters (4).

4. Notes

1. An ideal perfusate for in vivo brain dialysis should contain an exact same composition of the intercellular fluids around the probe insertion region. Therefore, although it may be possible to use also a Ringer solution, the better perfusate for in vivo neuropeptide microdialysis is usually aCSF. The aCSF must be sterile. Sterilize by autoclaving or filtration. 2. In measurement of neuropeptide release, the membrane of a microdialysis probe has large pores as neuropeptides are large molecules. Therefore, it is recommended to add BSA to the aCSF (5). BSA prevents perfusate loss via the large pore mem- brane because of its high osmolarity. Concentration of BSA is 0.5 or 0.25% (6). Addition of BSA is not necessary for microdi- alysis of small molecules. One millimolar pargyline, a mono- amine inhibitor, is instead included to prevent degradation of in case of norepinephrine measurement. 16 Microdialysis 269

3. If anesthesia given for surgery is not enough, one-third of the initial doses may be given additionally. However, sometimes animal will be dead because of excess anesthetics. The best solution for inadequate anesthesia is simply postponing surgery over 24 h. 4. Recovery rate of a molecule is depending on molecule and membrane size. The membrane for neuropeptide microdi- alysis should have a cut-off of about 100,000 Da of molecular weight. Commercial probes are recommended (3). For example, use the MA 12 Microdialysis Probe (available from CMA Microdialysis AB). 5. If animal is restless and it is difficult to insert a microdialysis probe, a light anesthesia can be applied. Ether inhalation is usually recommended, because of its rapid onset and short half-life. Do not use any device that can cause electrical dis- charge because ether is highly flammable. 6. It is necessary to wait for recovery as a consequence of tissue damage due to probe implantation. Typically, 1 or 2 h of recovery period are required, but the duration of recovery is depending also on the properties of substance under investigation.

References

1. Kalant, H. (1958) A microdialysis procedure for the determination of neurotransmitters. for extraction and isolation of corticosteroids Anal Chim Acta 653, 1–22. from peripheral blood plasma. Biochem J 69, 5. Trickler, W. J., and Miller, D. W. (2003) Use of 99–103. osmotic agents in microdialysis studies to improve 2. Benveniste, H., Drejer, J., Schousboe, A., and the recovery of macromolecules. J Pharm Sci Diemer, N. H. (1984) Elevation of the extracel- 92, 1419–27. lular concentrations of glutamate and aspartate 6. Jensen, S. M., Hansen, H. S., Johansen, T., and in rat hippocampus during transient cerebral Malmlof, K. (2007) In vivo and in vitro microdi- ischemia monitored by intracerebral microdialysis. alysis sampling of free fatty acids. J Pharm J Neurochem 43, 1369–74. Biomed Anal 43, 1751–6. 3. Clough, G. F. (2005) Microdialysis of large 7. Paxinos, G., Watson, C. (1996) The Rat Brain molecules. Aaps J 7, E686–92. in Stereotaxic Coordinate, Compact Third 4. Perry, M., Li, Q., and Kennedy, R. T. (2009) Edition. New York: Academic Press. Review of recent advances in analytical techniques

Chapter 17

Antibody Microprobes for Detecting Neuropeptide Release

Rebecca J. Steagall, Carole A. Williams† and Arthur W. Duggan

Abstract

Antibody-coated microprobes have been demonstrated to be useful for detecting the release of neuropeptide transmitters from discrete sites in the central nervous system (CNS). This technique uses glass micropipettes taken through a series of chemical coatings, starting with a G-aminopropyltriethoxysilane solution and end- ing with the antibody specific to the peptide transmitter of interest. The key to the reliability and repeat- ability of the technique is a uniform, even coating of the siloxane polymer to the glass micropipette. The microprobes, as they are called following the completion of the coating process, are inserted stereotaxically into a specific area of the CNS and the physiological intervention is performed. Tip diameters are around 5–10 Mm and, depending on the length of the pipette inserted into the CNS, diameters of the pipette shaft will approach 40–50 Mm. Once removed, the microprobe is then incubated with the radiolabeled peptide. Binding of the radiolabeled peptide will occur to the antibody sites not occupied by the endogenously released peptide. The images of the microprobes on sensitive autoradiographic film are analyzed for differ- ences in the optical density along a specified length of probe. Areas of lighter density signify sites along the microprobe where endogenous peptide was biologically released during the physiological intervention. Knowing the exact location of the probe tip in vivo in the CNS permits identification of neurophysiological sites corresponding along the length of the microprobe where the peptide was released.

Key words: Antibody-coated microprobes, Immobilized proteins, Neuropeptide transmitters, Autoradiographic analysis

1. Introduction

The preparation of antibody-coated microprobes and the computer- ized analysis of their autoradiographic images were first described by Duggan and colleagues some 22 years ago (1, 2). It is a method that has proved useful and reliable in detecting the release of neuropep- tide transmitters from discrete sites in the central nervous system (CNS) during varying physiological conditions. Identifying the neurotransmitters and modulators released in the CNS during a particular physiological process is essential to understanding the

Adalberto Merighi (ed.), Neuropeptides: Methods and Protocols, Methods in Molecular Biology, vol. 789, DOI 10.1007/978-1-61779-310-3_17, © Springer Science+Business Media, LLC 2011 271 272 R.J. Steagall et al.

mechanism(s) involved with its signaling. Furthermore, being able to determine the neural sites where these substances are released from provides critical insight into the pathways stimulated or inhib- ited in the CNS and the connectivity between them and the initiat- ing sensory signals. Although it is an invasive technique, there is documented evidence that the microprobes do minimal tissue damage upon insertion (1, 7). This is an advantage over other detec- tion techniques that rely on perfusion of fluids across semipermeable membranes in cannulae that are at least 100 times larger in diameter. Additionally, because the probe is inserted into the CNS with a micromanipulator to a precise depth, peptide release can be detected at multiple points along the length of the probe with a spatial resolu- tion of about 100 Mm (1). This is particularly important when considering the release or lack of release of a particular peptide as being a function of the physiological conditions or interventions imposed in vivo as opposed to local tissue damage because of the insertion and presence of the measuring probe. To date, using this technique has proven to be quite useful in furthering our under- standing of various pain (3–5) and cardiovascular regulatory mecha- nisms (6–8). A summary diagram illustrating the overall technique is presented in Fig. 1.

Fig. 1. Schematic diagram illustrating the major steps in the antibody-microprobe technique. (a) Silane-treated glass micropi- pettes are coated with antibody specific ( ) to the neuropeptide of interest. (b) The microprobe is inserted stereotaxically into the CNS where the peptide of interest (P), released in response to a physiological signal, binds to antibody sites along the probe. (c) The microprobe is removed from the CNS and then incubated with radiolabeled peptide (*P), which binds to antibody sites not occupied by the endogenously released peptide, P. (d) Image of the probe that had been inserted into the CNS following exposure to autoradiographic film. Note sites occupied by endogenously released P appear lighter than those occupied by the radiolabeled peptide, *P. Graphics program can then be used to display differences in optical density versus length of probe analyzed. See Subheading 3 for details. 17 Antibody Microprobes 273

2. Materials

2.1. Preparation 1. Corning Pyrex glass tubing 2 mm o.d. (Wilmad-LabGlass, of Glass Micropipettes Vineland, NJ). 2. Xylene. 3. 100% Ethanol or methanol. 4. 50% Nitric acid (see Note 1). 5. Vertical pipette puller. 6. Inverted stage microscope mounted with copper-coil attached to a variable resistor to generate heat. 7. Oven (temperature range up to 200°C).

8. Ultrapure type II H2O reagent-grade (2–10 M7 cm quality; <500 ppm TDS). 9. Covered plastic boxes to store micropipettes at various stages fitted with grooved trays to hold probes in place. 10. Portable small propane tanks 16.4 oz/468 g (as would be used in camping stoves; Coleman or other manufacturers). 11. Glass cylinders, nongraduated; 50 mL capacity, 25 mm o.d., 150 mm height.

2.2. Silane Coating 1. Round-bottom, ground glass-upper top, test tubes (o.d. 30 mm × i.d. 25 mm × length 160 mm) to fit ground-glass hollow-head stopper (24/25) (LabGlass, Kignsport, TN). 2. Custom-made Pyrex glass “buckets” (bucket depth 40 mm × i.d. 18 mm with 75-mm long hook handle; have three holes ~1.5 mm diameter in bottom of bucket) – see Fig. 2. 3. Toluene – for coating (use ACS spectranalyzed toluene for coating steps and a lesser quality for washing). 4. (3-Aminopropyl)-triethoxysilane in sure seal bottle (Sigma Chemicals, St. Louis, MO).

5. Molecular sieves, 4 Å (Sigma Chemicals, Na12 •[(AlO2)12(SiO2)12]

•xH2O; beads 4–8 mesh).

2.3. Antibody Coating 1. 50% Glutaraldehyde (available from Electron Microscopy Sciences, Hatfield, PA). 2. Protein A (from Staphylococcus aureus, soluble, Cowan Strain, available from Sigma Chemicals). 3. Drummond microcaps (micropipettes) 5 ML (Fisher Scientific). 4. PBS–azide solutions pH 5.4 and pH 7.4: Make stock solution

at 10× concentration: NaCl 80 g, KCl 2 g, NaH2PO4•(2H2O)

11.5 g, KH2PO4 2 g, NaN3 1 g, dissolved in 1 L ultrapure

H2O. Dilute 10× for working solutions; adjust pH to either 274 R.J. Steagall et al.

Fig. 2. Glass buckets to hold probes. The bucket should be made of Pyrex glass, with a vertical dimension of 12 cm and a width of 2 cm. The depth of the bucket should be 5–6 cm.

5.4 or 7.4, then filter through Millipore filter of 0.45-Mm pore size (see Note 2). Store at 4°C. 5. Borate stock solution: Boric acid 6.18 g in 500 mL ultrapure

H2O, adjust pH to 8.5, filter and store at 4°C.

6. Borohydride solution: 0.124 g NaBH4 in 50 mL borate stock solution. Cover immediately with Parafilm (solution should bubble). 7. Glass Petri dish, forceps. 8. Antibody to selected neuropeptide; dilutions determined by in vitro assays (see below). Radiolabeled I125-neuropeptide (dilute with PBS–azide, pH 7.4 containing 0.25 mg/mL BSA so that each 5 ML microcap has ~10,000 cpm). Bovine serum albumin (BSA) Fraction V, (Sigma Chemicals). Commonly available Bolton–Hunter labeled I125 peptide needs an antibody directed at the C-terminus. The method also works with I131 labeled peptide that has a high specific activity. 9. PBS–Tween solution: Add 5 mL of 10%Tween to 1 L PBS– azide, pH 7.4; filter and store at 4°C. 10. Polycarbonate manifolds (five ports/manifold – Cole-Palmer, Vernon Hills, IL). 17 Antibody Microprobes 275

11. Pieces of index card cut to 0.5 cm × 3.5 cm (Liquid Paper, Oak Brook, IL). 12. Kodak Bio-max MR-1 monoemulsion autoradiographic film (available from Sigma Chemicals). 13. Film cassettes.

2.4. In Vivo 1. Kodak GBX developer (available from Sigma Chemicals) and Experiments fixer (available from Sigma Chemicals) or suitable automated and Image Analysis film developer. of Autoradiographic 2. Computerized digitizing hardware and software that performs Film Microprobe high-resolution continuous linear measure of optical density Images along each autoradiographic probe image. 3. Processing software that can combine optical density results from several probes into groups, average the density, and per- form statistical tests for differences in optical densities along the length of the probe images. 4. Graphic software that can plot the group optical densities versus the length of probe analyzed.

3. Methods

Evenly coated microprobes are keys to obtaining reliable, useful information about the inhibition of binding of radiolabeled peptide along specific sites on the microprobe and, therefore, the release of the endogenous peptide from specific sites in the CNS. The pattern of inhibition should be repeatable and should be a function of the physiological intervention imposed during the in vivo experiment and not a function of nonuniform coating on individual micro- probes. This can be assured in part by visually inspecting each probe before it undergoes the antibody-coating step and by comparing a portion of each probe that does not reside in the CNS to the optical density of corresponding in vitro probes. Given the nature of the solutions used, it is essential to wear protective laboratory garments and gloves during the various coating steps.

3.1. Preparation 1. Cut enough of the 2 mm o.d. glass tubing in 4 in. lengths to of Glass Micropipettes fill a 250-mL beaker with the cut pieces of tubing. Soak the tubing pieces in 100% xylene overnight at room temperature, covering the beaker with a glass Petri dish. Discard xylene according to appropriate local regulations. 2. Wash the tubing pieces three times in 100% EtOH or MeOH for 30 min per wash. Discard the alcohol according to local regulations. 276 R.J. Steagall et al.

3. Rinse the tubing pieces in distilled H2O twice, making sure that all the alcohol and then the wash water is evacuated from the beaker each time. 4. Dry the pieces in the beaker overnight at 160–180°C. Cover the beaker once removed from the oven with Petri dish or foil until pieces are ready for pipette pulling. 5. Pull tubing piece into a micropipette, preferably using a vertical pipette puller. This should produce a pipette with the tapered shaft centered. This taper should be very gradual as the terminal 1 cm will be broken off subsequently and this is easier with finely tapering pipettes (see Note 3). Check that the shaft is perfectly straight. Pull the required number to accommodate several experiments. The pipettes can be stored in a covered plastic box on a Plexiglas tray with grooves to accommodate the pipettes. 6. Seal the large end of each micropipette with a propane flame without causing a bubble at the end. One can see the hollow of the tube disappear into the end as it is sealed (see Note 4). The tip end can be sealed using a heating coil connected to a rheostat-controlled variable voltage unit. Position the tip between two turns of the coil without touching any part of the coil. The unit is usually turned on and off with 1–2 s. This pro- vides enough heat that the micropipette tip is sealed. One can see this happening by watching the tip in position to the heat- ing coil under an inverted-stage microscope. The “feathering”- like appearance of the micropipette tip disappears. Be careful not to apply the heat for too long or the tip will bend and then the micropipette will have to be discarded. Store sealed probes in boxes until ready for the coating procedures. 7. When ready for silane coating, transfer 40–50 sealed probes to two glass buckets (about 20–25 probes/bucket). Fill two glass cylinders with approximately 30 mL 50% nitric acid, transfer the buckets with probes into the cylinders, and soak probes for 20 min. Remove the buckets with probes from cylinders, dis- card nitric acid according to local regulations. Wash the cylin-

ders with distilled H2O several times to clear remaining nitric

acid. Fill the cylinders with distilled H2O and place the buckets with probes into the cylinders again and rinse probes in dis-

tilled H2O three times, 5 min each time. Dry the probes in oven at 160–180°C overnight or for at least 2 h.

3.2. Silane Coating 1. Use micropipettes that have been heated in oven at least 2 h or overnight (20–25/bucket). 2. Add 34 mL toluene each to two clean 100-mL boiling tube with ground-glass top. Have boiling tube sitting in appropri- ate-sized test-tube rack in fume hood. 3. Withdraw 14 mL of the silane (i.e., 3-aminopropyl-triethoxysi- lane) from the air-tight (“sure-seal”) bottle with a disposable 17 Antibody Microprobes 277

20-mL syringe and 16G needle. Add 7 mL of silane to the toluene in each of the boiling tubes. Cover with the ground- glass top and invert the mixture 2–3 times. Remove top and wipe dry any of the silane–toluene solution from around the ground-glass rim of the test tube and from the top. This is critically important or else the silane mixture will coat the glass and seal the top to the test tube. Let the mixture sit in the fume hood for at least 30 min (see Notes 5 and 6). 4. Transfer molecular sieves into two buckets almost to the top and heat them in an oven for 30 min at 160–180°C. 5. Remove the buckets with sieves from the oven and slowly and gently lower into the test tubes containing the silane–toluene mixture. Again, wipe the top portion of the tubes and tops so that no solution is present. Let the sieves in the silane–toluene mixture stand in the fume hood for 30 min. 6. Remove the buckets with micropipettes from oven. Let stand in air for a few minutes; pass the tips of the pipettes through a head of steam for ~30 s, rotating the bucket in the steam so that all micropipettes are surrounded by the steam. A large

beaker with distilled H2O on a hot plate with foil placed over the top can be used to generate the steam. Hang the buckets with the micropipettes in a freezer for 1 h. 7. Place the test-tube rack holding the boiling tubes with the molec- ular sieves in the freezer at the same time and let stand in there for 1 h. It is useful if the freezer is close to the fume hood. 8. Remove the bucket with molecular sieves from the silane–toluene mixture and immediately place in the fume hood. Transfer the buckets with the micropipettes from the freezer and pass through the steam again for ~20–30 s, as described above, and then place in the tubes with the silane–toluene solution. Wipe the tops of the boiling tubes containing the solution and their respective tops. Replace top on the tubes with the silane solution contain- ing the micropipette buckets. Leave in freezer for 1 h. 9. Remove the test tubes containing the silane solution and micropipettes from the freezer and place in the fume hood. Once the tubes come up to room temperature and condensa- tion dissipates, remove tops of the boiling tubes and rewipe the tops of the boiling tubes and their tops to assure that no silane– toluene solution has coated these surfaces. Let the tubes stand in the hood overnight. 10. Remove the bucket with the probes and gently pass up and down in a cylinder containing toluene (for washing) for ~20 s. Remove the probes from bucket and spread out on a Plexiglas tray and let stand in the hood for 1–3 min. 11. Hand-wipe each probe with a Kimwipe by pulling the probe from the back end through the tissue wipe. It is best to turn 278 R.J. Steagall et al.

the probe as it is being pulled through the Kimwipe, keeping an even pressure on the tapered shaft as the point is pulled through the wipe. Pass through the wipe 3–4 times. Place wiped probes in a washed, dry coating bucket. 12. Hang the buckets with the probes in the oven at 160–180°C for at least 2 h. 13. At the 1 h mark after hanging the buckets with the probes in the oven, place the buckets with sieves in the oven for 30 min (see step 4 above). 14. After 30 min, remove the bucket with sieves and place them in the toluene–silane mixture and leave in the hood for 30 min. Repeat steps 6–9. 15. The next morning, rewipe the probes and repeat steps 10–14. Probes usually need to be coated and wiped about 4–5 times. The toluene–silane solution in the boiling tubes can be used two times for coating; thereafter, discard according to local regulations and replace with fresh solution. Use new molecular sieves after five coating procedures. 16. Probes need to be inspected visually for any clumps or uneven coating along the tapered shafts. Figure 3 displays how the probes should look after coating and some of the problems that commonly occur with uneven coating/wiping.

3.3. Antibody Coating 1. Use only probes that have been inspected and found suitable for use in experiments. 3.3.1. Protein A Step 2. Prepare a covered plastic container (such as a Rubbermaid 20 cm × 30 cm) that is large enough to hold a probe tray. Line the bottom of the container with paper towel soaked with dis-

tilled H2O to create a damp, humidified atmosphere. 3. Cure the required number of probes needed for an in vitro or in vivo experiment overnight in oven at 160–180°C. Remove and cool to room temperature. 4. Make a 1:20 dilution of 50% glutaraldehyde (1.5 mL in 30 mL

ultrapure H2O). Place cooled probes in bucket and immerse in solution contained in a suitable glass cylinder for 30 min at room temperature. Remove probes and then wash them in

ultrapure H2O for 5 min each, three times by placing the bucket with the probes in a fresh cylinder containing the water.

Discard water, rinse the cylinder, and replace H2O two more times for subsequent rinses. 5. Fill the appropriate number of 5 ML microcaps with a 1 mg/ mL Protein A solution (see Note 7). To make the solution, dilute 10 mg Protein A in 1 mL PBS–azide, pH 7.4. This can be stored at −20°C in 10-ML “stock” aliquots. When ready to use, take one 10 ML stock solution and dilute with 90 ML PBS–azide, pH 5.4 to use in the microcaps. 17 Antibody Microprobes 279

Fig. 3. Various stages of coated microprobes. Top panel, (a) probes schematically repre- sented; bottom panel, (b), actual probes. In each panel : (a) native (uncoated) micropipette shown from the tip to approximately 1.5 cm along the shaft; (b) an evenly well-coated probe; (c) an evenly coated probe that is not as densely coated at the one in (b). It is perfectly acceptable to use this; (d) a probe that is too-lightly coated to be used for experiments; (e) a probe that is densely coated, but clumps of silane have appeared along the shaft. This is due to uneven wiping, and this type of probe cannot be used for experiments. See http://extras. springer.com/ for the color version of this figure.

6. Insert probe tips into the filled microcaps using a dissecting microscope; incubate for 24 h at 4°C on a grooved tray that is placed in the plastic container. Loosely place lid on container and put in refrigerator. 280 R.J. Steagall et al.

3.3.2. Antibody Step 1. Remove the microcaps from the probes by turning probes with tip down; the microcaps should fall off. Discard the microcaps. Place probes in a glass Petri dish, back ends first, up against the back of the dish (so tips won’t break). Tilt the dish upright, back of probes in the downward position and slowly fill the dish with the borohydride solution. Make the solution in a graduated cylinder and cover with Parafilm immediately. The solution should bubble. Lower the Petri dish to the counter and keep probes in the solution for 5 min at room tempera- ture. Every 30 s or so lift the probe tips out of the solution with a pair of forceps to stop hydrogen bubbles from attaching to the probe surfaces. Ensure that the room is well ventilated. 2. Transfer probes to a clean glass bucket by tapping the blunt end of the probe on a Kimwipe to remove excess solution. Place the bucket with the probes in a glass cylinder containing about 30 mL PBS–azide, pH 7.4 and wash three times for 5 min each. 3. Remove a probe and use a small triangular file to cut off the sealed back-end of each probe. 4. Fill new 5-ML microcaps with properly diluted antibody and insert probe tips into microcaps under dissecting microscope (to determine the antibody dilution, see Subheading 3.3.3). 5. Return probes with antibody-filled microcaps to tray in plastic container and incubate for 24 h at 4°C. At the end of this 24 h period, fill new microcaps with fresh antibody solution and replace microcaps on probes, inserting the tips using a dissect- ing microscope. Probes at this stage can be labeled with a series of color-coded lines to delineate their function during inser- tion into the subsequent in vitro or in vivo experiment (for example, “rest probes” can be labeled with one, two, or three blue lines scribed around the circumference of the probe with a Sharpie permanent marker; “experimental” probes can be labeled with one, two, etc. red lines and so on).

3.3.3. Antibody Dilution 1. Fill 120 5-ML microcaps with varying dilutions of the selected antibody (i.e., 1:500; 1:1,000; 1:5,000; 1:10,000) and insert probes into each microcap. 2. Incubate ten probes at each of the dilutions for 5, 15, or 30 min at 37°C. Steps 1 and 2 can be done in separate batches (e.g., take the 1:500 probes and incubate at the three incubation times; this would require 30 total probes). 3. At the end of each incubation time, remove microcaps and replace with 5-ML microcaps filled with radiolabeled (125I) pep- tide diluted in PBS–azide with 0.25 mg/mL BSA to approxi- mate 2,000 cpm/ML. Have two of the microcaps filled with radiolabeled peptide and set aside at 4°C overnight to be used for counting the next day. 17 Antibody Microprobes 281

4. Incubate the probes at 4°C in a damp atmosphere in the covered box containing the grooved tray (see Subheading 3.3.1, step 6 above) for 24 h. 5. Remove the microcaps and dispose of appropriately according to radiation safety rules. Insert the back end of each of the microprobes in a manifold device that is connected to a vac- uum pump. The vacuum pump should be connected to a col- lection flask. Place the PBS–azide–Tween solution in a tray with a magnetic stir bar. Place the tray with the Tween solution into a larger tray containing ice water. This larger tray should sit atop a magnetic stir plate. Turn on the vacuum pump and lower the manifold containing the microprobes into the PBS– Tween solution so that at least 1 cm of the tip ends of each of the probes is immersed in the solution. Be careful that none of the tips are in close contact with the stir bar. Wash tips in PBS– azide with Tween for 15 min at 4°C with the Tween solution gently swirling from the magnetic stir bar. At the end of the 15 min, raise the probes out of the solution and let the pump run for another minute to make sure all the solution that may have been inside the probes is evacuated. This step is performed to assure that any radiolabeled solution that may have been inside the microprobes is removed. 6. Label a sheet copy paper with a place for each of the numbered probes. Carefully break off at least 1 cm of the microprobe from the tip end and secure to a piece of index card using Liquid Paper. This is done by holding the part farthest from the tip gently in place on the index card with fine forceps and applying just a small even dap of the Liquid Paper. Once the Liquid Paper dries, it secures the microprobe tip to the index card. The pieces of index card with the secured microprobe tips can then be inserted into appropriate counting tubes and the amount of radioactivity determined. Count the two 5-ML microcaps con- taining the radiolabeled solution with the probe tips. This will permit knowing how much radioactivity was available for bind- ing to the tips. Count each sample for 1 min. 7. After the counting is completed, secure each of the index card pieces with the probe tips facing downward to the labeled copy sheet of paper by using small pieces of Scotch Magic tape. Place the sheet in a film cassette and then load the cassette with a piece of Kodak Bio-max MR-1 film (emulsion side contacting the probe tips). Use the counts on the in vitro probe tips to determine the exposure time for the probe tips to the film (this can typically run from 1 to 3 days). 8. After the exposure time, develop the film and use an imaging program to determine the differences in the optical density along an appropriate length of probe. Use the same length for the in vitro probes as the total length of the in vivo probes to 282 R.J. Steagall et al.

be analyzed, e.g., 2 mm residing in situ, and 2 mm outside the CNS, thus a total of 4 mm analyzed. 9. Use the average counts for each dilution and each of the incubation times together with the differences in the optical density to determine the optimum dilution and in situ time for the peptide in question for the in vivo experiments.

3.3.4. Sensitivity Test 1. Once the optimum antibody dilution and exposure time has been determined, the sensitivity of the antibody can be deter- mined by incubating the selected dilution of antibody with known concentrations of the peptide in question. This can be done by incubating 5–10 microprobes each with concentra- tions ranging from 10−12 to 10−6 M. Repeat steps 3–9 as for the dilution procedure. Typically, 10−7 or 10−6 M peptide will result in 50% inhibition in radiolabeled binding compared to incuba- tion with just the radiolabeled peptide alone (see Note 8).

3.3.5. Specificity Test 1. After the appropriate dilution of antibody has been deter- mined, incubation with at least three concentrations of similar category of peptide should be performed to assure the specific- ity of the antibody. For example, probes coated with the anti- body to dynorphin should be incubated with 10−12, 10−9 and 10−6 M of both B-endorphin and met-enkephalin. Five to ten probes each for each of the concentrations and each of the competing neuropeptides can be used. Repeat steps 3–9 as for the Antibody Dilution procedure. Neither the B-endorphin nor met-enkephalin should bind to the dynorphin antibody- coated microprobes; thus, there should be no reduction in the counts of the probes incubated with these neuropeptides.

3.4. In Vivo 1. Insert probes into the specific CNS site designated for the Experiments in vivo experiment for the length of time found to be suitable and Image Analysis for the peptide binding to its antibody (see Subheading 3.3 of Autoradiographic above for control in vitro experiments). Irrigate the surface of Film Microprobe the CNS with sterilized, oxygenated ACSF as proteases can Images attack the bound antibody and mimic peptide binding. Remove probe from the CNS and place in a 5-ML microcap filled with radiolabeled 125I-peptide. Incubate for 24 h at 4°C, in a “damp” atmosphere in the covered tray. Microcaps containing the radi- olabeled peptide solution should also be placed on correspond- ing in vitro probes that were simultaneously prepared with the in vivo probes. These should also be incubated for 24 h at 4°C. At this step, fill two 5-ML microcaps with radiolabeled peptide solution and place in an appropriate gamma counting tube and store at 4°C. 2. Discard the microcaps containing the radiolabeled peptide into an appropriate radioactive waste container. In addition to spe- cific antibody binding, radiolabeled peptide could be trapped 17 Antibody Microprobes 283

in the interstices of the siloxane coating or may have entered the inside of the micropipette. Thus it is necessary to wash microprobes both inside and out. Insert the back end of each of the microprobes in a manifold device that is connected to a vacuum pump. The vacuum pump should be connected to a collection flask. Place the PBS–azide–Tween solution in a tray with a magnetic stir bar. Place the tray with the Tween solution into a larger tray containing ice-water. This larger tray should sit atop a magnetic stir plate. Turn on the vacuum pump and lower the manifold containing the microprobes into the PBS– Tween solution so that at least 1 cm of the tip ends of each of the probes is immersed in the solution. Be careful that none of the tips are in close contact with the stir bar. Wash tips in PBS– azide with Tween for 15 min at 4°C with the Tween solution gently swirling from the magnetic stir bar. At the end of the 15 min, raise the probes out of the solution and let the pump run for another minute to make sure that all the solution that may have been inside the probes is evacuated. This step is per- formed to assure that any radiolabeled solution that may have been inside the microprobes is removed. 3. Label a sheet copy paper with a place for each of the numbered probes. Carefully break off at least 1 cm of the microprobe from the tip end and secure to a piece of index card using Liquid Paper. This is done by holding the part farthest from the tip gently in place on the index card with fine forceps and applying just a small even dap of Liquid Paper. Once the Liquid Paper dries, it secures the microprobe tip to the index card. The pieces of index card with the secured microprobe tips can then be inserted into appropriate counting tubes and the amount of radioactivity is determined. Count the two 5-ML microcaps containing the radiolabeled solution with the probe tips. This will permit knowing how much radioactivity was available for binding to the tips. Count each sample for 1 min. 4. After the counting is completed, secure each of the index card pieces with the probe tips facing downward to the labeled copy sheet of paper by using small pieces of Scotch Magic tape. Place the sheet in a film cassette (no intensifying screens) and then load the cassette with a piece of Kodak Bio-max MR-1 film (emulsion side contacting the probe tips). Use the counts on the in vitro probe tips to determine the exposure time for the probe tips to the film (this can typically run from 1 to 3 days). 5. After the exposure time, develop the film and use an imaging program to determine the differences in the optical density along an appropriate length of the probe tip. This should include the length of the probe that resided in the CNS plus at least 1 mm of each probe that was outside of the CNS. This portion serves as an internal control and the optical density of this length should match the density of the in vitro probes. 284 R.J. Steagall et al.

4. Notes

1. Nitric acid may darken during storage. Keep in appropriate storage-code cabinet; purchase in smaller volumes. 2. Nalgene disposable sterilization filter units can be used to filter PBS solutions and store the solutions in 500 or 1,000 mL vol- umes, since these units can be directly connected to vacuum pumps. 3. Our pipettes are approximately 6 cm long before the taper begins, with the shaft approximately 1.7–1.8 cm long. It is important to have the tapered shaft part longer than a centime- ter, since this part of the probe will be broken off at about 1 cm from the tip for mounting against the autoradiographic film (see Subheading 3.1.5). 4. If using solid glass rods, the cleaning procedure will be the same as in Subheading 3.1, step 6, but the sealing process will not be needed. 5. Theoretically, the substituted silane (see Subheading 3.2) reacts with free hydroxyl groups on the glass surface to form a silox- ane polymer having free amino groups. There is evidence that chemisorbed water molecules on the glass surface contribute to this interaction (1). The procedure described above aims to have water adsorbed to the glass but not to be present in the silane–toluene solution, as polymerization in the latter is not wanted. 6. The entire coating process can be derailed if attention is not paid to thorough wiping of the ground-glass boiling tube top during the various steps described in Subheading 3.2, steps 3, 5, 8, and 9. Failing to ensure that there is no silane solution on the surfaces of the boiling tube top or the neck region of the boiling tube can result in the two pieces being irrevocably sealed with the loss of a glass bucket containing 20–25 probes in varying stages of the coating process. This has happened to just about every individual who learns this procedure, resulting in a certain level of dismay. 7. If one had a pure solution that only contained the antibody of interest, then the protein A step would be unnecessary. Protein A binds immunoglobulins but not other proteins present in antisera raised in rabbits and mice. Genetically engineered pro- tein G is suitable for antisera raised in sheep and goats. 8. It might be tempting to run a set of in vitro probes with vary- ing concentrations of the exogenous peptide with each in vivo experiment to determine the quantity of endogenous peptide released in response to a physiological intervention. This has proven to be impractical, since the number of probes required 17 Antibody Microprobes 285

for each in vivo experiment would be high (20–30 probes in addition to the number required for the in vivo protocol) and since the density of the coating between probes and among batches of probes will not be the same (remembering each probe must be inspected for the uniformity of the silane coat- ing). The advantage of the antibody-coated microprobe tech- nique is in the ability to determine sites of release from the CNS with specificity and not to quantitate the levels of neuro- peptide released.

Acknowledgment

This work was supported in part by AHA-SE grant 09GRN- T2250043.

References

1. Duggan, A. W., Hendry, I. A., Green, J. L., 6. Williams, C. A., Ecay, T., Reifsteck, A., Fry, B., Morton, C. R., and Hutchison, W. D. (1988) and Ricketts, B. (2003) Direct injection of The preparation and use of antibody micro- substance-P antisense oligonucleotide into the probes. J. Neurosci. Methods 23, 241–247. feline NTS modifies the cardiovascular 2. Hendry, I. A., Morton, C. R., and Duggan, A responses to ergoreceptor but not baroreceptor .W. (1988) Analysis of antibody microprobe afferent input. Brain Res. 963, 26–42. autoradiographs by computerized image pro- 7. Hua, F., Ricketts, B. A., Reifsteck, A., Ardell, J. cessing. J. Neurosci. Methods 23, 249–256. L., and Williams, C .A. (2004) Myocardial 3. Duggan, A. W. (1995) Release of neuropep- ischemia induces the release of substance P tides in the spinal cord. Prog. Brain Res. 104, from cardiac afferent neurons in rat thoracic 197–223. spinal cord. Am. J. Physiol. Heart Circ. Physiol. 4. Duggan, A. W. (2000) Neuropeptide spread in 286, H1654–H1664. the brain and spinal cord. Prog. Brain Res. 125, 8. Ding, X.-H., Hua, F., Sutherly, K., Ardell, J. L., 369–380. and Williams, C. A. (2008) C2 spinal cord stim- 5. Siddall, P. J., and Duggan, A. W. (2004) ulation induces dynorphin release from rat T4 Towards a mechanisms-based approach to pain spinal cord: potential modulation of myocardial medicine. Anesth. Analg. 99, 455–456. ischemia-sensitive neurons. Am J. Physiol. Regul. Integr. Comp. Physiol. 295, R1519–R1528.

Chapter 18

Neuropeptidases

Manuel Ramírez, Isabel Prieto, Inmaculada Banegas, Ana B. Segarra, and Francisco Alba

Abstract

The control of neuropeptide function is partially accomplished by aminopeptidases (neuropeptidases), which are the most abundant proteolytic enzymes in brain. Their analysis represents an important and quick tool to reflect the functional status of their endogenous substrates. Here, we describe an improved fluorometric method for the determination of neuropeptidase activities based on the fluorescence produced by B-naphthylamine when released from the artificial substrates aminoacyl-B-naphthylamides (arylamides) under the hydrolytic action of these enzymes.

Key words: Aminopeptidases, Neuropeptides, Arylamides, Brain, Aminoacyl-B-naphthylamides

1. Introduction

Hydrolytic enzymes localized in the cytosol, as a part of the cellular membranes or clustered into subcellular organelles, may regulate neuropeptide availability at synaptic level (1). Therefore, the con- trol of neuropeptide function is partially accomplished by several soluble and membrane-bound aminopeptidases (neuropeptidases), which are the most abundant proteolytic enzymes in brain (2). Although unspecificity is certainly a limitation in studies involving enzyme activities, their analysis represents a remarkable tool to reflect the functional status of the corresponding endogenous sub- strates. Knowledge of neuropeptidases is essential for a deep under- standing of neuropeptide function and would eventually offer the possibility of controlling the biological effects of peptides by a phar- macological manipulation. Notably, aminopeptidase inhibitors have been proposed as analgesic and antidepressant drugs (3), antihyper- tensive agents (4), or targets for drug development against different pathologies such as Alzheimer’s disease, epilepsy or ischemia (5).

Adalberto Merighi (ed.), Neuropeptides: Methods and Protocols, Methods in Molecular Biology, vol. 789, DOI 10.1007/978-1-61779-310-3_18, © Springer Science+Business Media, LLC 2011 287 288 M. Ramírez et al.

Based on previous work (6–10), here we describe an improved fluorometric method for the determination of neuropeptidase activities. This method relies on the fluorescence produced by B-naphthylamine when released from the artificial substrates amin- oacyl-B-naphthylamides (arylamides) under the hydrolytic action of these enzymes (see Note 1).

2. Materials

2.1. Collection and 1. Chloral hydrate. Treatment of Brain 2. Ethanol. Samples 3. Nembutal (Sigma Chemicals, St. Louis, MO). 2.1.1. Preparation of 4. Propylene glycol. Equithensin Anesthetic 5. Magnesium sulfate. 6. Hot magnetic shaker.

2.1.2. Animal Perfusion 1. Saline solution: NaCl 0.09%. 2. Basic surgical instruments (blades, scissors, forceps, fine pliers, etc.). 3. Dry ice.

2.1.3. Preparation 1. Homogenizer (Polytron, Kinematica AC, Lucerne, Switzerland). of Brain Samples 2. Ultracentrifuge. 3. Orbital rotor. 4. 0.2 M Tris (base): Adjust pH to 7.4 with concentrated HCl. Store at 4°C. 5. Triton X-100. 6. Polymeric adsorbent Bio-Beads SM-2 (Bio-Rad, Life Science Research, Hercules, CA).

2.2. Procedures for 1. L-Ala-B-naphthylamide (L-AlaNNap) (Sigma Chemicals). Enzymatic Assays 2. L-Arg-B-naphtylamide (L-ArgNNap) (Sigma Chemicals). 2.2.1. Determination 3. L-Glu-B-naphtylamide (L-GluNNap) (Sigma Chemicals). of Neuropeptidases 4. L-pGlu-B-naphtylamide (L-pGluNNap) (Sigma Chemicals). 5. B-naphthylamine (Sigma Chemicals). 6. 0.1 M phosphate buffer, pH 7.4: mix 95 mL of stock solution A (0.2 M sodium phosphate monobasic) with 405 mL of stock solution B (0.2 M sodium phosphate dibasic dodecahydrate). Add distilled water to less than 1,000 mL. Adjust pH to 7.4 with HCl. Make up to 1,000 mL with distilled water. Store at 4°C. 7. Ninety-six well (each one with a volume of 250 ML) black round bottom polystyrene not treated microplates. 18 Aminopeptidases in the Nervous System 289

8. 0.1 M acetate buffer, pH 4.2: Mix 368 mL of stock solution A (0.2 M acetic acid) with 132 mL stock solution B (0.2 M sodium acetate). Add distilled water to less than 1,000 mL. Adjust pH to 7.4 with HCl. Make up to 1,000 mL with dis- tilled water. Store at 4ºC. 9. Plate reader.

2.2.2. Determination 1. Coomassie Brilliant Blue G-250 (Sigma Chemicals). of Protein Concentration 2. Ethanol. 3. Orthophosphoric acid. 4. Bradford concentrate solution: Mix 50 mL ethanol, 100 mL orthophosphoric acid, and 50 mL distilled water. Add 100 mg Coomassie Brilliant Blue G-250. Filter to prepare stock solution. 5. Bradford stock solution (100 mL): Take 20 mL of concentrate solution and make up to 100 mL with distilled water (diluted five times). Store at 4°C.

3. Methods

3.1. Collection and 1. Dissolve in 9.88 mL ethanol 4.5 g chloral hydrate by gentle Treatment of Brain shaking. Samples 2. Add 0.972 g Nembutal and 16.2 mL distilled water. Shake to 3.1.1. Preparation of dissolve. Equithensin Anesthetic 3. Add 39.6 mL propylene glycol and 2.126 g magnesium sulfate. 4. Shake in hot magnetic shaker to dissolve and when cold, make up to 100 mL with distilled water. Store at 4ºC.

3.1.2. Animal Perfusion 1. Anesthetize animals (rats or mice) with equithensin (2 mL/kg body weight) administered intraperitoneally. 2. To avoid the interference of blood aminopeptidases, after opening the chest (see Note 2), perfuse the animal transcardi- ally with saline (see Note 3). 3. After perfusion, decapitate the animal quickly (less than 60 s) and remove the brain using a pair of fine pliers. 4. Cool the brain in dry ice. 5. To collect the selected tissue samples, defrost brain and, when it reaches the appropriate consistence, slice by hand with a blade. The selected areas are identified in the slices and their coordinates checked in the stereotaxic atlas of Paxinos and Watson (11). 290 M. Ramírez et al.

BRAIN SAMPLE

Homogenation Ultracentrifugation (100,000xg 30 min 4 °C)

Homogenation Ultracentrifugation + (100,000xg 30 min 4 °C) Supernatant Tritón-X-100 Pellet Pellet (discarded) SOLUBLE FRACTION Supernatant

+ BIO-BEADS MEMBRANE-BOUND (polymeric adsorbent) FRACTION shacked 2 h at 4 °C in an orbital rotor

Fig. 1. Protocol used to obtain the soluble and membrane-bound fractions from brain samples in which enzymatic activities and protein content are measured.

3.1.3. Preparation 1. Homogenize brain samples with a Polytron-aggregate in of Brain Samples 400 ML of 10 mM Tris–HCl buffer. Ultracentrifuge at 100,000 × g for 30 min at 4°C. 2. To solubilize membrane proteins, rehomogenize pellets in 10 mM Tris–HCl buffer plus 1% Triton X-100. 3. Centrifuge at 100,000 × g for 30 min at 4°C to remove deter- gent from the sample (12). 4. Shake supernatants in an orbital rotor for 2 h at 4°C with 100 mg/mL polymeric adsorbent Bio-Beads SM-2 (see Note 4). 5. After that, remove the Bio-Beads (see Note 5) and use the supernatants to measure in triplicate membrane-bound amino- peptidase activities and protein content (Fig. 1).

3.2. Procedures for The method relies upon the hydrolytic capacity of neuropeptidases Enzymatic Assays on artificial substrates, i.e., amino acid-B-naphthylamides (arylam- ides). The neuropeptidase recognizes the free amino-terminal 3.2.1. Determination group of the arylamide and separates, by hydrolysis, the B-naph- of Neuropeptidases thylamine from the adjacent amino acid (Fig. 2). Here, we describe, as an example, the standard method of determination for neutral (alanyl aminopeptidase; EC 3.4.11.14; aminopeptidase N; AlaAP), 18 Aminopeptidases in the Nervous System 291

Soluble or SUBSTRATE SAMPLE Membrane-bound SOLUTION (20 μL) fraction ACETATE BUFFER 100 (μL) (100 μL)

INCUBATION 30 min 37 °C START STOP REACTION REACTION

L-amino acid-B-Naphthylamide Aminopeptidase FLUOROMETRIC MEASUREMENT 345 wvl excitation 412 wvl emission

L-amino acid B-Naphthylamine

Fig. 2. Procedures for the enzymatic assay. 20 ML of sample from the corresponding source (soluble or membrane-bound fraction) is incubated for 30 min at 37°C with the corresponding substrate solution in microplates. The reaction is stopped by adding 100 ML of 0.1 M of acetate buffer, pH 4.2. The amount of B-naphthylamine released, as a result of the enzymatic activity, is measured fluorometrically at 412-nm emission wavelength with an excitation wavelength of 345 nm.

basic (arginyl aminopeptidase; EC 3.4.11.6; aminopeptidase B; ArgAP), acidic (glutamyl aminopeptidase; EC 3.4.11.7; amino- peptidase A; GluAP), and omega (pyroglutamyl aminopeptidase; EC 3.4.11.8; pGluAP pyrrolidonyl carboxypeptidase; PCP) (13) neuropeptidases. 1. For neuropeptidase determination, use 96-well (each one with a volume of 250 ML) black round bottom polystyrene untreated microplates. 2. Incubate 20 ML of supernatant of the corresponding tissue (from the soluble or membrane-bound fraction) for 30 min for AlaAP, ArgAP, GluAP, or pGluAP activities at 37°C with 100 ML of the corresponding substrate solution (Table 1). 3. Stop reactions by adding 100 ML of 0.1 M of acetate buffer, pH 4.2. 4. Measure the amount of B-naphthylamine, released because of the enzymatic activity fluorometrically at 412-nm emission wavelength with an excitation wavelength of 345 nm. The sensitivity of the method allows measurements of aminopepti- dases in the picomolar range (see Note 6). 292 M. Ramírez et al.

Table 1 Composition of the different substrate solutions

Substrate solutions

Enzyme Components Buffer

AlaAP L-AlaNNap 2.14 mg/100 mL 50 mM Phosphate (pH 7.4) BSA 210 mg/100 mL Dithiothreitol 210 mg/100 mL

ArgAP L-ArgNNap 23.35 mg/100 mL 50 mM Phosphate (pH 7.4) BSA 210 mg/100 mL Dithiothreitol 210 mg/100 mL

GluAP L-GluNNap 22.72 mg/100 mL 50 mM HCl–Tris (pH 7.4) BSA 210 mg/100 mL Dithiothreitol 210 mg/100 mL

CaCl2 20.555 g/100 mL pGluAP L-pGluNNap 22.54 mg/100 mL 50 mM Phosphate (pH 7.4) BSA 210 mg/100 mL Dithiothreitol 210 mg/100 mL EDTA 237.8 mg/100 mL BSA bovine serum albumin

5. Convert the fluorescence values obtained to pmol or nmol of the released B-naphthylamine by extrapolating to a standard curve previously obtained after determination of decreasing concentrations of B-naphthylamine in a medium equal to the corresponding substrate solution plus 100 ML of acetate buffer 0.1 M, pH 4.2.

3.2.2. Determination To determine the proteins in the soluble or membrane-bound of Protein Concentration fractions, the method of Bradford (14) is used. This method is based on the affinity of a dye (Coomassie Brilliant Blue G-250) for the proteins. The dye binds to proteins and causes a change in the absorption wavelength, which is measured spectrophotometrically at 595 nm. 1. Use the same type of microplates employed for fluorescence measurements. 2. Add 5 ML of the corresponding sample to each well, containing 200 ML of Bradford reagent, and then read absorbance in photometer. 3. Transform the obtained absorbance values in milligrams of proteins by extrapolation to a standard curve of bovine serum albumin (BSA) containing a range of 5–100 Mg protein in 200 ML of Bradford reagent. The blank consists in 200 ML of the reactive without sample. 18 Aminopeptidases in the Nervous System 293

4. Finally, express specific aminopeptidase activities as pmol or nmol of L-AlaNNap, L-ArgNNap, L-GluNNap, or L-pGluNNap hydrolyzed per min per mg of protein.

4. Notes

1. Arylamides have been reported as potential carcinogenic sub- stances and so care should be taken not to receive exposure. 2. Cut transversally the skin and muscle at the level of the xiphoid process and then perform parallel cuts at both sides of sternum to separate it and to expose the heart. Fix the xiphoid process to the inferior mandible with curved Kelly forceps. 3. To perfuse the animal, saline is injected with a hypodermic syringe through the left ventricle, cutting the right atrium to enable saline output. For rats ranging 250–300 g usually, 150 mL of saline is necessary. 4. Add a volume of approximately 0.5 mL of Bio-Beads to the tube containing the supernatant. 5. Permit the deposit of Bio-Beads and then gently remove the supernatant with a pipette. 6. Owing to autolytic process affecting these enzymes, the coef- ficient of variation between measurements is not reproducible. Therefore, all enzymatic determinations in each experimental group must be carried out in the same batch.

Acknowledgments

This work was supported by Junta de Andalucía through PAI CVI- 221 (Peptides and Peptidases) and BIO-221 (Neuroendocrinology and Nutrition). In Memorium of Francisco Alba.

References

1. Arechaga, G., Sánchez, B., Alba, F., de Dios 3. Noble, F. and Roques, B.P. (2007) Protection Luna, J., Luttenauer, L., Martínez, J.M., et al. of endogenous enkephalin catabolism as nat- (1995) Subcellular distribution of soluble and ural approach to novel analgesic and antide- membrane-bound Arg-beta-naphthylamide pressant drugs. Expert Opin Ther Targets. 11, hydrolyzing activities in the developing and 145–159. aged rat brain. Cell Mol Biol Res. 41, 4. Bodineau, L., Frugière, A., Marc, Y., 369–375. Inguimbert, N., Fassot, C., Balavoine, F. et al. 2. Checler, F. (1993) Methods in neurotransmit- (2008) Orally active aminopeptidase A inhibitors ter and neuropeptide research. Parvez, S.H., reduce blood pressure: a new strategy for treat- Naoi, M., Nagatsu, T., Parvez, S. (Eds), ing hypertension. Hypertension. 51, Elsevier, Amsterdam. 1318–1325. 294 M. Ramírez et al.

5. Stragier, B., De Bundel, D., Sarre, S., Smolders, determination of brain aminopeptidases. Arch I., Vauquelin, G., Dupont, A. et al. (2008) Neurobiol (Madr) 52, 169–173. Involvement of insulin-regulated aminopepti- 10. Ramírez, M., Prieto, I., Martinez, J.M., Vargas, dase in the effects of the -angiotensin F. and Alba, F. (1997) Renal aminopeptidase fragment angiotensin IV: a review. Heart Fail activities in animal models of hypertension. Rev. 13, 321–337. Regul Pept. 72, 155–159. 6. Greenberg, L.J. (1962) Fluorimetric measure- 11. Paxinos, G. and Watson, C. (1998) The rat ment of alkaline phosphatase and aminopepti- brain in stereotaxic coordinates. 4 th Ed. dase activities in the order of 10–14 mole. London: Academic Press. Biochem. Biophys. Res Commun. 9, 430–435. 12. Alba, F., Arenas, J.C., López, M.A. (1995) 7. Tobe, H., Kojima, F., Aoyagi. T. and Umezawa, Properties of rat brain dipeptidyl aminopepti- H. (1980) Purification by affinity chromatog- dases in the presence of detergents. Peptides. raphy using and properties of amino- 16, 325–329. peptidase A from pig . Biochim Biophys 13. Barret, A.J., Rawlings, N.D. and Woessner JF Acta. 613, 459–468. Eds. (1998) Handbook of proteolytic enzymes. 8. Schwabe, C. and McDonald, J.K. (1977) Academic Press, London. Demonstration of a pyroglutamyl residue at the 14. Bradford, M.M. (1976) A rapid and sensitive n-terminus of the B-chain of porcine relaxin. method for the quantification of microgram Biochem Biophys Res Commun. 74, 1501–1504. quantities of protein utilizing the principle of 9. Alba, F., Iríbar, F., Ramírez, M. and Arenas, protein dye binding. Anal Biochem. 72, C. (1989) A fluorimetric method for the 248–254. Chapter 19

Neuropeptide Autoantibodies Assay

Sergueï O. Fetissov

Abstract

This protocol describes an ELISA-based method for assaying serum levels of autoantibodies reactive with neuropeptides. The method allows for measuring relative amounts of free and bound, i.e., those present in immune complexes, autoantibodies using two types of sample buffers providing normal and dissociative conditions, respectively. This method can be applied to measure autoantibody levels directed against other than neuropeptide molecules and in a variety of biological fluids.

Key words: Autoantibodies, Neuropeptides, ELISA, Immunoglobulin, Immune complexes

1. Introduction

The immune system generates billions of immunoglobulin molecules, some having crucial functions in defending the body from patho- gens, some protecting against autoimmunity and cancer and some modulating chemical transmission. Eventually, no molecule could escape from having an immunoglobulin autoantibody partner which altogether will form an immune self-image of the organism (1). Besides their vital roles, some autoantibodies (autoAbs) can become pathogenic, and their involvement in the mechanisms of several autoimmune conditions as well as neurological and psychi- atric diseases is increasingly recognized (2–8). Neuropeptides are not an exception from this phenomenon, and autoAbs reactive with neuropeptides or peptide hormones were detected in both humans and rats (9–13). Since autoAbs are very versatile molecules, i.e., the same antigen can be bound by a variety of autoAbs with different functional properties, it is important to be able to assay such autoAbs to see if their production can be associated with a

Adalberto Merighi (ed.), Neuropeptides: Methods and Protocols, Methods in Molecular Biology, vol. 789, DOI 10.1007/978-1-61779-310-3_19, © Springer Science+Business Media, LLC 2011 295 296 S.O. Fetissov

given pathological condition. Neuropeptide-reactive autoAbs are represented by different classes of immunoglobulins similarly to any antibody generated by immunization, and therefore, their properties can be studied using classical immunological techniques developed for immunoglobulins. In particular, levels and affinity are the most important characteristic of any antibody, and as concerned neuropeptide autoAbs, they may be responsible for the modulation of biological effects of corresponding neuropeptides. Antibodies with high affinity are normally responsible for the neu- tralization of antigens and have tendency to form immune com- plexes, while low-affinity autoAbs do not usually display blocking properties but instead can bind antigens reversibly and hence may play peptide carrier role (14). Whereas affinity assays of antibodies are laborious immunological techniques, the present protocol describes a relatively simple approach for the detection of neuro- peptide autoAbs as well as their levels and relative affinity vs. control antibody, which can be used in any laboratory able to perform enzyme-linked immunosorbent assay (ELISA). In this technique, the neuropeptide of interest is coated to the ELISA microplate, and autoAbs reactive with this neuropeptide are detected in the biological fluids (plasma, serum, saliva, etc.) applied to the ELISA plates upon incubation with alkaline phosphatase- conjugated detection antibodies directed against the constant chains of autoAbs molecules and followed by routine detection of the chromogenic reaction. The method allows measuring relative amounts of free and bound, i.e., those present in immune complexes, autoAbs using two types of sample buffers creating normal or dissociative conditions, respectively, thereby giving immediately initial information about autoAbs levels and affinities. The present protocol provides the instructions for detection of neuropeptide IgG autoAbs in human serum, but it can be adapted to other species and other classes and subclasses of autoAbs molecules.

2. Materials

2.1. Buffers 1. Coating buffer: 0.5 M Na2CO3, 0.5 M NaHCO3, pH 9.6. Add 0.02% sodium azide. 2. 10× Phosphate-buffered saline (10× PBS): 1.37 M NaCl,

27 mM KCl, 81 mM Na2HPO4 7H2O, 15 mM KH2PO4, pH 7.4. 3. Wash buffer: 1× PBS + Tween 20, 0.05%, pH 7.4. 4. Sample normal buffer: 1× PBS + 0.02% sodium azide, pH 7.4 5. Sample dissociating buffer: 3 M NaCl, 1.5 M glycine, pH 8.9 (with 5 N NaOH). 19 Neuropeptide AutoAbs Assay 297

6. Detection buffer: 0.1 M Tris (base) – adjust pH, 0.1 M NaCl,

5 mM MgCl2 6H2O, pH 9.5. All buffers stored at 4°C and stable at 4°C for up to 2 months.

2.2. Reagents 1. Synthetic neuropeptide of interest (0.5 mg is sufficient for ten and Solutions 96-well plates). 2. Alkaline phosphatase (AP)-conjugated detection antibody directed against the constant chains of a specific class of immu- noglobulins (IgG, IgM, etc.) of the species in which autoAbs should be assayed. In this protocol, AP-conjugated anti-human IgG (Sigma Chemicals, St. Louis, MO). 3. Alkaline phosphatase substrate: 4-nitrophenyl phosphate diso- dium salt hexahydrate (pNPP, Sigma Chemicals). 4. Stop solution: 3 N NaOH.

2.3. Materials 1. ELISA microplates: For example, Maxisorp 96-well plates and Equipment (Nunc, Rochester, NY) or any others providing good adherence of peptide molecules and adapted for the detection system and the microplate reader. 2. Multichannel micropipette for 96-well microplates. 3. ELISA microplate reader, equipped with a filter corresponding to the detection system, ex 405 nm for AP detection.

3. Methods

1. Upon purchasing, dilute your lyophilized neuropeptide in coating buffer to make a stock solution of 2 mg/mL. Prepare aliquots of 10 ML (each aliquot will be used for coating of one ELISA plate). Store aliquots at −20°C. 2. Prepare one or several 96-well microplates: Draw schema with each sample in duplicate (external wells can be used for blank reading = no sample added). 3. Dilute one aliquot with the neuropeptide stock solution (10 ML, 2 mg/mL) 1,000× in coating buffer. 4. Add 100 ML of diluted peptide (2 Mg/mL in coating buffer) to each well of the microplate. 5. Wrap the plate in Saran wrap and incubate at 4°C overnight (see Note 1). 6. Dilute the samples in which neuropeptide autoAbs will be assayed (e.g., human serum, see Note 2) in sample normal buffer 1:200 and in sample dissociating buffer 1:200 (see Note 3) 298 S.O. Fetissov

considering that the volume of 100 ML will be added in each well in duplicate plus have some extra volume. 7. Discard coating solution from the plate. Wash three times with 300 ML of wash buffer per well. All residual wash buffers must be removed by tapping microplate gently over a filter paper or a towel. 8. Add diluted serum samples (100 ML per well) and wrap the plate in Saran wrap. 9. Incubate at 4°C overnight (see Note 4). 10. Dilute the detection antibody, e.g., AP-conjugated anti-human IgG 1:2,000 in sample buffer (see Note 5). 11. Discard sample solution from the plate. Wash three times with 300 ML of wash buffer per well. All residual wash buffers must be removed by tapping over a filter paper or a towel. 12. Add diluted detection antibody (100 ML) to wells, wrap the plate in Saran wrap, and incubate for 3 h at room temperature. 13. Prepare AP substrate (p-NPP) in detection buffer and keep out of light (see Note 6). 14. Discard detection antibody solution from the plate. Wash three times with 300 ML of wash buffer per well. All residual wash buffers must be removed by tapping over a filter paper or a towel. 15. Add diluted substrate (100 ML) to each well, wrap the plate in Saran wrap, and incubate for 40 min at room temperature, protected from light. 16. Add 50 ML of stop solution. Gently tap the plate to ensure thorough mixing. 17. Immediately determine optical density (OD) in a microplate reader at 405 nm. 18. From the mean OD values between duplicates subtract the mean OD blank values (see Note 7). 19. Analyze your data separately for (1) free autoAbs levels mea- sured in serum samples diluted in sample normal buffer and (2) total (dissociated) autoAbs levels measured in serum sam- ples diluted in sample dissociative buffer. 20. Percentage of complexed autoAbs can be calculated by the fol- lowing formula: 100 − (OD normal/OD dissociative 100×). 21. The obtained values can be compared with known standards or with values of autoAbs levels obtained from a control group studied using the same experimental protocol (see Note 8). 19 Neuropeptide AutoAbs Assay 299

4. Notes

1. Maximal saturation of peptide on the microplate is achieved by incubation between 48 and 72 h at 4°C. Plates can be stored for up to a week at 4°C. Alternatively, coat for 3 h at 37°C. 2. Serum samples should be aliquoted to avoid autoAbs degrada- tion from frequent thawing–refreezing. In fact, repeated autoAbs assay from the same sample following several thawing– refreezing cycles will result in a gradual increase of free autoAbs levels due to spontaneous dissociation of autoAbs present as immune complexes. It is an important confounding factor when comparing autoAbs levels from two sets of samples, which had different amount of thawing–refreezing cycles. 3. When deciding to select the optimal serum (or another bio- logical fluid) dilution for each new neuropeptide autoAbs and in a new set of samples, few samples should be tested with several serial dilutions in sample normal and dissociative buffers, e.g., 1:100, 1:200, 1:400, 1:800. The optimal dilutions are selected from the linear range of the optical density (from 0.2 to 2.5) with the detection antibody used at 1:1,000. Since OD of dissociated autoAbs are normally higher than OD of free autoAbs from the same serum sample diluted to the same levels, it is also important that OD of dissociated autoAbs would not be too high (OD < 3.0), i.e., should not reach the plateau level. In the serum or plasma, neuropeptide-reactive IgG autoAbs are normally detected with dilution 1:200 or 1:400 depending on the neuropeptide and the species; however, for the same neuropeptide when assayed in the cerebrospinal fluid, the dilu- tions should be reduced to 1:20–1:50. When optimal dilution of sera is determined with the detection antibody diluted at 1:1,000, the same serum dilution should be tested with the detection antibody at higher dilutions, e.g., 1:2,000 or 1:3,000. Obtaining OD values in the linear range with higher dilutions of detection antibody improves the specificity of the assay. As in any immunoassay, obtaining good signal level with the maximal antibody dilution represents the optimal experimental condi- tions improving the specificity. 4. As discussed in Note 3, prolonging incubations of maximally diluted sera samples at lower temperature allows for more specific interactions between neuropeptide and autoAbs. Alternatively, incubate for 3 h at 37°C. 5. Although in this example we used AP-conjugated anti-human IgGs, for the detection of autoAbs in other species (e.g., in the rat or mouse) one should use corresponding antibodies, i.e., anti-rat and anti-mouse, respectively. Additionally, other than 300 S.O. Fetissov

IgG classes of autoAbs can be detected with anti-IgM, anti-IgA, or anti-IgE detection antibodies or IgG subclasses can be detected with anti-IgG1-4 antibodies. In each new assay, an optimiza- tion of serum and antibody dilution should be performed as described in Note 3. 6. Detection systems other than AP can also be used, e.g., horse- radish peroxidase-conjugated detection antibodies or fluoro- phore-conjugated detection antibodies, with corresponding detection systems and equipment to read the signals. 7. Correct blank OD values are normally below 0.1. If higher, the problem is commonly related to the nonspecific binding of the detection antibody. To reduce it, higher dilutions of the detection antibody are recommended, or replacement with the antibody from another supplier. The variation between duplicate values should not exceed 5%. If this occurs, it usually indicates an error in sera dilution. To reduce the risk of such variations, serum samples can be diluted gradually. 8. Data interpretation includes the following: (1) the statement of the presence of autoAbs reactive with the neuropeptide of interest in the given sample and (2) relative levels and affinity of neuropeptide-reactive autoAbs. The OD values in the sample higher than blank OD values signify the presence of neuropeptide-reactive autoAbs. To further confirm the pres- ence of neuropeptide-reactive autoAbs, sera can be preadsorbed with the target peptide prior to ELISA. By doing that with different concentrations of the peptide, the OD signal will be modified depending on peptide concentrations, normally higher peptide concentration resulting in less OD signal in ELISA. However, sometime, the reverse relation can be observed, i.e., higher concentrations of peptide in the pread- sorption test will result in higher signal in ELISA. Although first type of absorptive effect of neuropeptide autoAbs is most common, both types of reactions can be found in sera of healthy individuals (10), the last phenomenon being related to the dissociation of immune complexes by increased concentra- tion of the antigen. The methods also permit estimation of the levels and affin- ity of neuropeptide autoAbs if compared with known standards or control autoAbs. Obviously, such controls should be ini- tially obtained by measuring levels of free and total autoAbs reactive with a given neuropeptide in a group of healthy subjects or animals using the same experimental protocol. Then, an increase of free autoAbs levels can be interpreted as increased carrier role of neuropeptide autoAbs and vice versa, while an increase in total autoAbs level would signify increased neutralization of the neuropeptide. Finally, by comparing the ratios between free and total autoAbs, the relative percentage of neuropeptide autoAbs present as immune 19 Neuropeptide AutoAbs Assay 301

complexes can also be compared with controls (15). An increased percentage of immune complexes versus controls would signify an increased affinity of neuropeptide autoAbs in the study sample. There is one caveat, however, that is related to the thermodynamics of antibody interaction with antigen present in the solid or liquid phases. The lower energy needed for the interaction of antibody on the solid-phase antigen is the underlying theory of the present protocol detecting total autoAbs levels in dissociative buffer. However, some immune complex-forming autoAbs would not be revealed with this approach of high stringency interaction. Therefore, sometime, the phenomenon of the dissociation of immune complexes by increased concentration of antigen, as discussed above, can be exploited to reveal high-affinity neuropeptide autoAbs. General methodological and functional aspects of neuropeptide autoAbs assay can also benefit from the knowledge gained in the field of insulin autoantibody, which has been used as a marker of dia- betes type 1 (16, 17).

Acknowledgements

The author is thankful to Dr. Danièle Gilbert, Inserm U519, Rouen University, France and to late Dr. Ann Kari Lefvert, Karolinska Institutet, Stockholm, Sweden, for help with protocol development, as well as to the members of the ADEN Laboratory, Rouen University, France whose contribution was crucial to repeat- edly reproduce this protocol.

References

1. Cohen, I.R. (2005) Tending Adam’s garden. 6. Fetissov, S.O., Hallman, J., Oreland, L., af Evolving the cognitive immune self. San Diego, Klinteberg. B., Grenbäck, E. et al. (2002) CA: Elsevier Academic Press. 266 p. Autoantibodies against A-MSH, ACTH, and 2. Bhat, R., and Steinman, L. (2009) Innate and LHRH in anorexia and bulimia nervosa Adaptive Autoimmunity Directed to the Central patients. Proc. Natl. Acad. Sci. USA 99, Nervous System. Neuron 64, 123–132. 17155–17160. 3. Fetissov, S.O., and Déchelotte, P. (2008) The 7. Fetissov, S.O., Harro, J., Jaanisk, M., Järv, A., putative role of neuropeptide autoantibodies in Podar, I. et al. (2005) Autoantibodies against anorexia nervosa. Curr. Opin. Clin. Nutr. neuropeptides are associated with psychological Metabol. Care 11, 428–434. traits in eating disorders. Proc. Natl. Acad. Sci. 4. Fetissov, S.O., Hamze Sinno, M., Coquerel, USA 102, 14865–14870. Q., Do Rego, J.C., Coëffier, M. et al. (2008) 8. Roy, B.F., Rose, J.W., Sunderland, T., Morihisa, Emerging role of autoantibodies against J.M., and Murphy, D.L. (1988) Antisomatostatin appetite-regulating neuropeptides in eating IgG in major depressive disorder. A prelimi- disorders. Nutrition 24, 854–859. nary study with implications for an autoimmune 5. Fetissov, S.O., Hallman, J., Nilsson, I., Lefvert, mechanism of depression. Arch. Gen. Psychiatry A.K., Oreland, L., et al. (2006) Aggressive 45, 924–928. behavior linked to corticotropin-reactive 9. Paul, S., Volle, D.J., Beach, C.M., Johnson, autoantibodies. Biol. Psychiatry 60, 799–802. D.R., Powell, M.J. et al. (1989) Catalytic 302 S.O. Fetissov

hydrolysis of vasoactive intestinal peptide by 14. Hamze Sinno, M., Do Rego, J.C., Coëffier, M., human autoantibody. Science 244, Bole-Feysot, C., Ducrotte P. et al. (2009) 1158–1162. Regulation of feeding and anxiety by 10. Fetissov, S.O., Hamze Sinno, M., Coëffier, M., A-MSH reactive autoantibodies. Psychoneu- Bole-Feysot, C., Ducrotté, P. et al. (2008) roendocrinology 34, 140–149. Autoantibodies against appetite-regulating pep- 15. Deloumeau, A., Bayard, S., Coquerel, Q., tide hormones and neuropeptides: putative Déchelotte, P., Bole-Feysot, C. et al. (2010) modulation by gut microflora. Nutrition 24, Increased Immune Complexes of Hypocretin 348–359. Autoantibodies in Narcolepsy. PLoS One 5, 11. Black, J.L., 3rd, Silber, M.H., Krahn, L.E., e13320. Fredrickson, P.A., Pankratz, VS. et al. (2005) 16. Franke, B., Galloway, T.S., and Wilkin, T.J. Analysis of hypocretin (orexin) antibodies in (2005) Developments in the prediction of type patients with narcolepsy. Sleep 28, 427–431. 1 diabetes mellitus, with special reference to 12. Sebriakova, M., and Little, J.A. (1973) A insulin autoantibodies. Diabetes Metab. Res. method for the determination of plasma insulin Rev. 21, 395–415. antibodies and its application in normal and 17. Radermecker, R.P., Renard, E., and Scheen, diabetic subjects. Diabetes 22, 30–40. A.J. (2009) Circulating insulin antibodies: 13. Roy, B.F., Rose, J.W., McFarland, H.F., influence of continuous subcutaneous or intra- McFarlin, D.E., and Murphy D.L. (1986) Anti- peritoneal insulin infusion, and impact on glu- beta-endorphin immunoglobulin G in humans. cose control. Diabetes Metab. Res. Rev. 25, Proc. Natl. Acad. Sci. USA 83, 8739–8743. 491–501. Chapter 20

Intranasal Delivery of Neuropeptides

Michael C. Veronesi, Daniel J. Kubek, and Michael J. Kubek

Abstract

A major barrier to entry of neuropeptides into the brain is low bioavailability and presence of the blood–brain barrier. Intranasal delivery of neuropeptides provides a potentially promising alternative to other routes of administration, since a direct pathway exists between the olfactory neuroepithelium and the brain. Use of the rat as an animal model in nose to brain delivery of neuropeptides allows for several advan- tages, including a large surface area within the nasal cavity dedicated to olfactory epithelium and robust neuronal pathways extending to and from most areas of the brain from the nose via the olfactory cortex. A major disadvantage to using rats for nose to brain delivery is the difficulty in selectively targeting the posterior olfactory epithelium (which facilitates delivery to the brain) over the more anterior respiratory epithelium (which facilitates delivery to the lungs and secondarily to the peripheral blood) in the nasal cavity. We have developed a novel delivery system that consists of surgically implanting stainless-steel can- nulas in the dorsal aspect of the nasal cavity overlying the olfactory neuroepithelium, thereby allowing neuropeptide compounds to bypass the respiratory epithelium.

Key words: Drug delivery, Intranasal delivery, Neuropeptides, Nasal cannula, Olfactory neuroepithelium

1. Introduction

Intranasal drug administration is gaining favor, especially for drugs targeting the central nervous system (CNS). Several studies have shown that intranasal administration of certain drugs result in cere- brospinal fluid (CSF) and olfactory bulb levels that are considerably higher than those following intravenous or subcutaneous adminis- tration (1–5). Another advantage of this route of delivery is that it appears to be a viable means of bypassing first-pass metabolism (6) and the blood–brain barrier, allowing for potentially lower concen- trations of the compound to achieve therapeutic efficacy. In addition, ease of intranasal administration could produce higher patient

Adalberto Merighi (ed.), Neuropeptides: Methods and Protocols, Methods in Molecular Biology, vol. 789, DOI 10.1007/978-1-61779-310-3_20, © Springer Science+Business Media, LLC 2011 303 304 M.C. Veronesi et al.

compliance relative to the discomfort experienced with intravenous or intramuscular injection and side effects by some orally adminis- tered drugs due to stomach upset (7). Finally, intranasal delivery may allow for enhanced concentrations in targets within the CNS by taking advantage of a more direct route to the brain by way of absorption across the olfactory neuroepithelium (7, 8). Administration by intranasal delivery appears to have a great potential for future application (9). There are currently over 40 neuropeptides that have been identified, and their role in disease has become an important area of research over the past 2 decades (10–12). Neuropeptides have been indicated as targets in several disorders, such as stroke (13, 14), obesity (15, 16), chronic pain (17), learning and memory (18), depression (19–21), and epilepsy (22–25). In order to extrapolate experimental findings between humans and rats, a brief description of nasal anatomical parameters is necessary. The surface area of the rat olfactory neuroepithe- lium is 15 cm2 and is represented by 15 million olfactory recep- tor cells, while in man it is 10 cm2 and is composed of 12 million cells (26).The nasal cavity of a 70-kg man has an average volume of 20 mL and an average surface area of 160 cm2. In comparison, the nasal cavity of a 0.25-kg rat has a volume of 0.4 mL and sur- face area of 14 cm2. The transport rate of olfactory particles that contact the mucus layer is roughly 14 mm/min in both man and rat. Additionally, in man, the clearance half-life is 15 min, while in rat the half-life is 5 min (27). Thus, the olfactory neuroepithe- lium is remarkably similar between man and rat in terms of recep- tor neurons and surface area, but the nasal cavity volume is significantly larger in man. These neuroanatomical similarities suggest that the rat is a reasonable animal for basic nose to brain research, but it must be kept in mind that beyond olfactory uptake, temporal relationships between rat nose to brain loci and human loci will significantly vary with size and distance from the olfactory bulb. Although intranasal delivery of bioactive neuropeptides offers several theoretical advantages over other forms of delivery, an abundance of peptidases found extracellularly in the nasal mucosa and intracellularly following neuronal uptake limits their transport to distal target brain regions from the nose. In addition, treatment of certain diseases may benefit from sustained, enhanced bioavail- ability at the pathologic site. Significant efforts to develop sus- tained-release biodegradable nanoparticle-sized carriers are underway, since they can enclose and, thus, protect neuropeptides and other therapeutic compounds from premature degradation before reaching their destination (8). However, despite the presence of the aforementioned barriers, evidence supports the feasibility of neuropeptide delivery to the brain using the nasal route. For instance, nerve growth factor (NGF), insulin-like growth factor (IGF-I), , and interferon 20 Neuropeptide Delivery 305 beta have all been shown to reach the CNS following intranasal administration (28–32). In addition, intranasal administration of NAP, an 8-amino acid peptide, has been shown to be neuroprotec- tive and exert cognitive enhancing properties in mice (33, 34), and melanocortin and insulin are found in the CSF following intranasal administration in humans (35). Intranasal delivery methods enabling drugs/neuropeptides to obtain direct contact with the olfactory neuroepithelium, while also bypassing the respiratory epithelium, would potentially maximize entry into the CNS rather than blood circulation or peripheral tis- sues (i.e., lungs, stomach, lymph nodes). Most studies in small ani- mals (i.e., rats, mice) utilize drug solutions, usually dissolved in saline, that are pipetted via drops (3–10 ML) into each nare while the animal is maintained in a supine position for a prolonged period of time (28, 30–32, 36–38). The advantages of this method of delivery are that it is noninvasive and the animal does not have to be anesthetized during the procedure. However, solution/drugs applied through the nares must first transgress the respiratory epi- thelium and vomeronasal organ (VNO) en route to the more pos- terior aspect of the nasal cavity where the olfactory neuroepithelium resides, increasing the likelihood of absorption into the blood, lungs, and/or lymph nodes as well as the VNO. In addition, the nasopalatine duct in rodents remains patent, allowing loss of the drug solution from the nasal cavity to the oral cavity and esopha- gus/stomach during application (39, 40). Unanesthetized animals are prone to struggling during administration and sneeze/blow the contents reflexively back out of the nasal cavity upon administra- tion, further reducing consistency and accuracy of the procedure. A second type of intranasal administration utilizes thin, flexible tubing attached to a 10 ML Hamilton syringe that can be inserted into the posterior aspect of the nasal cavity before application of the treatment (41). The advantage of the procedure is that the respiratory epithelium, nasopalatine duct and perhaps the VNO can be bypassed for more accurate administration onto the olfactory epithelium. However, the animal must be anesthetized and the tubing must be guided through the tightly spaced, delicate, highly vascular mucosa of the turbinates, which are especially susceptible to trauma/bleeding. Variability in the nasal anatomy and turbinates also create difficulty in consistent placement of the end of the tube into the region of the olfactory neuroepithelium, which is no longer visible during treatment. We have developed a third method of intranasal delivery involv- ing surgical placement of stainless-steel cannulas in close proximity to the cribriform plate and the olfactory neuroepithelium (42). Our approach provides more specific delivery to the olfactory neu- roepithelium, thereby reducing the issues related to the respiratory epithelium, nasopalatine duct, and the VNO, although the exis- tence of the latter in man is controversial (43). 306 M.C. Veronesi et al.

2. Materials

2.1. Nasal Cannula 1. TEC-4 Vaporizer w/O2 regulator (Paragon Medical, Coral Implantation Springs, FL). 2. Anesthesia induction chamber t3.5 cm × 6.5 cm × 9 cm. 3. Medipure® Oxygen, K-cylinder, 249 cu ft or smaller portable cylinders (Praxair Healthcare Services Inc., Danbury, CT). 4. 4% isoflurane (Abbott Laboratories, North Chicago, IL). 5. Stereotaxic apparatus with rat ear bars (Kopf Instruments, Tujunga, CA). 6. Nonrebreathing rat gas anesthesia mask with associated tubing (Harvard Apparatus, Holliston, MA). 7. Puralube eye lubricant 3.5-gm tube (Dechra Vet. Products, Overland Park, KS). 8. Cordless DREMEL© Tool (Robert Bosch Tool Corp., Racine, WI). 9. 2-mm round burr. 10. Cannulas – Vented 18–8 stainless-steel socket head cap screw, 2–56 thread × 1/4p thread length, 0.028p vent diameter (McMaster-Carr, Elmhurst, IL). 11. 5/64p t-handle hex wrench. 12. Heating pad. 13. Change-A-Tip Electrocautery (Aaron Medical, St. Petersburg, FL). 14. Standard surgical instrumentation – forceps, hemostats, scis- sors, etc. 15. Dental periosteal elevator. 16. Cranioplastic dental cement (Lang Dental, Wheeling, IL). 17. 5–0 black braided silk suture (Henry Schein Inc, Melville, NY). 18. Baytril injectable solution 2.27% – 20 mL bottle (Bayer, West Haven, CT). 19. Physiologic saline (0.9% NaCl) irrigation (Baxter Healthcare Corp., McGraw Park, IL). 20. 50-mL irrigation syringe. 21. Topical antibiotic ointment.

2.2. Intranasal 1. TEC-4 Vaporizer w/O² regulator (Paragon Medical, Coral Administration Springs, FL). 2. Anesthesia induction chamber t3.5 cm × 6.5 cm × 9 cm. 3. Medipure® Oxygen, K-cylinder, 249 cu ft or smaller portable cylinders (Praxair Healthcare Services Inc., Danbury, CT). 20 Neuropeptide Delivery 307

4. 4% isoflurane (Abbott Laboratories, North Chicago, IL). 5. Stereotaxic apparatus with rat ear bars (optional to aid in checking cannula patency) (Kopf Instruments, Tujunga, CA). 6. Nonrebreathing rat gas anesthesia mask with associated tubing (Harvard Apparatus, Holliston, MA). 7. Heating pad. 8. Hamilton Syringe, 100 ML (Hamilton Co. Inc., Whittier, CA). 9. 30 ga patency needle. 10. Puralube eye lubricant 3.5-gm tube (Dechra Vet. Products, Overland Park, KS).

3. Methods

3.1. Nasal Cannula 1. On the day of surgery, anesthetize using 4% isoflurane from a Implantation TEC-4 Vaporizer connected to the anesthesia induction cham- ber (see Note 1). 2. Once the animal is fully anesthetized (loss of corneal and right- ing reflex, approximately 1 min), remove it from the induction chamber and place the head in the stereotactic apparatus. Place a gas anesthesia mask over the snout to continue anesthesia at 1.75% isoflurane. Body temperature is maintained at 37°C via an electric heating pad. 3. Make a midline incision to expose the nasofrontal suture, which adjoins the nasal bone to the frontal bone on the dorsal aspect of the skull (Fig. 1a). 4. Drill bilaterally a 2.0-mm diameter hole immediately posterior to the nasofrontal suture (Fig. 1a) to access the olfactory epi- thelium within the posterior nasal cavity immediately anterior to the cribriform plate (Fig. 1b) (see Note 2). 5. Insert stainless-steel nasal ports into the holes to approximately 0.5 mm below the base of the boney plate. Anchor to the skull cap with cranioplastic cement to maintain patency of the holes during recovery and throughout the experiment. 6. Suture the skin incision with 5–0 black braided silk sutures. Treat rat with Baytril to prevent infection. 7. Allow animal to recover for a minimum of 1 week before attempting drug treatments (see Note 3). 8. In the week following surgery, in addition to monitoring weight gain, assess olfactory function in animals by observing preference for fruit-flavored snacks over normal chow. 9. At the end of the study, perform a gross examination of the nasal cavity on a representative number of animals to evaluate 308 M.C. Veronesi et al.

Fig. 1. Illustration of the delivery ports implanted into the nasal cavity of the rat. (a) Delivery ports (1) were implanted bilaterally, immediately posterior to the nasofrontal suture (2), allowing entry into the nasal cavity just anterior to the cribriform plate as shown from the dorsal view. (b) A mid sagittal view shows that the left nasal port (1) gains direct access to the nasal olfactory epithelium (3) from the outside when placed posterior to the nasof- rontal (2) suture. The olfactory epithelium allows access to the brain via the olfactory bulb (5) after passing through the cribriform plate (4). From ref. 42 with permission.

the placement of the nasal ports and look for evidence of olfac- tory epithelium pathology.

3.2. Intranasal 1. On the day of treatment, anesthetize both experimental and Administration control rats using 4% isoflurane from a TEC-4 Vaporizer con- nected to the anesthesia induction chamber. 2. Once the animals are fully anesthetized (loss of corneal and righting reflex, approximately 1 min), remove them from the induction chamber, and place on an electric heating pad to maintain body temperature at 37°C. Immediately place a non- rebreathing mask over the snout to continue anesthesia at 1.75% isoflurane. 3. Keep the animal in the supine position (Fig. 2) and administer treatments (25–50 ML), treatments into the posterior nasal 20 Neuropeptide Delivery 309

Fig. 2. Schematic of position of the rat during drug delivery. Note that the animal is supine allowing the olfactory neuroepithelium within the nasal cavity to be at the lowest point.

cavity through the implanted nasal ports using a sterile 100 ML Hamilton Syringe with 30 ga needle (see Notes 4 and 5). 4. Following delivery of the drug treatment, keep the animal in the supine position under anesthesia for 1–4 min to allow sufficient drug concentrations to absorb across the olfactory neuroepithelium (see Note 6). 5. Once the anesthetic is removed, place the animal back into its cage allowing a recovery time of about 3–5 min.

4. Notes

1. The use of general anesthetics may render our intranasal deliv- ery technique less than optimal if the goal is to assess physio- logical/behavioral effects with fast-acting drugs. However, we significantly attenuated seizure characteristics in the kindling animal model of epilepsy as early as 30 min after intranasal delivery of thyrotropin-releasing hormone (42). 2. The nasal cavity contains highly vascular mucosa and is subject to considerable bleeding with even minimal trauma. Care must be taken during surgery to avoid excessive trauma to the under- lying nasal mucosa upon drilling into the nasal cavity through the frontal bone just posterior to the nasofrontal suture. 3. Since the animals are rested for 1 week after surgery, the nasal cannulas may fill with hardened granulation tissue secondary to any mucous collection and bleeding that may have occurred because of the surgery. Removal of this tissue may elicit rebleed- ing similar to what happens when picking a scab off a wound. If this occurs frequently, we recommend placing a removable 310 M.C. Veronesi et al.

stop into the cannula immediately after surgery. Remember to run a test trial by removal of the stop 1 day prior to delivering drug treatments to ensure patency of the hole on the day of treatment. 4. During drug delivery, care must be taken to extend the syringe needle only slightly further than the length of the cannula not to penetrate the underlying nasal mucosa within the nasal cavity and thus cause trauma and bleeding. It may be best to customize the length of the syringe needle so that it can only extend at a fixed length of roughly 0.5 mm beyond the length of the nasal cannula. 5. Occasionally, reflux occurs with delivery of the drug treatment through a nasal cannula, especially in animals that have not received prior treatments. This occurs mostly when the can- nula hole is not patent either because the initial hole was not drilled full-thickness through the bone to gain entry into the nasal cavity or there is granulation material obstructing can- nula, preventing communication between the inside and out- side of the nasal cavity. 6. We found that delivery of no more than 50 ML of a substance containing the drug treatment allows the least chance for escape into the lungs/esophagus by way of the nasopalatine duct, which in supine animals lies superior to the cannula in the posterior nasal cavity.

References

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11. Hökfelt, T., Broberger, C., Xu, Z.Q., Sergeyev, 25. Veronesi, M.C., Aldouby, Y., Domb, A.J., and V., Ubink, R., and Diez, M. (2000) Kubek, M.J. (2009) Thyrotropin-releasing Neuropeptides--an overview. Neuropharmaco- hormone d,l polylactide nanoparticles (TRH- logy 39, 1337–1356. NPs) protect against glutamate toxicity in vitro 12. Strand, F.L. (2005) The neuropeptide concept and kindling development in vivo. Brain Res. and the evolution of neuropeptides In 1303, 151–160. Neuropeptides: Regulators of Physiological 26. Purves, D., Augustine, G.J., Fitzpatrick, D., Processes, ed. FL Strand, pp. 3–18. Cambridge: Hall, W.C., LaMantia, A.-S., McNamara, J.O., MIT Press. and White, L. (2008) Neuroscience. Sunderland, 13. Liu, X.F., Fawcett, J.R., Thorne, R.G., DeFor, MA. Sinauer Assoc. T.A., and Frey, W.H, 2nd. (2001) Intranasal 27. Gizurarson, S. (1990). Animal models for administration of insulin-like growth factor-I intranasal drug delivery studies. A review arti- bypasses the blood-brain barrier and protects cle. Acta Pharm. Nord. 2, 105–122. against focal cerebral ischemic damage. 28. De Rosa, R., Garcia, A.A., Braschi, C., Capsoni, J. Neurol. Sci. 187, 91–97. S., Maffei L, et al. (2005) Intranasal adminis- 14. Semkova, I., and Krieglstein, J. (1999) tration of nerve growth factor (NGF) rescues Neuroprotection mediated via neurotrophic recognition memory deficits in AD11 anti- factors and induction of neurotrophic factors. NGF transgenic mice. Proc. Natl. Acad. Sci. Brain Res. Brain Res. Rev. 30, 176–188. USA 102, 3811–3816. 15. Bjorbaek, C., and Kahn, B.B. (2004) Leptin 29. Frey, W. (2002) Intranasal delivery: bypassing signaling in the central nervous system and the the blood brain barrier to deliver therapeutic periphery. Recent Prog. Horm. Res. 59, agents to the brain and spinal cord. Drug Deliv. 305–331. Technol. 151, 66–77. 16. Gale, S.M., Castracane, V.D., and Mantzoros, 30. Ross, T.M., Martinez, P.M., Renner, J.C., C.S. (2004) Energy homeostasis, obesity and Thorne, R.G., Hanson, L.R., and Frey, W.H., eating disorders: recent advances in endocri- 2nd. (2004) Intranasal administration of inter- nology. J. Nutr. 134, 295–298. feron beta bypasses the blood-brain barrier to 17. Gentilucci, L. (2004) New trends in the devel- target the central nervous system and cervical opment of opioid peptide analogues as advanced lymph nodes: a non-invasive treatment strategy remedies for pain relief. Curr. Top. Med. Chem. for multiple sclerosis. J. Neuroimmunol. 151, 4, 19–38. 66–77. 18. Lim, K.C., Lim, S.T., and Federoff, H.J. (2003) 31. Thorne, R.G., and Frey, W.H., 2nd. (2001) Neurotrophin secretory pathways and synaptic Delivery of neurotrophic factors to the central plasticity. Neurobiol. Aging 24, 1135–1145. nervous system: pharmacokinetic consider- 19. Claes, S.J. (2004) Corticotropin-releasing hor- ations. Clin. Pharmacokinet. 40, 907–946. mone (CRH) in psychiatry: from stress to psy- 32. Thorne, R.G., Pronk, G.J., Padmanabhan, V., chopathology. Ann. Med. 36, 50–61. and Frey, W.H., 2nd. (2004) Delivery of insulin- 20. Datar, P., Srivastava, S., Coutinho, E., and like growth factor-I to the rat brain and spinal Govil G. (2004) Substance P: structure, func- cord along olfactory and trigeminal pathways tion, and therapeutics. Curr. Top. Med. Chem. following intranasal administration. 4, 75–103. Neuroscience 127, 481–496. 21. Strohle, A., and Holsboer, F. (2003) Stress 33. Alcalay, R.N., Giladi, E., Pick, C.G., and Gozes, responsive neurohormones in depression and I. (2004) Intranasal administration of NAP, a anxiety. Pharmacopsychiatry 36, S207–214. neuroprotective peptide, decreases anxiety-like behavior in aging mice in the elevated plus 22. Balasubramaniam, A. (2002) Clinical potentials maze. Neurosci Lett. 361, 128–131. of neuropeptide Y family of hormones. Am. J. Surg. 183, 430–434. 34. Gozes, I., Giladi, E., Pinhasov, A., Bardea, A., and Brenneman, D.E. (2000) Activity- 23. Binaschi, A., Bregola, G., and Simonato, M. dependent neurotrophic factor: intranasal (2003) On the role of somatostatin in seizure administration of femtomolar-acting peptides control: clues from the hippocampus. Rev. improve performance in a water maze. Neurosci. 14, 285–301. J. Pharmacol. Exp. Ther. 293, 1091–1098. 24. Kubek, M.J., and Garg, B.P. (2002) 35. Born, J., Lange, T., Kern, W., McGregor, G.P., Thyrotropin-releasing hormone in the treat- Bickel, U., and Fehm, H.L. (2002) Sniffing ment of intractable epilepsy. Pediatr. Neurol. neuropeptides: a transnasal approach to the 26, 9–17. human brain. Nat. Neurosci. 5, 514–516. 312 M.C. Veronesi et al.

36. Capsoni, S., Giannotta, S., and Cattaneo, A. 40. Vaccarezza, O.L., Sepich, L.N., and Tramezzani, (2002) Nerve growth factor and galantamine J.H. (1981) The vomeronasal organ of the rat. ameliorate early signs of neurodegeneration in J. Anat. 132, 167–185. anti-nerve growth factor mice. Proc. Natl. 41. Gao, X., Tao, W., Lu, W., Zhang, Q., Zhang, Acad. Sci. USA 99, 12432–12437. Y. et al. 2006. Lectin-conjugated PEG-PLA 37. Chen, X.Q., Fawcett, J.R., Rahman, Y.E., Ala, nanoparticles: preparation and brain delivery T.A., and Frey, I.W. (1998) Delivery of nerve after intranasal administration. Biomaterials growth factor to the brain via the olfactory 27, 3482–3490. pathway. J. Alzheimers. Dis. 1, 35–44. 42. Veronesi, M.C., Kubek, D.J., and Kubek M.J. 38. Gozes, I., Bardea, A., Reshef, A., Zamostiano, (2007) Intranasal delivery of a thyrotropin- R., Zhukovsky, S. et al. (1996) Neuroprotective releasing hormone analog attenuates seizures strategy for Alzheimer disease: intranasal in the amygdala-kindled rat. Epilepsia 48, administration of a fatty neuropeptide. Proc. 2280–2286. Natl. Acad. Sci. USA 93, 427–432. 43. Meredith, M. (2001) Human vomeronasal 39. Illum, L. (1996) Nasal delivery. The use of ani- organ function: A critical review of best mal models to predict performance in man. and worst cases. Chem. Senses 26, J. Drug Target 3, 427–442. 433–445. Chapter 21

Prodrug Design for Brain Delivery of Small- and Medium- Sized Neuropeptides

Katalin Prokai-Tatrai and Laszlo Prokai

Abstract

The blood–brain barrier (BBB) represents multiple barriers for drug delivery from the circulation. Peptides potentially useful to treat maladies of the brain are especially limited in their ability to cross the BBB due to several shortcomings. Specific delivery strategies have been conceived to outwit the BBB to target neuropeptides into the brain. It should be noted, however, that no unified method is possible for true brain-targeting of these fascinating biomolecules due to their structural features, properties, and intricate interplays among factors governing their entrance into and retention within the brain. In most brain- targeting prodrug approaches, a lipophilic and bioreversible moiety(ies) is covalently attached to the peptide that results in the complete loss of the innate biological activity of the parent peptide (prodrugs are inactive per definition) but significantly improves brain uptake and metabolic stability in the plasma and the inter- stitial fluid. Once the peptide prodrug has crossed the BBB, specific enzymes liberate the parent agent from its prodrug in the brain. To illustrate the applicability of the prodrug strategy for brain delivery of 2 small neuropeptides, pGlu-Glu-Pro-NH2, [Glu TRH], a thyrotropin-releasing hormone (TRH) analogue with a vast array of central activities, was chosen as an example. An ester prodrug provided significantly improved brain delivery compared to the unmodified parent peptide. The synthesis, in vitro and in vivo evaluations of this prodrug as specific examples are given for typical exploratory prodrug validation.

Key words: Blood–brain barrier, Neuropeptide, Prodrug, Solid-phase peptide synthesis, Lipophilicity, Immobilized artificial membrane chromatography, Bioactivation, Neuropharmacology, Analeptic effect, Thyrotropin-releasing hormone analogue

1. Introduction

Neuropeptides have extraordinary potential to treat various diseases and disorders of the brain; many of them are significant unmet medical needs (1). Our inability to effectively deliver these agents into and/or maintain therapeutic concentration within the central nervous system (CNS), however, prevents practical drug development for neuropeptides. Neuropeptides, or drugs in general, face a

Adalberto Merighi (ed.), Neuropeptides: Methods and Protocols, Methods in Molecular Biology, vol. 789, DOI 10.1007/978-1-61779-310-3_21, © Springer Science+Business Media, LLC 2011 313 314 K. Prokai-Tatrai and L. Prokai

formidable obstacle in reaching the brain mainly due to the existence and peculiar nature of the blood–brain barrier (BBB) that is a vital element in the regulation of the inner sanctuary of the brain (2). The existence of the blood–cerebrospinal fluid barrier (CSFB) as an additional barrier for drug transport from the systemic circula- tion should also be recognized; however, since the surface area of the BBB is significantly larger than CSFB, the BBB represents the most important barrier between the brain and the blood for drug candidates (3). The BBB essentially behaves like a continuous lipid bilayer and thus exhibits low permeability to hydrophilic and/or large substances that do not have specific transport mechanisms. Consequently, hydrophilic molecules such as peptides potentially useful for combating diseases affecting the brain cannot cross the BBB in pharmacologically significant amounts. Highly active enzymes also represent a metabolic component that contributes to the homeostatic balance regulated by the BBB (4). Most peptides are especially susceptible for recognition by peptidases and thus for enzymatic degradation in the BBB, e.g., in the cytosolic endothelial space; therefore, their biological half-lives are very short, which further limit their practical drug development. Efficient efflux pumps are also present in BBB that transport a wide range of structurally diverse compounds out of the CNS and may also contribute to the exclusion of certain drug candidates from the brain (5). In all, BBB represents a multitude of barriers for drug entry into the brain, and consequently only less than 2% of small molecule drugs and practically no large-molecule drugs can penetrate through the BBB (6). To overcome or, rather, outwit the BBB, invasive and noninva- sive approaches have been developed for drug delivery (2). The high-risk (neurosurgical) invasive strategies go around the BBB or alter/modify it to provide entry into the interstitial fluid of the brain. Noninvasive techniques either exploit various transport processes that exist in the brain capillary endothelium to ferry therapeutic agents into the brain after systemic administration or transiently manipulate drug properties for enabling its BBB trans- port. The latter approach is the so-called prodrug strategy (7) that offers perhaps the most versatile chemical manipulation strategy for drug delivery into the brain. This technique relies on the bioreversible, thus, transient alteration of the target peptide with “pro” moiet(ies) to produce its inactive/inert (bio)precursor having no intrinsic biological activity but improved physicochemical characteristics for BBB-transport compared to those of the parent peptide (8). Prodrugs are almost exclusively aimed at increasing a peptide’s attraction to the lipoidal BBB (i.e., making the peptide prodrugs “lipophilic” or “neutral”) to reach the brain via diffusion, also referred to as passive transport, although “promoieties” that rely on carrier-mediated (active) transport have also been reported (9). Once the prodrug has crossed the BBB, conversion by “post barrier” 21 Brain Delivery of Neuropeptides 315

CNS-enzyme(s) is utilized to remove the “pro” moiety(ies) to regenerate the parent peptide at the site of action. The restriction of the prodrug approach to small neuropeptides is a practical hint; the capacity of chemical modifications to modify physicochemical properties decreases significantly as the size of the peptide increases, and therefore, the impact of making prodrugs to enhance peptide transport across the BBB may diminish progressively with increasing molecular size. Bioreversible alteration of the poorly CNS-available neuropep- tides to make their prodrugs involves organic/peptide chemistry taking advantage of the inherently present functional group(s) in a peptide, such as the amino- and carboxyl-termini or side-chain functional groups (e.g., amino-, hydroxyl-, or carboxyl group) (8). Therefore, typically we can create esters, ethers, phosphates, carbam- ates, amides, imines, oximes, N-Mannich bases, etc. of a peptide to obtain potential prodrugs. Sometimes uncommon functional groups can also be utilized for prodrug creation. The appropriate transient masking of these polar groups by a “promoiety” also results in decreased hydrogen-bonding capacity and renders the prodrug “neutral” at physiological pH, which is a prerequisite for passive transport across the BBB. Additionally, precise placement and choice of the cleavable “promoieties” can also provide protection against exo- and endopeptidases present in the blood where many small peptides with free N- and C-termini are degraded primarily by exopeptidases usually within a few minutes. Protection against peptidase recognition in blood is important because even if peptide prodrugs can cross the BBB, they can only sustain adequate concentration in the brain if their blood concentration is maintained at sufficiently high levels by preventing their systemic degradation (10). The synthesis of a peptide prodrug can be entirely solid-phase based (the desired product is assembled while anchored to a solid support such as polystyrene beads with 1% divinylbenzene, as a cross- linking agent known as “resins”) (11) or solution-phase based; in this case, immobilized reagents can also be used (12) or the combi- nation of solid-(assembly of the peptide) and solution-phase (preparation of the prodrug from the peptide) (13). Altogether, a rich array of chemistry can be considered to create many potential prodrugs from a neuropeptide (8). Frequently, however, not only the chemistry driving prodrug design is complicated but also the in vivo prodrug activation for the liberation of the parent neu- ropeptide relies on several enzymes by sequential metabolism (8, 13, 14). Therefore, a simple example chosen for this chapter to recommend methods and protocols to create and evaluate prodrugs is intended to those new to the field. Not all intended peptide “prodrugs” would qualify as prodrugs. By definition, prodrugs should be inactive (7), which should be tested, if possible, in an appropriate receptor binding, enzyme inhibition or a similar biological assay in vitro. However, many 316 K. Prokai-Tatrai and L. Prokai

neuropeptides have pleiotropic action, and their cognate receptors, neurobiological pathways, and structure–activity relationships have been often poorly understood to unequivocally accomplish or assess complete lack of activity by their presumed prodrugs synthesized through selected chemical modifications. Strategies directed to discover prodrug-amenable neuropeptide analogues (often implicating “pro” moieties that would promote retention in the CNS after in situ prodrug to drug conversion while accelerating elimination from the systemic circulation) deserve consideration (8, 11, 13, 15), although the detailed coverage of principles and experimental methods of this approach was beyond the scope of the present introductory chapter on the use of neuropeptide prodrugs. Experimental evaluation of prodrugs designed and synthesized for delivering a peptide into the CNS should address their BBB transport. Computational (in silico) methods (16), predicting measurements in models distributing the prodrug between a membrane-mimicking and an aqueous phase (17, 18), in vivo techniques measuring transport across artificial membranes (19) or monolayers of cultured brain capillary endothelial cells (20), and in vivo experiments determining access of the neuropeptide to the brain versus peripheral tissue by, e.g., a brain uptake index (BUI) (21) or actually measuring peptide concentrations reached in the CNS and in the circulating blood (16, 22) have been employed. This order of methods reflects their increasing experimental requirements (in time and costs, as well as specialized expertise and experience necessary) and decreasing throughput. At present, in silico methods do not predict BBB transport reliably for peptide prodrugs (16, 17), and therefore, the use of experimental tech- niques is needed. Passive transport through the BBB is controlled by several physicochemical parameters including molecular size (more exactly molecular volume), charge, and hydrogen-bonding (donor or acceptor) capacity. Nevertheless, lipophilicity (frequently expressed as the logarithm of the n-octanol–water partition coefficient, logP) has been generally considered the most important indicator for BBB penetration and it also plays a pivotal role in predicting the absorption, distribution, metabolism, and elimination (ADME) of therapeutic drugs. A logP value of around 2 (i.e., 100-times higher affinity to the lipid-mimicking n-octanol than to water) is believed to be an optimal value for CNS-delivery by passive trans- port (23, 24). However, immobilized artificial membrane chroma- tography (IAMC) mimics membrane interactions better than partitioning in the isotropic n-octanol–water system when logP is used (18), and the technique has been applied to the assessment of BBB penetration for structurally diverse compounds including prodrugs of CNS-active peptides (8, 12, 13, 16). 21 Brain Delivery of Neuropeptides 317

Scheme 1. A simplified mechanism of CNS-enhanced peptide delivery by the prodrug approach after systemic adminis- tration (2).

An additional criterion of a prodrug is the need for its in vivo conversion at the site of action to elicit the desired biological activity (7, 8). When this bioactivation occurs only at a negligible rate, a prodrug may not realize the promise of improving drug delivery. When the intended site of action is the CNS, the prodrug should also be more resistant to conversion to the biologically active mol- ecule in the systemic circulation compared to that in the central compartment, as illustrated in Scheme 1. The assessment of these desired metabolic properties is usually performed in vitro using tissue sections or homogenates, body fluids such as plasma etc., commonly obtained from experimental animals (10). Chemical stability in a suitable pharmaceutical vehicle is also required for practical applications. In vivo testing of CNS-mediated pharmacological effect(s) after systemic administration of the synthesized prodrugs has nevertheless remained an essential tool to evaluate their potential for practical applications. Designing these experiments requires the selection of pharmacological paradigms appropriate to reflect the CNS activity of the target (parent) neuropeptide to substantiate the successful delivery of the parent peptide into the CNS via its prodrug(s). A typical poorly CNS-available peptide with a vast

array of central effects is pGlu-Glu-Pro-NH2, also known as [Glu2]TRH. This endogenous peptide is structurally related to 318 K. Prokai-Tatrai and L. Prokai

thyrotropin-releasing hormone (TRH, pGlu-His-Pro-NH2), and although originally identified from rabbit prostate, it has been shown to occur also in the mammalian brain (25). TRH and struc- turally related compounds have been implicated in the manage- ment of various neurological and neuropsychiatric disorders such as depression, epilepsy, brain injury, Alzheimer’s disease, and schizo- phrenia, as well as stimulation of spinal-cord motor neurons (26). The best-known neuropharmacological effect of these molecules is analepsia manifested by the reversal of drug-induced sleeping time. The analeptic effect is a frequently used paradigm for testing TRH-related compounds in early-phase drug development (27). [Glu2]TRH shares many beneficial central effects of TRH, but does not evoke endocrine response. The pharmacological responses after treatment with [Glu2]TRH are also more robust and/or longer lasting compared to the mostly transient response exerted by TRH. These advantages are due, in part, to the increased resistance of [Glu2]TRH to serum enzymes responsible for the rapid degrada- tion of TRH in vivo (28). [Glu2]TRH is a small highly hydrophilic compound and mainly ionized at physiological pH on the J-carboxyl group of the central Glu residue, which hinders its diffusion into the CNS. Therefore, the prodrug approach to render this molecule “neutral” and more lipophilic for adequate CNS-transport is an obvious choice (12). This neuropeptide also represents probably one of the simplest examples through which methods and protocols to create and evaluate prodrugs for brain delivery of neuropeptides can be introduced to those aspiring to become familiar with the subject. The side-chain carboxyl group of the central Glu offers an easy handle for a transient chemical modification, especially for ester type of prodrug creation using various alcohols. This, under physi- ological conditions negatively charged side chain of the central

residue in pGlu-Glu-Pro-NH2 has been believed to be important to confer the peptide its characteristic neurochemical and neu- ropharmacological properties (29, 30); hence, blocking the charge by forming an ester on the side chain of the Glu residue and thereby eliminating charge conceivably creates a prodrug (12). Many esters of [Glu2]TRH can be prepared using aliphatic and aromatic alcohols as potential prodrugs. However, we have previ- ously found that the n-hexyl ester of this peptide afforded the most

promising compound (kcIAM = 16.0 versus kcIAM = 0 for the unmodi- fied peptide), as far as membrane affinity predicting BBB transport is concerned (12). In vitro stability studies showed that this pep- tide ester’s half-life in mouse brain homogenate was 22 min and, upon its intravenous administration (i.v., into the tail vein of mice), a statistically significant reduction in the pentobarbital-induced sleeping time (a measure of analeptic effect) was measured that was the most profound among all the studied peptide esters. Moreover, administration of this prodrug brought about a tenfold increase 21 Brain Delivery of Neuropeptides 319

in efficacy compared to the unmodified parent peptide (12). The effective dose giving 50% of the maximum analeptic response

(ED50) was 2.4 ± 1.1 Pmol/kg for the prodrug, while EC50 for the parent [Glu2]TRH was 23.2 ± 5.6 Pmol/kg. Therefore, even a sim- ple prodrug such as the hexyl ester of this peptide may be a useful agent for potential neurotherapy utilizing [Glu2]TRH.

2. Materials

2.1. Prodrug Synthesis 1. N,N c-dimethylformamide (DMF), reagent or peptide synthesis grade. 2. Methanol, reagent or peptide synthesis grade. 3. Trifluoroacetic acid (TFA), peptide synthesis grade. 4. Piperidine, peptide synthesis grade. 5. Anhydrous dichloromethane. 6. Fmoc-Pro-Rink-MBHA resin (loading: 0.48 mmol/g or similar). 7. N-D-Fmoc-N-J-allyl-glutamic acid (Fmoc-Glu(All)-OH; All

denotes “allyl”; CH2=CH–CH2-group). 8. pGlu. 9. Benzotriazole-1-yloxy-tris-pyrrolidinophosphonium hexafluo- rophosphate (PyBOP). 10. 1-Hydroxybenzotriazole (HOBt). 11. N,Nc-diisopropyl-ethylamine (DIPEA).

12. Pd(PPh3)3.

13. PhSiH3. 2 14. [Glu ]TRH (pGlu-Glu-Pro-NH2) as reference peptide, from commercial vendors (e.g., Sigma Chemicals) or custom syn- thesized (see Subheading 3.1 and Note 8). 15. Manual synthesis reaction vessel (15 mL) (Anaspec Inc.) (see Note 1). 16. Analytical balance AL104 (Mettler-Toledo, Inc., Columbus, OH) or similar. 17. Argon or nitrogen tank.

2.2. High-Performance 1. Preparative high-performance liquid chromatography (HPLC) Liquid column: Reversed-phase (RP) RCM DeltaPack C18, Chromatography and 100 mm × 25 mm i.d., 5-Pm particles (Waters, Milford, MA). Immobilized Artificial 2. Analytical RP-HPLC column: Econosphere C18 Membrane (150 mm × 4.6 mm i.d., 5-Pm particles with 80 Å pore size) Chromatography (Grace, Deerfield, IL) fitted with an Econosphere All-Guard 320 K. Prokai-Tatrai and L. Prokai

(7.5 mm × 4.6 mm i.d., 5-Pm particles) C18 cartridge (Grace). 3. IAMC: 30 mm × 4.6 mm i.d. IAM.PC.DD2 column (12-Pm particles with 300 Å pore size) (Regis Technologies, Morton Grove, IL) protected with an IAM.PC.DD2 guard cartridge, 10 mm × 3.0 mm i.d. (Regis Technologies). 4. Water, HPLC-grade.

5. Acetonitrile (CH3CN), HPLC-grade. 6. TFA high purity, in 1-mL ampoules (Thermo Fisher Scientific, Waltham, MA) (see Note 2).

7. Ammonium acetate (NH4OAc, HPLC-grade) (Thermo Fisher Scientific). 8. Glacial acetic acid, HPLC-grade (Thermo Fisher Scientific). 9. Wilmad-Labglass Mobile Phase System Filter/Degas, 1 L (available from Thermo Fisher Scientific), or similar vacuum filter/degas system. 10. Nalgene® Aspirator Vacuum Pump (available from Thermo Fisher Scientific), or similar oil-free vacuum pump. 11. Barnstead® Economy Filters and Holders, 0.45-Pm pore size (available from Thermo Fisher Scientific), or equivalent. 12. Wilmad-Labglass HPLC Mobile Phase Reservoir (Single Cavity), 1,000 mL capacity (available from Thermo Fisher Scientific), or equivalent. 13. Blunt-tip syringes: 1 mL (1001RNR, Hamilton, Reno, NV) and 100 PL volume (1710N, Hamilton), or equivalent, for manual HPLC injections. 14. Mass spectrometer: LCQ (Thermo Fisher Scientific) or similar routinely available mass spectrometer configured for manual sample injections. 15. Lyophilizer: FreeZone 4.5-L Benchtop Freeze Dry System (available from Labconco Corp., Kansas City, MO), or similar system. 16. HPLC pump (e.g., SpectraSystem P1000 available from Thermo Fisher Scientific).

2.3. In Vitro Metabolic 1. Typically, rodents (rats or mice) are used for initial in vitro met- Stability Studies abolic stability studies and in vivo neuropharmacological evalu- ations. The procedures below assume the use of male Swiss Webster mice (30 ± 2 g body weight) obtained from Charles River Laboratories (Wilmington, MA) or similar vendors. The animals should be housed no more than five per cage with free 21 Brain Delivery of Neuropeptides 321

access to water and food with a normal day/night cycle. Each animal should be tested only once (see Notes 3 and 4). 2. Classic guillotine and surgical scissors (available from Kent Scientific Corp., Torrington, CT). 3. 6-mL plasma determination tubes containing sodium heparin (BD Vacutainer, Preanalytical Solutions, Franklin Lakes, NJ). 4. Potter-Elvehjem tissue grinder (Wheaton, Millville, NJ). 5. Motor-driven overhead stirrer (Wheaton). 6. General laboratory centrifuge (e.g., GLC-1 from Sorwal, Newtown, CT). 7. Precision balance (to weigh the animals) and analytical balance (available from Mettler-Toledo, Inc.). 8. Phosphate-buffered saline solution (PBS, pH = 7.4) (available from Sigma). 9. Shaking water bath with temperature control (e.g., Precision Dubnoff Reciprocal Shaking Baths, from Thermo Fisher Scientific). 10. Eppendorf tubes: e.g., Plastibrand Microcentrifuge Tubes (Sigma). 11. Digital single-channel pipette and disposable pipette tips. 12. Vortex mixer. 13. Analytical RP-HPLC with UV detection (O = 215 nm). 14. Water, HPLC-grade.

15. CH3CN, HPLC-grade. 16. RP-HPLC column (see Subheading 2.2, item 2). 17. TFA (high purity, in 1 mL ampoules, Thermo Fisher Scientific) (see Note 2).

2.4. Measuring 1. Broome Rodent Restrainers (Harvard Apparatus, Holliston, of Analeptic Effect MA) or similar. 2. Heat lamp. 3. Disposable insulin syringe 26 or 27 G. 4. Timer, such as count up/down timer clock (Sigma). 5. Alcohol swabs or spray bottle. 6. Paper towel. 7. Small boxes to keep the sleeping animal during measurement of the sleeping time (e.g., Thermo Scientific freezer fiberboard storage boxes; 5¼ L × 5¼ W × 2 in. H; from Thermo Fisher Scientific). 8. Sodium pentobarbital (see Note 5). 322 K. Prokai-Tatrai and L. Prokai

3. Methods

An ester prodrug of pGlu-Glu-Pro-NH2, a peptide structurally related to thyrotropin-releasing hormone (TRH, pGlu-His-Pro- 2 NH2) and also known as [Glu ]TRH, is used to illustrate the appli- cability of the prodrug strategy for CNS-delivery of small neuropeptides (12). Specific methods address synthesis, predictive evaluation of transport properties and bioactivation, and assessment of CNS-delivery through a pharmacological paradigm. Solid-phase peptide synthesis (SPPS) has become the most successful method to synthesize peptides since its introduction by Bruce Merrifield in 1963 (31). SPPS also offers a convenient way to produce small peptide prodrugs. SPPS involves (1) chain assembly, (2) cleavage from resin (and concurrent removal of side-chain protecting group(s)), if present, (3) purification, (4) additional chemical reac- tion (if for example prodrug is prepared in solution phase from the parent peptide obtained by SPPS), and (5) characterization. It should be noted that steps (3) and (4) can be switched, since, in most cases, the crude peptide cleaved from the resin is sufficiently pure for prodrug creation. In SPPS, a peptide is assembled from the C-terminus towards the N-terminus while the D-carboxyl group of the amino acid is attached to a solid support (resin). For most amino acids, resins with a protected amino acid already anchored to them (“loaded”) are available commercially. Otherwise, the C-terminal amino acid needs to be “loaded” to the resin. In any case, the D-amino group of the amino acid to be coupled to the C-terminal amino acid immobilized to a solid support (or to the resin if “load- ing” is needed) must be protected during this reaction. Most fre- quently, Fmoc (9-fluorenylmethoxycarbonyl) group is used for this purpose that can be removed via base-induced E-elimination, usu- ally in the presence of secondary amines, such as piperidine (32). Before coupling, the carboxyl group of this N-protected amino acid must be “activated.” While various agents can be used for this pur- pose, the use of “onium” salt-based couplings (as described in Subheading 3.1) is especially popular (33). SPPS is also suitable to directly generate simple prodrugs of small peptide on a solid sup- port (11), such as the hexyl ester of [Glu2]TRH (see Scheme 2). This is a specific example that enhances CNS-delivery of a small neuropeptide via a simple yet effective ester-type prodrug that undergoes in vivo enzymatic hydrolysis by esterases to afford the parent peptide in the brain, the site of action. Standard Fmoc-based SPPS using PyBOP/HOBt/DIPEA (1:1:2) activation is used to construct the model peptide prodrug. Washings between synthetic steps (deprotection and coupling) are done with DMF (5× 30 s), methanol (MeOH, 5× 30 s), and DMF (5× 30 s), successively, using 6 mL of solvent/g resin each time. Solvents and soluble reagents are removed by suction. Removal of 21 Brain Delivery of Neuropeptides 323

the Fmoc protecting group at the beginning of each coupling is done with 20% (v/v) piperidine in DMF. For “standard” coupling step, the corresponding Fmoc-protected amino acid (4 eq), PyBOP (4 eq), and HOBt (4 eq) dissolved in DMF (6 mL/g resin) are sequentially added to the resin, followed by DIPEA (8 eq). The mixture is allowed to react with intermittent manual stirring for 2 h. The solvent is then removed by suction and the resin is washed thoroughly, and for the qualitative monitoring of the coupling reaction a color test, most frequently the Kaiser test (34), is applied. If necessary, the coupling procedure is repeated again (“double coupling”) (see Note 6). IAMC measures the partitioning into monolayers of cell mem- brane phospholipids immobilized by covalent binding on silica

particles. The chromatographic capacity factor (kcIAM) for a compound obtained by IAMC is directly related to its partition coefficient between the aqueous phase and the chemically bonded membrane

phase and, ultimately, to the Km value representing its fluid-membrane

partition coefficient (20). Essentially, an increase in the kcIAM indi- cates an increase in the membrane permeability of the compound and may also be used to predict BUI (16). The selected simple model peptide, [Glu2]TRH, evokes a dose- dependent analeptic activity (12, 31) whose assessment is described as an example for a specific procedure involving neurophar-macology.

3.1. Prodrug Synthesis (Example, see Scheme 2 for illustration of the synthetic procedure.) 1. Resin conditioning/swelling: Place 500 mg of dry Fmoc-Pro- Rink Amide-MBHA resin (0.48 mmol/g, 0.24 mmol, 1 eq) into the reaction vessel and wash it with DMF (3 mL, 5× 30 s) to “swell” the resin. Use intermittent stirring or agitate the reaction vessel to assure a complete mixing. Remove solvent. 2. Deprotection: Add 5 mL of 20% (v/v) piperidine in DMF to the resin for 2× 1 min, then for 1× 15 min to remove Fmoc. Use intermittent stirring or agitate the reaction vessel. 3. Wash the resin as specified above (DMF, 5× 30 s, MeOH, 5× 30 s and DMF, 5× 30 s) to remove piperidine. The last washing must be neutral. 4. Coupling: Add Fmoc-Glu(All)-OH (393 mg, 0.965 mmol,

4 eq), PyBOP (502 mg, 0.965 mmol, 4 eq) and HOBt × H2O (148 mg, 0.965 mmol, 4 eq) dissolved in 3 mL of DMF to the resin, followed by DIPEA (253 mg, 341 PL, 8 eq). Allow the mixture to react with intermittent manual stirring for 2 h. 5. Monitor the coupling efficiency; repeat the same coupling (double coupling) as above, without removing the Fmoc group, if it is necessary. Then, remove the solvent (see Note 6). 6. Perform deprotection and wash. 324 K. Prokai-Tatrai and L. Prokai

2 Scheme 2. Solid-phase synthesis of pGlu-Glu(Hex)-Pro-NH2, a prodrug of [Glu ]TRH. (i) 20% (v/v) piperidine in DMF,

(ii) PyBOP:HOBt:Fmoc-Glu(All):DiPEA in DMF (1:1:1:2); (iii) PyBOP:HOBt:pGlu:DiPEA in DMF (1:1:1:2); (iv) Pd(PPh3)3:PhSiH3

(1:100) in DCM; (v) n-Hexanol: DIPCDI: DMAP (10:10:1) in DMF, (vi) TFA:H2O (98:2, v/v).

7. Coupling: Add the C-terminal amino acid, pGlu (125 mg, 0.965 mmol, 4 eq), PyBOP (502 mg, 0.965 mmol, 4 eq) and

HOBt × H2O (148 mg, 0.965 mmol, 4 eq) dissolved in 3 mL of DMF to the resin, followed by DIPEA (253 mg, 341 PL, 8 eq). Allow the mixture to react with intermittent manual stirring for 2 h. 8. Monitor the coupling efficiency with ninhydrin test. If it is necessary, use double coupling and then wash (see Note 6). 21 Brain Delivery of Neuropeptides 325

9. Remove “All” protecting group of central Glu: add Pd(PPh3)4

(28 mg, 0.024 mmol, 0.1 eq) and PhSiH3 (295 PL, 2.41 mmol, 10 eq) in anhydrous dichloromethane (DCM, 3 mL) under inert atmosphere (argon or nitrogen) for 15 min. Repeat the procedure two more times. Wash the resin (see Note 7). 10. Perform esterification of the central Glu: add n-hexanol (302 PL, 2.41 mmol, 10 eq), 1,3-diisopropylcarbodiimide (DIPCDI, 373 PL, 2.41 mmol, 10 eq) and 4-(dimethylamino) pyridine (DMAP, 29 mg, 0.24 mmol, 1 eq) dissolved in DMF (3 mL) to the resin. Allow the mixture to react with intermit- tent manual stirring for 30 min. Repeat the procedure one more time (see Note 8). 11. Wash the resin with DMF (5× 30 s), MeOH (5× 30 s) and finally with DCM (5× 30 s). Dry the resin with suction and a gentle stream of nitrogen (or in vacuo). 12. Remove the peptide ester from the solid support: place the dried peptide-resin in a small flask equipped with a micro stirbar. Add 5 mL of TFA–water–triisopropylsilane 95:2.5:2.5 (v/v) (see Notes 2 and 9). Stir the reaction mixture for 1 h. Filter the reaction mixture through a fritted disk funnel of medium porosity. Wash the remaining resin with approximately 1 mL TFA and filter into the reaction mixture. Concentrate the solu- tion with a gentle stream of nitrogen and precipitate the peptide with ice-cold diethyl ether. 13. Isolate the peptide by filtration or centrifugation. Wash the solid with ether and, after drying, analyze the crude product by mass spectrometry (MS) and purify it by semipreparative RP-HPLC (see Note 10). 14. After RP-HPLC purification (35) combine the fractions con- taining the product and obtain solid by lyophilization. 15. Product identification can be done by mass spectrometry (36) and NMR spectroscopy. 16. The purity of peptide can be checked by combustion analysis, analytical RP-HPLC, LC-MS, and NMR spectroscopy. Obtain

formula (CxHyNzOw × nCF3OOH × mH2O) from combustion analysis, and use formula weight for calculating quantities nec- essary to make stock solutions used in the procedures (see Note 11).

3.2. Immobilized These instructions assume the use of atmospheric-pressure chemical Artificial Membrane ionization (APCI) MS detection on a quadrupole ion trap instru- Chromatography ment, such as the LCQ. The assay is readily adaptable to electro- spray ionization (ESI) (37) that also enables the coupling of LC-based separations and MS, as well as for using mass spectro- meters equipped with ESI and APCI sources but sold by other vendors. Autosampler may also be used and samples in this case should be 326 K. Prokai-Tatrai and L. Prokai

supplied in appropriate vials, well-plate, etc., required by the instru- ment. Alternatively, ultraviolet (UV) spectrophotometric detection at 215 nm can be used, and some changes in the IAMC conditions may be made, e.g., using PBS as a mobile phase, albeit addition of acetonitrile may be required to obtain acceptable chromatographic behavior (38). However, coupling IAMC with MS involving sensi- tive and selective atmospheric-pressure ionization (API) methods allows for the analysis of compounds without adequate UV chro- mophore, for screening crude products, and for a simultaneous analysis of several prodrugs to increase throughput (38). The method uses isocratic conditions; therefore, a relatively inexpensive HPLC pump will suffice. Except for purging and flow control on the pump, all other operations are performed via the mass spec- trometer’s personal computer-based tuning and data acquisition (Tune Plus), as well as data processing programs (Xcalibur). Tuning and calibration of the instrument should be done according to the manufacturer’s instruction. 1. Preparation of the mobile phase: dissolve 771 mg ammonium acetate in 900 mL of HPLC-grade water in a 1-L volumetric cylinder. 2. After the white crystals dissolve upon gentle shaking, fill up the volume to about 990 mL and bring pH to 5.4 by drop wise addition of glacial acetic acid. 3. Fill up the volume to 1 L, and filter the solution through Barnstead® economy filter in a Wilmad-LabGlass mobile phase filter/degas system connected to an aspirator vacuum pump attached directly to a sink faucets for pumping. 4. Save a small volume (e.g., 25 mL) of the filtered/degassed mobile phase to make solutions of the samples, fill the rest into its reservoir (1-L single-cavity Wilmad-LabGlass) and submerge the inlet line of the pump (fitted with a 0.2-Pm in-line stainless- steel filter) into the mobile phase. Observe guidelines for the care of IAM columns, which are available online (38). 5. Connect the column to the appropriate port of the built-in valve of the instrument (configured for injection), purge the pump by operating the purge valve according to the instructions by the manufacturer, and turn the pump to “run” mode at 1.0 mL/min flow without connecting the column outlet to the mass spectrometer’s ion source. 6. While the column is equilibrating (10 min recommended), prepare the instrument to run the samples through the data acquisition module (Xcalibur) according to the instructions by the manufacturer, assuming a 10-min total run time initially (the latter can be shortened or extended, when required by high or low prodrug affinity to the immobilized lipid of 21 Brain Delivery of Neuropeptides 327

the column). Mass range should be selected to cover the molecular-weight range of the compounds to be analyzed (e.g., m/z 300–600 for [Glu2]TRH and its ester prodrugs). In the tune file associated with the APCI method, set the vaporizer and capillary temperature to 450 and 150°C, respec- tively, the discharge current to 5 PA, the sheath and auxiliary gas (nitrogen) flow at 40 and 10 arbitrary units, respectively (see parameters in ref. 38 for ESI conditions). 7. Connect the column to the mass spectrometer’s ion source and let the system stabilize for 5 min. Prepare an approximately 0.1% (w/v) solution of [Glu2]TRH and the prodrug(s) using the saved portion of the mobile phase as the medium. With the injector valve in the “load” position, fill aliquots of the sample solution into the loop in the valve using the blunt-tip 100 PL Hamilton syringe (see Note 12) and, then, initiate the run by the instrument’s acquisition program (Xcalibur). 8. From the acquired raw data file, reconstruct the selected-ion monitoring (SIM) chromatogram(s) for the compound of interest(s) by using the “Qual Browser” function of Xcalibur. Supply the molecular mass (monoisotopic, one decimal point accuracy on an ion trap instrument) plus 1.0 to obtain the SIM-trace for the protonated molecule (MH+) of the peptide or prodrug analyzed (see Note 13). Employ digital smoothing of the chromatograms, if necessary, and obtain retention

times (tR) from them. Calculate IAMC capacity factors (kcIAM)

as follows: kcIAM = (tR(X) − tR(V))/tR(V), where tR(X) and tR(V) are the retention times for the compound of interest and the void volume marker, [Glu2]TRH, respectively (see Note 14). An example is shown in Fig. 1.

3.3. In Vitro Metabolic 1. Euthanize the animals with cervical dislocation (see Note 15). Stability Studies 2. Decapitate the animals by putting the head of the animal 3.3.1. Preparation between the guillotine blades and then separate the head from of Tissues the trunk using a rapid movement of the guillotine arm. Alternatively, the head can be removed with a surgical scissor. 3. Collect the trunk blood into a 6-mL plasma determination tube containing sodium heparin. 4. Prepare plasma without delay by centrifugation at 1,500 × g for 10 min using a general laboratory centrifuge. 5. Open the skull with a surgical scissor and remove the brain, weigh it and transfer it into a 15-mL glass Potter-Elvehjem tis- sue grinder placed in an ice bath. 6. Add an ice-cold PBS volume in mL equivalent to four times of the weight of the brain in grams to make a 20% w/v brain homogenate. 328 K. Prokai-Tatrai and L. Prokai

Fig. 1. Illustration of the procedure for obtaining capacity factors by IAMC coupled with APCI mass spectrometric detection 2 through postacquisition processing of recorded full-scan mass spectra: (a) [Glu ]TRH (k cIAM = 0; no affinity to immobilized 2 lipid phase); (b) the prodrug, [Glu(Hex) ]TRH (k cIAM = 16.0 indicating a significant increase in such affinity). The SIM traces + + were extracted for the molecular ions ([M+NH4] and MH , respectively) circled on the APCI mass spectra.

7. Prepare brain homogenate by running the ball-shaped Teflon pestle of the grinder, with the steel shaft connected to a motor- driven overhead stirrer, for 80 s at 1,500 rpm. 8. Use the brain homogenate and plasma immediately for the in vitro stability studies.

3.3.2. In Vitro Stability 1. Pre-warm the brain homogenate or plasma for 3 min in a Studies temperature-controlled, shaking water bath set at 37°C. 21 Brain Delivery of Neuropeptides 329

2. Add approximately 100 PM; thus, 100 nmol of test compound to 1 mL of mouse plasma or brain homogenate (20%, w/w, pH 7.4, in phosphate buffer). For example, in case of the prodrug (M approximately 438, see Subheading 3.1, step 15), this concentration roughly corresponds to 50 Pg prodrug/mL tissue. Accordingly, make a 10 mg/mL stock solution of the prodrug in saline (e.g., dissolve 1 mg prodrug in 100 PL saline) then add 5 PL of stock solution into the tissue and vortex. 3. Incubate the mixture at 37°C in a shaking water bath. Take aliquots (100 PL) after 5, 15, 30, 60, and 120 min of incuba- tion, and transfer them into separate 1.5-mL Eppendorf tubes containing 200 PL of ice-cold solution of 5% (v/v) acetic acid in acetonitrile. Vortex and centrifuge the samples, then remove the supernatant for analyses by RP-HPLC to record the decline in the area under the chromatographic peak versus incubation time for the prodrug added (see Note 16). 4. To obtain calibration samples, first incubate brain homogenate and plasma (6 mL each; tissue pulling is necessary) at 90°C for 30 min in, e.g., a 15-mL polypropylene centrifuge tube with the cap tightly closed. After cooling, vortex and aliquot 990 PL each into five 1.5-mL Eppendorf tubes, followed by the addition of 10 PL from 20, 15, 10, 5, or 2 mM stock solutions of the prodrug into the respective tubes to make 200-, 150-, 100-, 50-, and 20-PM calibration samples. After thoroughly vortexing the mixtures, remove 100 PL aliquots from each into separate 1.5-mL Eppendorf tubes containing 200 PL of ice-cold solution of 5% (v/v) acetic acid in acetonitrile. Perform sample preparation and RP-HPLC analyses as described in the previous paragraph. 5. From the analyses of the calibration samples, perform assay calibration through the appropriate data processing module of the HPLC system, or by tabulating concentration (Y ) versus chromatographic peak area (X) in a Microsoft Excel spread- sheet and using linear regression (Y = aX+b). Then, calculate concentrations of prodrug in the samples removed during the incubation from results of their respective HPLC analyses. 6. Tabulate prodrug concentration (Y ) versus time (X) profiles in a Microsoft Excel spreadsheet and, assuming a pseudo-first-order degradation, employ exponential fitting (“Add trendline” option) to Y = ae−kX in the “scatter” (X–Y ) chart created from the data (select “Display equation on chart” option). Half-lives

(t1/2) are calculated from the rate constants (k) as 0.693/k.

3.4. Analeptic Effect 1. Divide animals [Swiss Webster mice (30 ± 2 g body weight)] into groups of 10–15 mice/group. In the control group animals will receive vehicle only. In the treatment groups, animals will receive test compounds [one compound (parent peptide or prodrug) per one concentration per one treatment group]. 330 K. Prokai-Tatrai and L. Prokai

2. Give the animals identification numbers, mark them on their back (e.g., by permanent marker), weigh them before starting the in vivo assessment and, then, return them into the cage until you start the procedure. Before gaining substantial expe- rience with this method, use one animal at a time, and do not inject another animal with the test compound before finishing an ongoing experiment. 3. Calculate the necessary dose; in the example given use 10 Pmol/kg body weight of test compound corresponding to approximately 0.3 Pmol of test compound per mouse. 4. Dissolve test compounds in saline calculating for around 60 PL of injection volume (see Note 17). 5. Restrain one animal in a Broome rodent restrainer. 6. Keep the animal warm by using a heat lamp and insert the needle of the syringe into the vein in about 15° angle from the horizon. Slide the syringe carefully into the vein (withdraw a small amount of blood to verify the position of the needle), then inject the whole volume of test compound at once (see Note 18). 7. After 10-min post-drug injection, remove the animal from the cage and inject 60 mg/kg sodium pentobarbital intraperitone- ally (i.p.) (see Note 5). 8. Put the animal into a container (e.g., small box, specified in Subheading 2.4, item 6); it will fall in sleep rapidly (within 1 min of injection). Record this time as the onset of loss of righting reflex and measure the time elapsed until the animal regains this reflex by turning its body into upright position; determining this way the sleeping time. 9. Repeat the entire experiment with the next animal from the same group. 10. After the last animal was tested; calculate the sleeping times as average ± SEM (in min). 11. Use proper statistical analysis among groups such as analysis of variance (ANOVA) followed by post hoc Tukey test for multiple comparisons to establish statistically significant differ- ences between the control and treated groups (see Note 19). An example for the results is summarized in Table 1.

4. Notes

1. SPPS can also be carried out in a polypropylene syringe (10 mL) fitted with polyethylene porous disk. The reaction vessel can be put on a shaker or shaken/stirred intermittently. 21 Brain Delivery of Neuropeptides 331

Table 1 Analeptic effect of [Glu2TRH] obtained upon i.v. administration of the unmodified parent peptide ([Glu2TRH]) or its prodrug [Glu(Hex)2TRH]

Test compound Sleeping time ± SEM (min)

Control 80 ± 2 [Glu2TRH] 65 ± 3* [Glu2TRH] 10×a 56 ± 3*,** [Glu(Hex)2TRH] 50 ± 2*,** Ten min after i.v. injection of the test compounds at equimolar doses of 10 Pmol/kg body weight (or saline vehicle for the control group), pentobar- bital (i.p., 60 mg/kg body weight) was given a[Glu2TRH] at 100 Pmol/kg dose (10×) Data are standard errors of the mean (SEM, n = 10–18); *statistically signifi- cant differences (ANOVA followed by post hoc Tukey test, p < 0.05) from the control group (vehicle), **statistically significant differences (p < 0.05) from both the control group and [Glu2TRH] at a dose of 10 Pmol/kg body weight (12, 28)

2. TFA is extremely corrosive and must be handled with great caution. Use safety glasses, proper rubber gloves, and safety clothing. Handle it under a chemical fume hood. Refer to the Material Safety Data Sheets (MSDS) to familiarize yourself with its (and any other chemicals used during the studies pre- sented here) hazards. 3. Animal protocols pertaining to in vitro and in vivo substantia- tions of the designed prodrug(s) must to be conducted in accordance with the guidelines set forth in the Declaration of Helsinki and the Guiding Principles in the Care and Use of Animals (DHEW Publication, NIH 80–23) by properly trained personnel in animal handlings. 4. Mice are hard to grasp. Care should be taken to avoid getting bitten by or causing harm to the animal. One method of mov- ing mice from one cage to another is by gently grasping the skin behind the neck with a pair of forceps. Restraining the mouse can be done by grasping the base of the tail with one hand and also grasping the loose skin behind the animal’s neck with the other hand. Slow, deliberate movements will avoid exiting the animals. Noise should also be kept to a minimum. 5. Sodium pentobarbital or any other anesthetics are controlled substances and proper permit for obtaining them and their use must be obtained prior carrying out the studies. See institutional 332 K. Prokai-Tatrai and L. Prokai

guidelines on compliance requirements including permission requirements, approval of procedures and proper storage and handling. 6. Double coupling may not be necessary; the efficiency of the coupling reaction can and should be monitored. A range of color tests for the qualitative monitoring of the coupling reaction has been developed that is reliable for small peptides (for very long peptides the detection of the remaining free amino group will usually become difficult and double coupling and capping are performed). (a) Ninhydrin (Kaiser) Test (35): based on the reaction of ninhydrin with amines. The presence of a primary amine produces an intense blue color mainly on the beads. This test is somewhat less suitable for secondary amines such as Pro that produces a brownish red color. The Kaiser test consists of three solutions: Solution 1: 5 g ninhydrin in 100 mL ethanol; Solution 2: 80 g phenol in 20 mL ethanol and Solution 3: 2 mL 0.001 M aqueous KCN in 98 mL pyridine. The test kit is available commercially from, e.g., Anaspec. To perform a color test, place a few washed resins in a small test tube and add 2–5 drops of each solu- tion. Place the test tube in an oven and leave the reaction to develop for 5 min at 100°C. Positive result produces is blue color of the resin and solution, if the resin and the solution are colorless or slightly yellow, then the coupling was complete. (b) For secondary amines (e.g., Pro or N-alkylamines) various color tests have been developed. One of the frequently used tests for these amines (but also applicable for primary amines) is the chloranil test (39), that requires two solu- tions [2% (v/v) acetaldehyde in DMF and 2% (w/v) chlo- ranil in DMF]: add a few beads of resin to a small test tube and 2–5 drops of each solution. After a short mixing leave the mixture at room temperature for 5 min and check on the colors of the beads. Dark blue to green beads: posi- tive; colorless to yellowish beads: negative result. The two test solutions should be kept refrigerated and made fresh monthly. 7. pGlu does not possess N-teminal amino group therefore, there is no deprotection step when this amino acid is used to “cap” a peptide chain. Otherwise, before TFA cleavage, the D-amino terminal of the resin-bound peptide should be removed. 8. Alternatively, the ester prodrug can also be prepared in solution phase via direct esterification of [Glu2TRH] (1 eq) in the presence of commercially available immobilized reagents; N-cyclohexyl- carbodiimide, N c-methyl polystyrene (loading: 1.3 mmol/g, 21 Brain Delivery of Neuropeptides 333

2 eq) and N-(methylpolystyrene)-4-(methylamino)pyridine (loading: 1.49 mmol/g, 0.1 eq) in DCM (3–6 mL) using fivefold excess of alcohol at room temperature via overnight stirring (12). In this case, either the commercially available (e.g., Bachem) or the synthesized [Glu2TRH] is used. In the latter case, use Fmoc-Glu(OtBu)-OH instead of Fmoc- Glu(All)-OH. The acid-sensitive side chain protecting group (tBu) will be hydrolyzed during the cleavage of the peptide from the resin. 9. Depending on the peptide sequence, “scavengers” are also fre- quently used to minimize side reactions that can occur during cleavage (see e.g., http://www3.appliedbiosystems.com/cms/ groups/psm_marketing/documents/generaldocuments/ cms_040654.pdf ). 10. Peptide product cleaved from the solid support is purified by semipreparative RP-HPLC using either isocratic or gradient elution. Typical mobile phase is mixed from 0.1% (v/v) TFA in

H2O (solvent A) and 0.1% (v/v) of TFA in CH3CN (solvent B) with a flow rate recommended by the manufacturer of the chromatographic column used. RP-HPLC columns such as the one specified in Subheading 2.2 work optimally at 5.0 mL/min flow rate and suitable for product isolation at low mg-scale. Therefore, dedicated equipment configured for semipreparative work (large-displacement pump heads for the gradient solvent delivery system, mL-size injector and increased- volume UV-detector’s flow cell) is recommended. After about 15 min of equilibration typically at 95% A and 5% B, keep this composition for 3 min (isocratic) after injection of the solution containing the sample dissolved in solvent A (some solvent B may be added to achieve complete dissolution). Program the solvent delivery system to increase component B then by a linear gradient to 55% in 25 min (+2% per min). Depending on the lipophilicity of the prodrug, starting and final percentage of B in the mobile phase may be adjusted to achieve optimal elution. Collect fractions based on monitoring UV absorption presuming that the major chromatographic peak usually corresponds to the desired product. After eluting the com- pound of interest, ramp to 80% B at 10% per min and maintain the this mobile phase composition for 10 min to clean the column before returning to the initial conditions (95% A and 5% B) changing 10% per min for equilibration before another injection is made. 11. RP-HPLC analyses can be performed with ultraviolet UV spectrophotometric detection (215 nm) using commercially available equipment, which includes hardware and software for data acquisition and processing. See recommended column in Subheading 2.2 (item 2) for which the suggested mobile phase 334 K. Prokai-Tatrai and L. Prokai

flow rate is 0.5 mL/min, mixed from 0.1% (v/v) TFA in H2O

(solvent A) and 0.08% (v/v) of TFA in CH3CN (solvent B). Solvent gradient may be similar to that specified for semi- preparative HPLC (see Note 10). For MS, LC-MS, NMR, and combustion analyses, use core or commercial laboratories that usually offer services including instrumentation and personnel to furnish data necessary to confirm the structure of the com- pound and purity of the product. 12. Withdraw into the syringe at least twice the volume of the (usually 5- or 20-PL) loop connected to the injector valve. + 13. NH4OAc in the mobile phase may yield intense [M + NH4] ions (see Fig. 1a) for certain peptides upon APCI that may also result in excessive breakdown and thus low-abundance molecular ions. In such cases, consider using ESI (38), which is an inherently “soft” method for producing gas-phase ions from biomolecules to enable MS. However, count on the appearance of multiply charged [M + 2H]2+ and [M + 3H]3+ ions for peptides with molecular weight above 800 Da and containing basic amino- acid residues with unprotected side chain. 14. IAMC retention of [Glu2]TRH is actually lower than that of citric acid, a routinely employed reference as a compound with essentially no affinity to a lipid membrane. Therefore, for the

determination of kcIAM capacity factors, we suggest the use of [Glu2]TRH as a void volume marker (12).

15. The use of CO2 asphyxiation or overdose to euthanize the animals may induce changes in brain tissue and body fluids that interfere with drug metabolism. Therefore, cervical dislo- cation is the preferred method of euthanasia for this type of studies performed using small rodents. The method confirms the guidelines set forth in the Declaration of Helsinki and the Guiding Principles in the Care and Use of Animals (DHEW Publication, NIH 80–23). 16. RP-HPLC analyses can be performed with UV spectrophoto- metric detection (215 nm) using commercially available equipment. See recommended column in Subheading 2.2 (item 2) and conditions in Note 11. 17. For example, dissolve 1.8 mg of [Glu2]TRH (M = 354) in 1 mL saline. If the animal weighs exactly 30 g, then inject exactly 60 PL of [Glu2]TRH solution that contains 0.108 mg [Glu2]TRH/animal corresponding to 10 Pmol/kg body weight dose. 18. Injection into the tail vein of a mouse requires practice. After you restrained the animal, rotate the tail slightly to visualize it. Warming a mouse by putting it under heating light or warming its tail directly by soaking it in hot water (40–45°C) will dilate the veins to facilitate the injection. Grab the tail vein firmly 21 Brain Delivery of Neuropeptides 335

with your nondominant hand. Disinfect the injection site and insert needle (25 or smaller gauge, 1 mL syringe) into the vein at a slight angle (15–30°) from the horizon. Inject slowly and smoothly and watch for clearing of the lumen. Incorrect needle positioning will result in a slight bulge in the tail. Upon completion remove the needle and apply gentle pressure to injection site.

19. For dose–response studies, ED50 values can be calculated by using, e.g., the Scientist software (Micromath, St. Louis, MI), by fitting the results of the dose–response experiments to an equation similar to the one introduced by Cheng and Prusoff (40):

'max ' h ªº§·ED «»1  50 ¨¸D ¬¼«»©¹i

where ' and 'max are the average decrease and maximal mea- sured average decrease in sleeping time (min) compared to

control, respectively, Di is the dose (Pmol/kg body weight), and h is the Hill coefficient.

References

1. Strand, F.L. (2003) Neuropeptides: general 8. Prokai-Tatrai, K., and Prokai, L. (2003) characteristics and neuropharmaceutical poten- Modifying peptide properties by prodrug tial in treating CNS disorders In: Prokai, L., design for enhanced transport into the CNS. and Prokai-Tatrai, K. (eds) Peptide transport In: Prokai, L., and Prokai-Tatrai, K. (eds) and delivery into the central nervous system, Peptide transport and delivery into the central Vol. 61, Birkhäuser, Basel. nervous system, Vol. 61, Birkhäuser, Basel. 2. Prokai, L. (2002) Targeting drugs into the cen- 9. Wong, A., and Toth, I. (2001) Lipid, sugar and tral nervous system. In Muzykantow, V.R., and liposaccharide based delivery systems. Curr. Torchilin, V. (eds) Biomedical aspects of drug Med. Chem. 8, 1123–1136. targeting, Kluwer Academic Publishers, 10. Powell, M.F. (1993). Peptide stability in drug Dordrecht. development: in vitro peptide degradation in 3. Kusuhara, H., and Sugiyama, Y. (2001) Efflux plasma and serum. Ann. Rep. Med. Chem. 28, transport systems for drugs at the blood–brain 285–294. barrier and blood–cerebrospinal fluid barrier 11. Prokai-Tatrai, K., Teixido, M., Nguyen, V. et al (Part 1). Drug Discov. Today 6, 150–156. (2005) A pyridinium-substituted analog of the

4. Begley, D.J., and Brightman, M.W. (2003) TRH-like tripeptide pGlu-Glu-Pro-NH2 and its Structural and functional aspects of the blood– prodrugs as central nervous system agents. brain barrier. In: Prokai, L., and Prokai-Tatrai, K. Med. Chem. 1, 141–152. (eds) Peptide transport and delivery into the 12. Prokai-Tatrai, K., Nguyen, V., Zharikova, A.D. central nervous system, Vol. 61, Birkhäuser, et al (2003) Prodrugs to enhance central ner- Basel. vous system effects of the TRH-like peptide

5. Brownlees, J., and Williams, C.H. (1993) pGlu-Glu-Pro-NH2. Bioorg. Med. Chem. Lett. Peptidases, peptides at the mammalian blood– 13, 1011–10144. brain barrier. J. Neurochem. 60, 793–803. 13. Prokai-Tatrai, K., and Prokai, L. (1996) Brain- 6. Pardridge, W.J. (2002) Drug and gene target- targeted delivery of a -enkephalin ana- ing to the brain with molecular Trojan horses. logue by retrometabolic design. J. Med. Chem. Nature Rev. Drug Discovery 1, 131–139. 39, 4775–4782. 7. Albert, A, (1958) Chemical aspects of selective 14. Prokai-Tatrai, K., Kim, H.S., and Prokai, L. toxicity. Nature 182, 421–422. (2008) The utility of oligopeptidase in 336 K. Prokai-Tatrai and L. Prokai

brain-targeting delivery of an enkephalin 27. Prokai-Tatrai, K., Prokai, L. (2009) Prodrugs analogue by prodrug design. Open Med. of thyrotropin-releasing hormone and related Chem. J. 2, 97–100. peptides as central nervous system agents. 15. Prokai, L., Prokai-Tatrai, K., Zharikova, A.D. Molecules 14, 633–654. et al. (2004) Centrally acting and metabolically 28. Kelly, J.A., Slator, G.R., Tipton, K.F. et al. stable thyrotropin-releasing hormone analogues (2000) Kinetic investigation of the specificity of by replacement of histidine with substituted porcine brain thyrotropin-releasing hormone- pyridinium. J. Med. Chem. 47, 625–6033. degrading ectoenzyme for thyrotropin-releas- 16. Klon, A.E. (2009) Computational models for ing hormone-like peptides. J. Biol. Chem. 275, central nervous system penetration. Curr. Med. 16746–16751. Chem. 5, 71–89. 29. Nguyen, V., Zharikova, A.D., and Prokai, L. 17. Danielsson, L.G., and Zhang, Y.H. (1996) (2007) Evidence for interplay between pGlu- Methods for determining n-octanol-water par- Glu-Pro-NH2 and thyrotropin-releasing hor- tition constants. TRAC–Trends Anal. Chem. mone in the brain. Neurosci. Lett. 415, 15, 188–196. 64–67. 18. Ong, S., Hanlan, C., and Pidgeon, J. (1996) 30. Nguyen, V., Zharikova, A.D., Prokai-Tatrai, K. Immobilized-artificial-membrane chromatog- et al. (2010) [Glu2]TRH dose-dependently raphy: Measurements of membrane partition attenuates TRH-evoked analeptic effect in the coefficient and predicting drug membrane per- mouse brain. Brain Res. Bull. 82, 83–86. meability. J. Chromatogr. 728, 13–128. 31. Merrifield, R.B. (1963) Solid phase peptide 19. Faller, B. (2008) Artificial membrane assays to synthesis. Synthesis of a . J. Am. assess permeability. Curr. Drug Metab. 9, Chem. Soc. 85, 2149–2154. 886–892. 32. Carpino, L.A., and Han, G.Y. (1972) 20. Deli, M.A., Abraham, C.S., Kataoka, Y. et al. 9-Fluorenylmethoxycarbonyl amino-protecting (2005) Permeability studies on in vitro blood- group. J. Org. Chem. 37, 3404–3409. brain barrier models: Physiology, pathology, and 33. Albericio, F., Bofill, J.M., El-Faham, A. et al. pharmacology. Cell Mol. Neurobiol. 25, 59–129. (1998) Use of onium salt-based coupling 21. Bradbury, M.W.B., Patlak, C.S., and Oldendorf, reagents in peptide synthesis. J. Org. Chem. 63, W.H. (1975) Analysis of brain uptake and loss 9678–9683. of radiotracers after intracarotid injection. Am. 34. Kaiser, E., Colescott, R.L., Bossinger, C.D. J. Physiol. 229, 1110–1115. et al. (1970) Color test for detection of free 22. Prokai, L., Zharikova, A.D., Janaky T et al. (2000) terminal amino groups in the solid-phase Exploratory pharmacokinetics and brain distribu- synthesis of peptides. Anal. Biochem. 34, tion study of a neuropeptide FF antagonist by 595–598. liquid chromatography/atmospheric pressure 35. Mant, C.T.; and Hodges, R.S. (1991). High- ionization tandem mass spectrometry. Rapid performance liquid chromatography of peptides Commun. Mass Spectrom. 14, 2412–2418. and proteins; CRC Press: Boca Raton, Fl. 23. Hansch, C., and Clayton, J.M. (1973) Lipophilic 36. Prokai, L., Zharikova, A., Janáky, T. et al. character and biological-activity of drugs. 2. (2001) Integration of mass spectrometry into Parabolic case. J. Pharm. Sci. 62, 1–21. early-phase discovery and development of cen- 24. Summerfield, S.G., Read, K., Begley, D.J. tral nervous system agents. J. Mass Spectrom. et al.(2007) Central nervous system drug 36, 1211–1219. disposition: The relationship between in 37. Braddy, A.C., Janaky, T., and Prokai, L. (2002) situ brain permeability and brain free frac- Immobilized artificial membrane chromatogra- tion. J. Pharmacol. Exp. Ther. 322, phy coupled with atmospheric pressure ioniza- 205–213. tion mass spectrometry. J. Chromatogr. A 966, 25. Del Rio-Garcia, J., and Smyth, D.G. (1990) 81–87. Distribution of pyroglutamylpeptide amides 38. http://www.registech.com/InfoPages/ related to thyrotropin-releasing hormone in the IamInfo/IamCareAndUse.html central nervous system and periphery of the rat. 39. Vojkovsky, T. (1995) Detection of secondary J. Endocrinol. 127, 445–450. amine on solid phase. Pept. Res. 8, 236–237. 26. Prokai, L. (2002) Central nervous system 40. Cheng, Y., and Prusoff, W.H. (1973) effects of thyrotropin-releasing hormone and Relationship between the inhibition constant its analogues: opportunities and perspectives (Ki) and the concentration of inhibitor which

for drug discovery and development. In: Jucker, causes 50 percent inhibition (I50) of an enzy- E.M. (ed) Progress in drug research, Vol 59, matic reaction. Biochem. Pharmacol. 22, Birkhäuser, Basel. 3099–3108. Chapter 22

Measurement of Phosphorothioate Oligodeoxynucleotide Antisense Transport Across the Blood–Brain Barrier

William A. Banks

Abstract

Phosphorothioate oligodeoxynucleotides (PODNs) can act as antisense molecules, knocking down proteins. Many PODNs have the unusual characteristic of being transported across the blood–brain barrier by a saturable system. This means that PODNs injected intravenously can accumulate in the central nervous system in quantities sufficient to knock down proteins in brain and the blood–brain barrier. A critical step in the development of PODNs that can be administered peripherally and knockdown proteins in the cen- tral nervous system is to determine the relation to the blood–brain barrier, specifically, does the PODN cross the blood–brain barrier and, if so, how fast and to what degree.

Key words: Blood–brain barrier, Antisense, Transport, Oligonucleotides, Central nervous system, Drug delivery

1. Introduction

Knockdown of proteins in brain with the various RNA-related inhibitors of translation offers great promise for the study of protein function and as possible therapeutics (1, 2). However, the delivery of the various inhibitors to the central nervous system is highly problematic (3, 4). An important exception is that of the phospho- rothioate oligodeoxynucleotides (PODNs). These molecules offer several advantages for targeting the central nervous system, the two major advantages being that they are highly resistant to degradation by enzymes found in peripheral tissues, the blood–brain barrier, and the central nervous system and that many of them are transported across the blood–brain barrier by a saturable system (5).

Adalberto Merighi (ed.), Neuropeptides: Methods and Protocols, Methods in Molecular Biology, vol. 789, DOI 10.1007/978-1-61779-310-3_22, © Springer Science+Business Media, LLC 2011 337 338 W.A. Banks

Several examples of the ability of the blood–brain barrier to take up and transport into the central nervous system significant quantities naked PODN antisenses have been published. In some cases, transporters of the blood–brain barrier have been targeted with resulting alterations in brain function (6) or resistance to stroke (7). In other cases, a brain protein has been targeted with delivery of antisense sufficient to decrease the targeted protein and alter behavior (5, 8, 9). An important step in the development of PODN antisense molecules that are capable of entering the central nervous system after peripheral administration in sufficient quantities to knock down brain proteins is the determination of whether they cross the blood–brain barrier and, if so, to what degree. The following chapter outlines the major steps in determining the quantitative uptake of PODNs by the blood–brain barrier.

2. Materials

2.1. Labeling and 1. PODN of choice (available from Midland Certified Reagents, Purification Midland, TX, see Note 1). 2. Kinase buffer, 10× (New England Biolabs, Ipswich, MA). 3. T4 Polynucleotide kinase (New England Biolabs). 4. 32P-ATP, 6,000 Ci/mmol (Perkin Elmer NEN, Waltham, MA). 5. Ethanol. 6. Sodium acetate, 3 M. 7. Microcentrifuge. 8. Water bath. 9. Vortex mixer. 10. Scintillation counter.

2.2. Small Animal 1. 32P-labeled PODN. Surgery 2. Animals (e.g., CD-1 mice). 3. 1.5-mL polypropylene tubes. 4. Surgical instruments used include Metzenbaum tissue scissors, 7Ȍ sharp-blunt dissecting scissors, two each fine point, curved 3Ȍ forceps, iris scissors. 22 Blood Brain Barrier Transport of PODNs 339

3. Methods

3.1. Labeling and 1. Add in the following order the following components: 5 Mg Purification PODN (1.3 ML of 3.85 Mg/ML), 1.5 ML of 10× Kinase buffer, 10 ML gamma 32P-ATP (6,000 Ci/mmol), and 1.5 ML T4 Polynucleotide Kinase. 2. Mix by knocking with finger; if necessary spin for 10 s to bring to the bottom of tube. 3. Incubate in a 37°C water bath for 45 min. 4. Incubate in a 65°C (can be hotter) water bath for 5 min to inactivate the enzyme. 5. Add in the following order the following components: 80 ML

of dH2O, 10 ML of 3 M sodium acetate, and 300 ML of cold ethanol. 6. Vortex, then spin if necessary, to bring to the bottom of tube. 7. Incubate in a dry ice–alcohol bath for 20 min. 8. Let warm up for approximately 5 min, then spin at 5,000 × g, 4°C, for 20 min. 9. Decant solution, saving for radioactive disposal (see Note 2). 10. Add 500 ML of cold ethanol, vortex, and then spin again for 20 min. 11. Decant solution to radioactive disposal. 12. Add 500 ML of cold ethanol, vortex, and then spin again for 20 min. 13. Decant solution to radioactive disposal. 14. Add 100 ML of cold ethanol, vortex, and then spin for 20 min. 15. Decant solution to radioactive disposal. 16. Air-dry the remaining pellet or dry in speed vac (no heat) for about 5 min. 17. Count tube in scintillation counter (no scintillation fluid required). 18. May have to reconstitute and dilute the sample if level of radio- activity is too high. To reconstitute, use phosphate buffer or other solution in which the label will be diluted for experiments. 19. Store stock 32P-PODN at 4°C until needed.

3.2. Small Animal 1. In an anesthetized animal, expose the left jugular vein and the Surgery right carotid artery (see Note 3). 2. Inject 200 ML of the labeled 32P-PODN to be studied at spe- cific time intervals, i.e., 1, 2, 3, 4, 5, 7.5, 10, 12, 15, 20, 25, 340 W.A. Banks

and 30 min (see Note 4). Typically, one mouse is used for each time point. 3. At the specified postinjection time interval, collect whole blood by severing the right carotid artery (see Note 5) and collecting the blood in a 1.5-mL polypropylene tube. 4. Decapitate the mouse with scissors, excise the cranium, and remove the whole brain, placing it in a 12 mm × 75 mm glass tube. 5. Centrifuge the blood at 3,000 × g for 10 min. 6. Pipette 50 ML serum into new 12 mm × 75 mm tubes. 7. Count the serum and whole brain in scintillation counter. Be sure to use the same type of tubes for the brain and serum, i.e., all glass tubes or all polypropylene tubes to get comparable counts. 8. Calculate the exposure time as described below.

3.3. Calculations The unidirectional influx rate measures the rate at which the study substances cross the blood–brain barrier. 3.3.1. Blood-to-Brain Unidirectional Influx Rate 1. Plot time (min) vs. the log of serum CPM. (K ) i 2. Fit curve to points, extending the curve to Time 0. 3. Read new log values from curve for each time point, including time 0. This is called the Graph Data or Theoretical Data from Graph. 4. Average the first two graph data values. 5. Calculate the antilog value of the average. 6. Multiply by the change in time of the first two values. This equals the first integration value and represents the area under the curve from Time 0 to the first time point, i.e., 1 min. 7. Repeat steps 4–6, adding the new integrated value to the previ- ous integrated value. 8. Calculate the Exposure Time (in minutes) (ExpT) by dividing each integrated value by the antilog of the second Graph Data number used to calculate that integrated value. 9. Calculate the brain-to-serum ratio as: 50Pu L Serum brain CPM Brain - to - serum ratio . Brainwt.u serum CPM 10. Plot each brain–serum ratio against its respective ExpT. Use regression analysis to calculate slope, but use only the data where the relation between the brain–serum ratio and ExpT is linear.

The slope yields the blood-to-brain unidirectional influx rate (Ki)

in units of ML/g min. The intercept of this relation yields the Vi in units of ML/g and is used in calculating %Inj/g (see below). 22 Blood Brain Barrier Transport of PODNs 341

3.3.2. Percent of This value estimates the percent of the peripherally administered Intravenous Dose Taken Up dose that enters the brain at each time point (see Note 6). It is useful by Brain (%Inj/g) in calculating dosages and is dependent on both the unidirectional influx rate and peripheral pharmacokinetics.

1. First calculate for each time point the percent of the injected dose that is present in a microliter of serum (%Inj/ML) as follows: %Inj CPM /P L serum 100u . PL Serum CPM / injection

2. Then, the percent injected dose can be calculated using either

Ki (step 2a) or brain–serum ratios (step 2b) (see Note 7).

(2a) Using Ki:

(K i )(ExpT)(%Inj /P L serum) (P L / g min)(min)(%Inj / P L) %Inj / g.

(2b) Using Vi and brain–serum ratios:

100(Brain / serum ratio at timetVPi )(%Inj / L serum) (PP L/g L/g)(%Inj/ P L) %Inj/g.

4. Notes

1. See refs. 5–9 for examples of antisenses that target brain or blood–brain barrier proteins and whose pharmacokinetics has been measured using this method. 2. Radioactive materials must be disposed of according to local regulations. 3. The type of anesthesia and route of administration depend on the species. Although some anesthetics can interfere with BBB transportation measurements (e.g., pentobarbital and the mea- surement of amino acid flux), no such interaction is currently documented for measurement of antisenses. 4. Selected times can vary. As a rule, shorter time intervals and more frequent sampling are chosen for antisenses that come into rapid equilibrium between blood and brain and longer time intervals and less frequent sampling chosen for antisenses that more slowly come into equilibrium. Pilot studies may be needed to select the ideal time curve. 5. Arterial blood is preferred to venous blood, as the latter is lower according to tissue extraction. Because tissues vary in their extraction rates, venous blood from different sources can have different concentrations of material in them. All arteries, except for the pulmonary arteries, contain the same concentration 342 W.A. Banks

of materials, and any artery, except the pulmonary arteries, may be used for sampling. The carotid artery is used here, as it is a convenient source in mice for the studies designed here. 6. More than one mouse per time point may be needed to define this parameter accurately. This is because each time point acts

as its own statistical cell, whereas in the calculation of Ki, the entire set of points used to define the regression curve act as a statistical cell.

7. In general, calculations based on Ki are technically more con-

venient, whereas those based on Vi and brain–serum ratios yield more conservative estimates.

References

1. Phillips, M.I., and Gyorko, R. (1997) Antisense 6. Jaeger, L.B., Dohgu, S., Hwang, M.C., Farr, oligonucleotides: New tools for physiology. S.A., Murphy, M.P., Fleegal-DeMotta, M.A. News Physiol. Sci. 12, 810–817. et al. (2009) Testing the neurovascular hypoth- 2. Morley, J.E., Farr, S.A., Kumar, V.B., and esis of Alzheimer’s disease: LRP-1 antisense Banks, W.A. (2002) Alzheimer’s disease reduced blood-brain barrier clearance, increases through the eye of a mouse: Acceptance lecture brain levels of amyloid beta protein, and impairs for the 2001 Gayle A. Olson and Richard D. cognition. J. Alzheimers Dis. 17, 553–570. Olson prize. Peptides 23, 589–599. 7. Dogrukol-Ak, D., Kumar, V.B., Ryerse, J.S., 3. Agrawal, S., Zhang, X., Lu, Z., Zhao, H., Farr, S.A., Verma, S., Nonaka, N. et al. (2009) Tamburin, J.M., Yan, J. et al.(1995) Absorption, Isolation of peptide transport system-6 from tissue distribution and in vivo stability in rats of brain endothelial cells: therapeutic effects with a hybrid antisense oligonucleotide following antisense inhibition in Alzheimer’s and stroke oral administration. Biochem. Pharmacol. 50, models. J. Cereb. Blood Flow Metab. 29, 571–576. 411–422. 4. Boado, R.J., Tsukamoto, H., and Pardridge, 8. Banks, W.A., Jaeger, L.B., Urayama, A., Kumar, W.M. (1998) Drug delivery of antisense mole- V.B., Hileman, S.M., Gaskin, F.S. et al. (2006) cules to the brain for treatment of Alzheimer’s Preproenkephalin targeted antisenses cross the disease and cerebral AIDS. J. Pharm. Sci. 87, blood-brain barrier to reduce brain 1308–1315. enkephalin levels and increase voluntary drink- 5. Banks, W.A., Farr, S.A., Butt, W., Kumar, V.B., ing. Peptides 27, 784–796. Franko, M.W., and Morley, J.E. (2001) 9. Poon, H.F., Joshi, G., Sultana, R., Farr, S.A., Delivery across the blood-brain barrier of anti- Banks, W.A., Morley, J.E. et al. (2004) sense directed against amyloid: reversal of Antisense directed at the A-beta region of APP learning and memory deficits in mice overex- decreases brain oxidative markers in aged senes- pressing amyloid precursor protein. J. cence accelerated mice. Brain Res. 1018, Pharmacol. Exp. Ther. 297, 1113–1121. 86–96. Chapter 23

Liposome-Encapsulated Neuropeptides for Site-Specific Microinjection

Frédéric Frézard, Robson A.S. dos Santos, and Marco A.P. Fontes

Abstract

This paper describes an experimental approach based on nanotechnology for assessing the chronic actions of short-lived neuropeptides at specific sites of the brain. This methodology combines the advantages of two different techniques: the microinjection of a suspension of peptide-containing liposomes into a spe- cific site of the brain, and the use of liposomes as a local and sustained release nanosystem of the peptide.

Key words: Liposomes, Neuropeptides, Microinjection, Site-specific, Brain

1. Introduction

The central nervous system in mammals is made up of approximately 100 billion neurons. Therefore, studying the neurochemical and functional properties of a specific population of neurons is an extremely complex task. On the other hand, this is undoubtedly a critical preliminary step to understand and treat central nervous system-related diseases. Despite the enormous progress made by neuroscience in the last decades, still there are several problems to technically address this subject in different neuroscience fields. For example, a major difficulty encountered in the study of the physiol- ogy of short-lived neuropeptides is the lack of appropriate method- ology for assessing their chronic actions. This is particularly critical when these actions have to be evaluated at a specific site of the brain and in freely moving awake animals. Indeed, the rapid in vivo metab- olism of the peptide results in biological actions of very short duration. In the case of endogenous neuropeptides, site-specific microinjection of an aqueous solution fails to mimic the physiological conditions of their chronic local production. Moreover, conventional

Adalberto Merighi (ed.), Neuropeptides: Methods and Protocols, Methods in Molecular Biology, vol. 789, DOI 10.1007/978-1-61779-310-3_23, © Springer Science+Business Media, LLC 2011 343 344 F. Frézard et al.

methodology does not allow studies on the interactions between different brain areas, which would require multiple microinjections in different brain sites. This paper describes a novel experimental approach based on nanotechnology for assessing the chronic actions of short-lived neuropeptides at specific sites of the brain. This methodology com- bines the advantages of two different techniques: site-specific microinjection into the brain and the use of liposomes as a local and sustained release nanosystem of the peptide. This methodol- ogy has been successfully applied by our group to the study of the chronic cardiovascular actions of angiotensinergic peptides at the rostral ventrolateral medulla (1–3).

2. Materials

2.1. Preparation 1. 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC – obtained of Peptide-Containing from Lipoid GmBH, Ludwigshafen, Germany) (see Note 1). Liposomes 2. 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine- N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG(2000) – obtained from Lipoid GmBH) (see Note 1). 3. Cholesterol (CHOL – Sigma St. Louis, MO). 4. Chloroform. 5. Methanol. 6. Vacuum pump. 7. Round bottom flask. 8. Water bath. 9. 3-mm probe tip sonicator. 10. Argon gas cylinder. 11. 15-mL Falcon conical tubes. 12. Freeze dryer. 13. Vortex. 14. Phosphate-buffered saline (PBS): 0.15 M NaCl, 10 mM

Na2HPO4 (adjust to pH 7.2 with HCl or NaOH if necessary) (see Note 2). May be stored at 4°C for up to 2 weeks. 15. 10-mL Extruder (Lipex™, Northern Lipids Inc., Burnaby, BC). 16. Polycarbonate membranes with 25 mm diameter and 200 nm pore size (GE Water & Process Technologies, Trevose, PA). 17. SpectraPor® Biotech regenerated cellulose dialysis membrane MWCO 15 kDa, 16-mm flat (Spectrum Laboratories, Rancho Dominguez, CA). 18. Millex-GV, 0.22-Pm sterilizing syringe filters (Millipore, Billerica, MA). 23 Liposome-Encapsulated Neuropeptides 345

2.2. Characterization 1. Microcon YM-50 with MWCO 50 kDa (Millipore). of Peptide-Containing 2. Particle size analyzer, allowing dynamic light scattering analysis Liposomes (e.g. Zetasizer Nano ZS90 available from Malvern, Worcestershire, UK). 3. PBS supplemented with 0.02% (w/v) sodium azide as a preser- vative (antimicrobial agent) for in vitro stability assay.

2.3. Brain 1. Stereotaxic apparatus (available from David Kopf, Tujunga, Microinjection CA). 2. Dentistry needles (30 G). 3. Polyethylene tube I.D. 28 mm O.D. 61 mm (PE10 Clay Adams, Becton Dickinson, Franklin Lakes, NJ). 4. Hamilton syringe (5 or 10 PL). 5. Syringe pump. 6. Fluorescently labeled liposomes: 1,1c-Dioctadecyl-3,3,3c,3c- tetramethylindocarbocyanine perchlorate (DiI) (Invitrogen, Carlsbad, CA). Dissolve in ethanol at 2 mg/mL and store solution at −20°C.

3. Methods

We describe below the three major steps of the proposed experi- mental technique: the preparation of peptide-containing lipo- somes, the characterization of the resulting liposome formulation, and the microinjection of the formulation into a specific site of the brain.

3.1. Preparation The technique proposed for the preparation of peptide-containing of Peptide-Containing liposomes consists of the formation of dehydration–rehydration Liposomes vesicles (DRVs) (4) followed by liposome sizing through extru- sion (5). This technique yields a high efficiency of encapsulation of hydrophilic water-soluble peptides in liposomes having defined and narrow size distribution. Figure 1 summarizes the main steps involved in the technique. The preferred lipid composition is: DSPC, CHOL, and DSPE- PEG(2000) at a molar ratio of 5:4:0.3 (see Note 3). Table 1 dis- plays the typical volumes and amounts of lipids and peptide to be used at each step of the method of liposome preparation. 1. Weigh lipids and dissolve them in an organic solvent to assure a homogeneous mixture of lipids. The intent is to obtain a clear lipid solution for complete mixing of lipids. This process is carried out using chloroform or chloroform:methanol mix- tures (as organic solvent). 346 F. Frézard et al.

Evaporation Hydration

of organic solvent Mechanical agitation

Lipids in Thin lipid film Multilamellar vesicles organic solvent in water

Sonication

Addition of an aqueous solution Freeze-drying of the peptide

Freeze-dried liposomes Small unilamellar vesicles in water Rehydration with water/saline

Extrusion through polycarbonate Dialysis Membranes

(200-nm pore) Dehydration-rehydration Calibrated Peptide-containing vesicles unilamellar large unilamellar vesicles (100-2000 nm) vesicles (200 nm)

Fig. 1. Schematic representation of the method used for preparation of peptide-containing liposomes.

Table 1 Typical amounts of lipids and peptides and volumes used in the preparation of peptide-containing liposomes

Step Lipids Peptide Volume

Formation of lipid film 0.191 g DSPC – 20 mL of organic solvent 0.075 g CHOL 0.040 g DSPE-PEG Hydration of lipid film Lipid concentration – 5 mL of water (formation of MLVs) of 90 mM Mixing of liposome Final lipid Final peptide 1 mL of liposome suspension suspension with concentration concentration 90 mM + 1 mL of 2 g/L peptide solution of 45 mM of 1 g/L peptide, 30 mM NaCl solution Rehydration Lipid concentration Final peptide 0.2 mL of water of freeze-dried of 450 mM concentration mixture – 1st step of 10 g/L Rehydration Lipid concentration Final peptide 0.2 mL of PBS of freeze-dried of 225 mM concentration mixture – 2nd step of 5 g/L 23 Liposome-Encapsulated Neuropeptides 347

2. Transfer the organic lipid solution to a round bottom flask and remove the organic solvent by rotary evaporation yielding a thin lipid film on the sides of the flask (see Note 4). 3. Thoroughly dry the lipid film by placing the flask on a vacuum pump overnight to remove residual organic solvent. 4. Hydrate the dry lipid film by first adding water to the round bottom flask so as to achieve a final lipid concentration of 90 mM (see Note 5). 5. Downsize multilamellar vesicles (MLVs) by submitting the suspension to ultrasounds. This process typically produces small, unilamellar vesicles (SUVs) with diameters in the range of 20–100 nm. Accomplish sonication by placing the tip of the sonicator in a test tube containing the MLVs suspension and sonicate for 5–10 min above the gel–liquid crystal phase transi-

tion temperature (Tc) of DSPC phospholipid (+55°C) (see Note 6). To prevent lipid oxidation and hydrolysis, expose the liposome suspension to argon gas (instead of air) during the sonication process. Immerse the test tube in a water-bath

(at about the Tc) during sonication, to maintain the liposome membrane in the fluid liquid–crystal phase and to prevent overheating. The lipid suspension should then begin to clarify and yield a slightly hazy transparent solution. 6. Just after the sonication step, annealing is recommended for

liposomes made from high-Tc phospholipid in order to dissi- pate eventual defects in membrane lipid organization. To do

so, incubate the sonicated suspension for 15 min above the Tc. 7. First centrifuge the suspension at 3,000 × g for 10 min and then filter using sterile 0.22-Mm syringe filters. This step is nec- essary to remove residual large vesicles and contaminating tita- nium particles released from the sonicator tip. 8. Mix 1 mL of the SUVs suspension with 1 mL of 2 g/L peptide 0.03 M NaCl aqueous solution (see Note 7). 9. Immediately freeze the mixture in liquid nitrogen (in 15-mL Falcon conical tubes) and then dry overnight using a freeze- dryer. 10. Rehydrate the lyophilized powder as follows: Add 0.2 mL of distilled water, and incubate and repeatedly vortex the mixture for 30 min at 55°C. Add 0.2 mL of PBS and continue vortex- ing as above. Finally, after homogenization (vortex) and incu- bation of the suspension for 30 min at 55°C, add 0.8 mL of PBS. DRVs are formed during this step. 11. Filter the resulting liposome suspension repeatedly (ten times) through two stacked polycarbonate membranes with 200 nm pore size at 60°C under a pressure of 200–500 psi, using an Extruder (Lipex™) (5). This technique typically leads to large 348 F. Frézard et al.

unilamellar vesicles (LUVs) having a diameter close to the pore size of polycarbonate membrane (see Note 8). 12. Separate the liposomes from non-encapsulated peptide by dial- ysis against PBS using dialysis tubing with MWCO higher than the peptide molecular weight. Perform dialysis during 24–36 h at 4–25°C with constant stirring of the receiver buffer and three buffer exchanges (see Note 9). 13. Sterilize the lipid suspension by filtration using sterile 0.22-Mm syringe filters. 14. Obtain a suspension of empty liposomes, to be used as control, using the same procedure as described above, but omitting the peptide in the preparation.

3.2. Characterization 1. Analyze the vesicle size distribution in the resulting formula- of Peptide-Containing tion by quasi-elastic laser light scattering (also known as Liposomes dynamic light scattering or photon correlation spectroscopy) using a nanoparticle size analyzer or nanosizer. The results of this analysis are the mean hydrodynamic diameter (Z-average diameter) and the polydispersity index (PdI). Prepare sample by adding a 10- to 50-ML aliquot of the vesicle suspension to 2 mL of isotonic buffer (typically PBS). The liposome suspen- sion is considered as monodisperse when PdI is inferior to 0.3 (see Note 10). 2. The modern equipments used to analyze the particle size dis- tribution of liposome suspensions often allow the measurement of the zeta potential (named zetasizer) that is related to the surface membrane potential of the membrane (see Note 11). 3. To determine the amount of encapsulated peptide in the final liposome preparation, add a 50-PL aliquot to 1,950 PL of methanol to disrupt the liposomes and release the encapsu- lated peptide. In the case of peptides that contain either Trp or Tyr, the amount of encapsulated peptide can be determined exploiting its intrinsic fluorescence (see Note 12). 4. From the knowledge of the amount of encapsulated peptide in the liposome suspension, two parameters are derived: the peptide encapsulation efficiency corresponding to: (amount of encapsu- lated peptide/amount of peptide initially added) × 100; and the encapsulated peptide-to-lipid ratio corresponding to: amount of encapsulated peptide/amount of lipid (see Note 13). 5. (Optional) Perform an in vitro peptide release assay to evaluate the kinetic of peptide release from the liposomes. Typically, incubate the liposome suspension at 37°C in PBS containing 0.02 (w/v) sodium azide, and, after different time intervals, remove a 50-ML aliquot and submit to ultrafiltration using Microcon YM-50 ultrafilters. Determine the concentration of released peptide in the ultrafiltrate. 23 Liposome-Encapsulated Neuropeptides 349

3.3. Brain 1. Find the stereotaxic coordinates for the brain site to be targeted Microinjection using the atlas from Paxinos and Watson (6). 2. After the appropriate general anesthesia, the rat must be care- fully and correctly placed in the stereotaxic apparatus. For example, for rats weighting 280–320 g the coordinates for reaching paraventricular nucleus of hypothalamus (PVN) are: 1.8 mm posterior, 0.5 mm lateral, and 7.8 mm ventral. 3. Using bregma as a reference, perform a craniotomy 1.8 mm posterior and 0.5 mm lateral from bregma to allow the inser- tion of the microinjection needle into the brain, i.e. 7.8 mm ventral, aiming the PVN. 4. Perform microinjections of liposomes with a microinjection needle (30 G) connected to a 5- or 10-PL Hamilton syringe with a polyethylene tube (Fig. 2). The syringe is mounted in a syringe pump that is used to deliver 100 nL of injectate over 30 s. The microinjection is considered successful if, immediately

Fig. 2. Schematic representation of the method used for microinjections into the rat brain. (a) The polyethylene tube filled with liposome solution is connected to the microinjection needle. The injection volume is administered using a 10-PL syringe controlled by the pump. (b) Schematic drawing of a brain section at the level of rat hypothalamus. Using the three- dimensional system of stereotaxic coordinates (see atlas of Paxinos and Watson), microinjection needle is positioned into the brain nucleus. In the example, needle targets the paraventricular nucleus of hypothalamus (PVN), which corresponds to the level 1.8 mm posterior to the bregma. 350 F. Frézard et al.

after removal of the microinjector, flow appear within 3 s after the pump is reactivated, indicating that the injector was not clogged (see Note 14). 5. After suturing the skin, and antibiotic/analgesic treatment, return rats to their home cages and allowed to rest before start- ing the experimental approach. At the end of experimental procedures, perform histological analysis to confirm spreading of the injectate and the injection site (see Note 15).

4. Notes

1. Store at −20°C in a desiccator (items 1 and 2). 2. Unless stated otherwise, all solutions should be prepared in water that has a resistivity of 18.2 M: cm and total organic content of less than five parts per billion. This standard is referred to as “water” in this text. 3. This lipid composition is proposed as a first option, because it was highly effective in prolonging the duration of ’ actions at the rostral ventrolateral medulla (2, 3). Preferred lipids are natural lipids or their derivatives, because of higher

biocompatibility (7). The high-Tc phospholipid, DSPC, is selected as the main lipid, because it yields rigid and low-per- meability membrane and confers high membrane stability in biological fluids (7). Its use implies that most of the preparation steps must be performed at temperature between 55 and 65°C

(above 55°C, the Tc of DSPC). If this temperature range is not compatible with the stability of the selected peptide, DSPC may

be substituted for a lipid with lower Tc, for instance, dioleylphos-

phatidylcholine (DOPC, Tc = +0°C), dimyristoylphosphatidyl-

choline (DMPC, Tc = +23°C), or dipalmitoylphosphatidylcholine

(DPPC, Tc = +42°C). In the case of liposomes made from high-

Tc phospholipid (membrane in gel phase at physiological tem- perature), CHOL helps in dissipating the structural defects in the membrane lipid organization. In the case of liposomes

made from low-Tc phospholipid (membrane in liquid–crystal phase), CHOL improves the membrane stability in biological fluids. The addition of the synthetic lipid derivative of poly(ethylene glycol), DSPE-PEG(2000) at 3 mol%, seems essential to achieve a long-lasting biological action, as demon- strated previously with liposome-encapsulated angiotensins (2, 3). PEG-lipid promotes colloidal stability (prevents vesicle aggregation and membrane fusion) through steric stabilization, reduces the binding of plasma protein to the membrane surface and slows down the endocytosis/phagocytosis of liposomes, which ultimately leads to liposome degradation by lysosomal 23 Liposome-Encapsulated Neuropeptides 351

enzyme and to peptide release (8, 9). DSPE-PEG(2000) may be substituted by DSPE-PEG(5000) and/or its proportion may be increased up to 5 mol% to achieve maximal effective- ness. When DOPC is used, the recommended PEG-lipid is DOPE-PEG (to prevent lipid phase separation and membrane instability); when DMPC or DPPC is selected, recommended PEG-lipid is DMPE-PEG or DPPE-PEG. It is noteworthy that

liposomes made from low-Tc phospholipid are expected to be degraded and to release their content more rapidly (10, 11) and, according to own experience, produce shorter biological response (2, 3). The proposed methodology relies on the assumption that the peptide is not inactivated or transformed by lysosomal enzymes. In the case of peptides susceptible to lysosomal enzymes, the extent of peptide degradation may be reduced through the use of pH-sensitive liposomes that are destabilized and become fusogenic during the acidification of endosomes (12). Fusion of liposomes with the endosomal membrane leads to peptide release into the cytosol before the activation of lysosomal enzymes. The bioavailability of the peptide then depends on its ability to cross the cell plasma membrane. pH-sensitive liposomes are classically obtained by combining the hexagonal II phase-forming phospholipid, dio- leoylphosphatidylethanolamine (DOPE), with the pH-sensi- tive cholesteryl hemisuccinate (CHEMS). Steric stabilization, for instance with PEG lipid, is also necessary to prevent the binding protein to the membrane and preserve the pH-sensi- tivity of the membrane (12, 13). 4. Glass beads (5 mm average diameter, 20 g beads per 1 g phos- pholipid) may be added to the round bottom flask prior to solvent evaporation to increase the surface area of the dried lipid film, thus enhancing the rate of solvent evaporation, increasing the lipid contact with the aqueous phase, and facili- tating lipid hydration. 5. The temperature of the hydrating medium should be above

the Tc of the lipid with the highest Tc (+55°C in the case of DSPC). The hydration step may be accomplished by placing the flask on a rotary evaporation system without a vacuum. Spinning the round bottom flask in the warm water bath main-

tained at a temperature above the Tc allows the lipid to hydrate in its fluid phase with adequate agitation. A hydration time of 1 h with vigorous shaking, mixing, or stirring is recommended. The product of hydration is a suspension of large, MLVs analo- gous in structure to an onion, with each lipid bilayer separated by a water layer. 6. The preferred volume of the liposome suspension is 2–3 mL, and the preferred test tubes for sonication are the more resis- tant Pyrex 4- to 5-mL tubes with a thick wall. The sonication 352 F. Frézard et al.

probe should be immersed as deep as possible in the test tube, without hitting the tube wall or bottom. The energy applied should be sufficient to promote homogenization and progres- sive clarification of the lipid suspension, without promoting excessive cavitation. 7. The proposed procedure assumes that the peptide is highly sol- uble in aqueous saline solution and does not aggregate or pre- cipitate when hydrating the freeze-dried powder with a reduced volume of water. The pH and/or composition of the original peptide solution can be manipulated in order to achieve maxi- mum solubility. The concentration of the peptide should be chosen as a function of solubility and pharmacological require- ments. Finally, the whole composition of the peptide solution should also be adjusted to prevent osmotic shock and subse- quent release of encapsulated peptide during steps 10–12. For this purpose, the osmolarity of the solution of the first hydra- tion step of the freeze-dried powder should be close to plasma osmolarity (0.3 Osm/L). Ultimately, the water-soluble peptide is encapsulated within the aqueous phase of the vesicles. In the case of water-insoluble peptides that are soluble in organic solvents such as chloroform or chloroform:methanol mixture, the method of preparation may be modified as follows. The lipids and the peptide are first co-dissolved in the organic solvent and the organic solvent was removed by rotary evapo- ration, as described in Subheading 3.1, steps 1–2. The lipid– peptide film is then hydrated with PBS (as described in Subheading 3.1, step 4), and the resulting MLVs are down- sized by extrusion through polycarbonate membranes with 200-nm pore size (as described in Subheading 3.1, step 5). Accordingly, such peptides are expected to be incorporated in the vesicle membrane. Non-encapsulated peptides usually form precipitates, which are retained by the polycarbonate mem- brane during the extrusion process. The occurrence of peptide precipitation is usually detected during the extrusion process when the filtration becomes slow (peptide aggregates clog the polycarbonate filters). To proceed with the filtration process, it is recommended to change the polycarbonate membrane. The encapsulation efficiency of water-insoluble peptides depends on their affinity of binding to the liposome membrane and is influenced by the peptide characteristics, the lipid composi- tion, and the peptide-to-lipid ratio. We recommend a specific study to select the most appropriate lipid composition. According to our own experience, rigid membranes made from

a high-Tc-saturated phospholipid (DSPC or DPPC) often exhibit lower a binding capacities for hydrophobic or amphi- philic compounds, when compared to fluid membranes made

from a low-Tc-unsaturated phospholipid (DOPC, natural PC). 23 Liposome-Encapsulated Neuropeptides 353

Furthermore, the presence of CHOL often reduces the extent of peptide binding to the membrane. On the other hand, fluid vesicles prepared without CHOL are expected to be unstable in biological fluids (7). 8. The extrusion process allows the transformation of polydis- perse large oligolamellar vesicles into monodisperse unilamel- lar vesicles of reduced and well-defined size. The liposome size is an important parameter that influences the vesicle pharma- cokinetics (14, 15). Furthermore, large vesicles with a diameter on the order of 1 Pm may clog blood capillaries. Thus, particle size reduction is strongly recommended when liposomes have to be administered intravenously. However, it may not be nec- essary when the liposome suspension has to be microinjected locally into specific sites of the brain. We observed that empty large oligolamellar vesicles produced mechanical stimulation after microinjection at the rostral ventrolateral medulla (2). This effect was not observed with a liposome suspension cali- brated with 200-nm polycarbonate membrane. Therefore, we recommend the size calibration of liposomes, even though this process also reduces the peptide encapsulation efficiency. On the other hand, we do not recommend size reduction below 200 nm (for instance, using polycarbonate membranes with 100 nm pore size) as such small liposomes may extravasate more readily to the neighboring areas of the microinjection site. Although the factors influencing the in vivo fate of liposomes have not been investigated specifically in the brain tissue, previ- ous studies performed with liposomes administered subcuta- neously in the dorsal side of the paw of rats have shown that the size of liposomes is a critical parameter that determines their ability to migrate from the injection site (16, 17). 9. To guarantee that the gradient of peptide concentration cre- ated across the liposome membrane during the dialysis process will result in minimal release of encapsulated peptide, the rec- ommended temperature of the assay is 4°C to maintain a low fluidity and permeability of the liposome membrane. 10. When angiotensins were encapsulated in liposomes according to the proposed method, the mean hydrodynamic diameter was about 200 nm and PdI was inferior to 0.2 (1, 3). 11. Colloidal particles dispersed in a solution are electrically charged due to their ionic characteristics and dipolar attributes. Each particle dispersed in a solution is surrounded by oppo- sitely charged ions called the fixed layer. Outside the fixed layer, there are varying compositions of ions of opposite polarities, forming a cloud-like area. This area is called the diffuse double layer, and the whole area is electrically neutral. When a voltage is applied to the solution in which particles are dispersed, par- ticles are attracted to the electrode of the opposite polarity, 354 F. Frézard et al.

accompanied by the fixed layer and part of the diffuse double layer, or internal side of the “sliding surface.” Zeta potential is considered to be the electric potential of this inner area includ- ing this conceptual “sliding surface.” The zeta-potential will be influenced by the ionic strength of the aqueous medium. Measurement of this parameter in empty and peptide-contain- ing liposomes may be used to reveal the binding of the peptide at the membrane surface. 12. As another alternative method for the determination of the peptide encapsulation efficiency, the formulation coming from the extrusion process (before dialysis) may be submitted to ultrafiltration using Microcon YM-50 ultrafilters to separate the non-encapsulated peptide from liposomes. Determination of peptide concentration in the ultrafiltrate allows deducing the amount of peptide incorporated in liposomes from the known initial amount of peptide. 13. According to the proposed methodology, typical peptide encapsulation efficiency is in the range of 15–20%. Lipid loss is minimal during the process of preparation of the liposomes. However, if a precise determination of lipid concentration is needed, this can be performed through colorimetric assays for phospholipid (18, 19). 14. Hamilton syringe can be filled with distilled water, and a small air bubble must be present between the water and the lipo- some solution into the polyethylene tube. Observing the movement of the air bubble, when the pump is activated helps to indicate a successful injection. In our experience, a volume of 100 nL is the minimum for a safe microinjection into the brain of conscious rats using this method. 15. To evaluate the spreading of liposomes from the microinjec- tion site, empty liposomes labeled with non-exchangeable lipophilic fluorescent marker, DiI may be prepared and used according to the method proposed by Claassen (20). Briefly, DiI dissolved in ethanol at 2 mg/mL is added to the liposome suspension at a final concentration of 10 Pg/mL and incubated for 60 min at 60°C. The preparation of labeled liposomes is then dialyzed against PBS for 24 h at 25°C to eliminate resid- ual ethanol. Following microinjection of DiI-labeled lipo- somes, animals are euthanized at different time intervals. Frozen brains are cut into 16-Pm sections and DiI fluorescence is observed as red with an Axioplan Zeiss fluorescence micro- scope, using BP 545 excitation filter and O 570 emission filter. When applied to the study of the localization of DSPC/ CHOL/DSPE-PEG(2000) liposomes with 200-nm mean diameter after microinjection into the rostral ventrolateral medulla, this methodology demonstrated the permanence of the liposomes in this region for at least 7 days (1). 23 Liposome-Encapsulated Neuropeptides 355

Acknowledgments

This work was supported by FAPEMIG (PPM, REDE 221/08), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and the National Institute of Science and Technology in Nanobiopharmaceutics. FF, RASS, and MAPF are recipients of CNPq research scholarship.

References

1. Silva-Barcellos, N.M., Frézard, F., Caligiorne, 11. Moghimi, S.M., and Patel, H.M. (1998) S. et al (2001) Long-lasting cardiovascular Serum-mediated recognition of liposomes by effects of liposome-entrapped angiotensin-(1–7) phagocytic cells of the reticuloendothelial at the rostral ventrolateral medulla. Hypertension system—the concept of tissue specificity. Adv. 38, 1266–1271. Drug Deliv. Rev. 32, 45–60. 2. Silva-Barcellos, N.M., Caligiorne, S., Santos, 12. Slepushkin, V.A., Simões, S., Dazin, P. et al R.A.S. et al (2004) Site-specific microinjection (1997) Sterically stabilized pH-sensitive lipo- of liposomes into the brain for local infusion of somes. Intracellular delivery of aqueous con- a short-lived peptide. J Control Release 95, tents and prolonged circulation in vivo. J. Biol. 301–307. Chem. 272, 2382–2388. 3. Silva-Barcellos, N.M., Frézard, F., and Santos, 13. Momekova, D., Rangelov, S., Lambov, N. R.A.S. (2007) A novel approach based on nan- (2010) Long-circulating, pH-sensitive lipo- otechnology for investigating the chronic somes. Methods Mol. Biol. 605, 527–544. actions of short-lived peptides in specific sites 14. Allen, T.M., Austin, G.A., Chonn, A. et al of the brain Regul. Pep. 138, 59–65. (1991) Uptake of liposomes by mouse bone 4. Kirby, C., and Gregoriadis, G. (1984) marrow macrophages: influence of liposome Dehydration–rehydration vesicles: a simple composition and size. Biochim. Biophys Acta method for high yield drug entrapment in lipo- 1061, 56–64. somes. Biotechnology 2, 979–984. 15. Senior, J., Crawley, J.C.W., and Gregoriadis, G. 5. Naya, R., Hope, M.J., and Cullis, P.R. (1989) (1985) Tissue distribution of liposomes exhib- Generation of large unilamellar vesicles from iting long half-lives in the circulation after long-chain phosphatidylcholines by extrusion in vivo injection. Biochim. Biophys Acta 839, technique. Biochim Biophys Acta 986, 200–206. 1–8. 6. Paxinos, G., and Watson, C. (2007) The Rat 16. Allen, T.M., Hansen, C.B., and Guo L.S.S. Brain in Stereotaxic Coordinates. Academic (1993). Subcutaneous administration of lipo- Press, Amsterdam. somes: a comparison with the intravenous and 7. Frezard, F. (1999) Liposomes: from biophysics intraperitoneal routes of injection. Biochim to the design of peptide vaccines Braz. J. Med. Biophys Acta 1150, 9–16. Biol. Res. 32, 181–189. 17. Oussoren, C., Zuidema, J., Crommelin, D.J.A. 8. Allen, T.M., Hansen, C., Martin, F. et al (1991) et al (1997) Lymphatic uptake and biodistribu- Liposomes containing synthetic lipid deriva- tion of liposomes after subcutaneous injection. tives of poly(ethylene glycol) show prolonged II. Influence of liposomal size, lipid composi- circulation half-lives in vivo. Biochim Biophys tion and lipid dose. Biochim Biophys Acta 1328, Acta 1066, 29–36. 261–272. 9. Oussoren, C., and Storm, G. (1999) Role of 18. Stewart, J.C. (1980) Colorimetric determina- macrophages in the localization of liposomes in tion of phospholipids with ammonium ferro- the lymph nodes after subcutaneous adminis- thiocyanate. Anal. Biochem. 104, 10–14. tration. Int. J. Pharmacol. 183, 37–41. 19. Bartlett, G.R. (1959) Colorimetric assay meth- 10. Derksen, J.T.P., Baldeschwieler, J.D., and ods for free and phosphorylated glyceric acids. Sherphof, G.L. (1988) In vivo stability of ester- J. Biol. Chem. 234, 469–471. and ether-linked phospholipid-containing lipo- 20. Claassen, E. (1992) Post-formation fluorescent somes as measured by perturbed angular labelling of liposomal membranes. In vivo correlation spectroscopy. Proc. Natl. Acad. Sci. detection localisation and kinetics. J. Immunol. USA 85, 9768–9772. Meth. 147, 231–240.

Chapter 24

Recombinant Adeno-Associated Viral Vectors

Marijke W.A. de Backer, Keith M. Garner, Mieneke C.M. Luijendijk, and Roger A.H. Adan

Abstract

Recombinant adeno-associated viral (rAAV) vectors can be used to locally or systemically enhance or silence gene expression. They are relatively nonimmunogenic and can transduce dividing and nondividing cells, and different rAAV serotypes may transduce diverse cell types. Therefore, rAAV vectors are excellent tools to study the function of neuropeptides in local brain areas. In this chapter, we describe a protocol to produce high-titer, in vivo grade, rAAV vector stocks. The protocol includes an Iodixanol gradient, an anion exchange column and a desalting/concentration step and can be used for every serotype. In addi- tion, a short protocol for rAAV injections into the brain and directions on how to detect and localize transduced cells are given.

Key words: Recombinant adeno-associated virus, rAAV, Vector production, Vector purification, Serotype, Central nervous system

1. Introduction

Recombinant adeno-associated viral (rAAV) vectors are single- stranded DNA vectors that are able to deliver genes to both divid- ing and nondividing cells. For this reason, they are used as tools to study gene function as well as to develop gene-based therapies. In addition, rAAV vectors can establish stable long-term expression, are nonpathogenic and show no to little immunogenicity (1, 2). rAAV vectors are derived from wild-type AAV by removing most of the viral genome (the rep and cap genes) and replacing it with a promoter and sequence of interest. Thus, only the inverted termi- nal repeats (ITRs) on both ends of the AAV genome are kept in the vector. These ITRs are necessary for replication and packaging of the rAAV vector. To produce infective rAAV particles in cells, the

Adalberto Merighi (ed.), Neuropeptides: Methods and Protocols, Methods in Molecular Biology, vol. 789, DOI 10.1007/978-1-61779-310-3_24, © Springer Science+Business Media, LLC 2011 357 358 M.W.A. de Backer et al.

rep and cap genes, but also helper genes from adenovirus, are necessary. The helper genes are necessary because wild-type AAV is a Dependovirus, which requires helper viruses, such as adenovirus or herpes simplex, to replicate and assemble viral particles. The first generation of rAAV vectors was helper-virus-based (produced by cotransfection of a rAAV plasmid with a plasmid containing AAV rep and cap genes, followed by an infection with a helper virus such as an adenovirus) (3), but later “helper-free” systems were devel- oped, where several genes of a helper virus were provided on a plasmid (4–8). The AAV rep and cap genes and the helper genes can be provided by transfection (located on 1 or 2 plasmids) or by stable cell lines (4, 7, 9–11). These newer production methods improve rAAV vector yield and reduce/eliminate detectable con- tamination with replication competent AAV (4, 5, 7, 12). There are several ways to encapsidate rAAV plasmids. The rAAV genome can be packaged with his “true” capsid (ITR, rep and cap genes are derived from same wild-type virus, e.g., AAV2), but it can also be pseudotyped with capsids from other AAV sero- types (ITR and rep gene are from one serotype and cap gene is from another serotype) or with genetically altered capsids (reviewed in refs. 13, 14). The different AAV serotypes have distinct cell specificities, because they probably use different receptors and entry pathways to introduce their genome into cells. For several serotypes, the entry receptors and coreceptors are known. However, for most serotypes the entry receptor remains to be determined (15–20). Until now, the most widely used AAV serotype is AAV2; however, recent studies have shown that other serotypes can be more efficient in transduction of several rat brain areas, such as striatum, hippocampus, midbrain, and hypothalamus (21–26). Nevertheless, the optimal serotype in one brain area is not neces- sary the most efficient in another brain area or at another develop- mental time point. Thus, for every “new” injection area or developmental time point, several serotypes may have to be tested to determine the most optimal method for rAAV-mediated gene delivery to a specific area/cell type. Cell-type specific expression can of course also be achieved by the use of cell-type specific pro- moters to drive transgene expression. One limitation of rAAV is that it has a small packaging size; maximal 4.8 kb can be packaged without loss of infectivity (27, 28). However, this is not a problem when small genes, such as neuropeptides or short hairpin RNAs are overexpressed. In addi- tion, rate limiting steps in rAAV mediated gene expression are the slow uncoating rate and the conversion from single-stranded DNA to double-stranded DNA. This last step, which is necessary for onset of gene expression, may be circumvented by the use of self- complementary rAAV (scAAV) vectors (29–33). During replica- tion, the sense and the antisense strand of scAAV cannot dissociate due to a mutation in one of the ITRs. These scAAV vectors show 24 rAAV Vectors 359

improved onset of expression and increased expression levels; however, the size of the rAAV cassette is reduced from 4.8 to 2.4 kb. When the optimal method to transduce a certain brain area, at a certain developmental stage, in a specific species is known, the method can be used to overexpress or suppress a desired gene. rAAV vectors have also been used to stimulate or reduce recep- tor signaling, for instance by overexpression of neuropeptide genes. However, there are two problems with the long-term overexpres- sion of neuropeptide genes via viral vectors. First, the precursor gene encodes for multiple peptides, which may serve different functions (see above). Therefore, overexpression of the precursor gene may reflect the effects of multiple peptides. The second prob- lem occurs when one is interested in the effects of a neuropeptide in a target area. When a neuropeptide is overexpressed in a target area, it can potentially be released also in a projection area to which the transduced neuron projects, due to anterograde transport of vector derived mRNA and protein, rather than being released locally at the site of transduction (25, 34, 35). To overcome these problems other groups showed that it is possible to use other pre– pro signals to overexpress neuropeptides in other secretion routes (36–39). We recently have overexpressed a minigene cDNA

(AgRP83–132) and showed that it is constitutively secreted in vitro and resulted in behavioral changes in vivo (de Backer et al., accepted BMC Neuroscience). We usually inject rAAV vectors pseudotyped with AAV1 in a nucleus of the adult rat hypothalamus via a microinfusion pump at a fixed speed and at a titer of 1 × 109 genomic copies (g.c.). This transduces almost the whole brain nucleus in which we are inter- ested, such as lateral hypothalamus or the paraventricular hypo- thalamus (26). However, when larger brain areas need to be transduced, the titer can be increased, multiple injections can be done, or viral spread can be enhanced by convection-enhanced delivery (40, 41), coinjection of heparin (for AAV2 serotype, (42), or coinjection of mannitol (43, 44)). This chapter describes a protocol to produce high-titer, in vivo grade, rAAV vectors. In addition, we describe how to establish and analyze rAAV-mediated gene expression in the brain.

2. Materials

2.1. rAAV Production 1. Human Embryonic Kidney 293T cells.

2.1.1. Cell Culture 2. Dulbecco’s Modified Eagle Medium (DMEM) (Gibco – and Transfection Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (Lonza, Basel, Switzerland), 1× nonessential amino acids (n.e.a.a., 100×, Gibco), 1× glutamine (100×, Gibco), 360 M.W.A. de Backer et al.

and 1× penicillin–streptomycin (pen/strep, 100×, PAA Laboratories, Pasching, Austria). 3. Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 2% FBS, 1× nonessential amino acids, 1× glutamine, and 1× penicillin–streptomycin. 4. 1× trypsin (10× PAA Laboratories). 5. Solution of polyethyleneimine [PEI, Mw 25 kDa (Polysciences Inc., Warrington, PA) dissolved in water (0.323 g/L, pH = 8.0)]. This solution has to be freeze-thawed at least four times before use. 6. 1.5 M NaCl. 7. The desired rAAV plasmid (see Notes 1 and 2) and helper plasmid(s) (see Note 3).

2.1.2. Harvesting 1. Cell scrapers (available from Corning Life Sciences, Lowell, of Transfected 293T Cells MA or Sigma Chemicals, St. Louis, MO). 2. Ice-cold phosphate-buffered saline (dPBS, Gibco) with 5 mM ethylenediamine tetraacetic acid (EDTA) (Sigma Chemicals). Store at 4°C. 3. Ice-cold buffer containing: 150 mM NaCl and 50 mM Tris, pH 8.4. Store at 4°C.

2.2. rAAV Purification 1. Dry ice–100% ethanol.

2.2.1. Purification 2. Benzonase (Sigma Chemicals). by Ultracentrifugation 3. Polyallomer Quick-seal tubes 25 × 89 mm (Beckman Coulter, in Iodixanol Gradient Brea, CA). 4. OptiPrep (60% iodixanol, Lucron Bioproducts, Sint Martens- Latem, Belgium). 5. 10× Dulbecco’s phosphate-buffered saline (10× dPBS, Gibco).

6. 1 M MgCl2. 7. 1 M KCl. 8. 0.5% phenol red: dissolve 0.25 g in 50 mL 50% ethanol. 9. 50 mL 15% Iodixanol: 12.5 mL 60% iodixanol, 5 mL 10× PBS,

10 mL 5 M NaCl, 50 PL 1 M MgCl2, 125 PL 1 M KCl, 75 PL

0.5% phenol red, H2O up to 50 mL. 10. 50 mL 25% Iodixanol: 20.83 mL 60% iodixanol, 5 mL 10×

PBS, 50 PL 1 M MgCl2, 125 PL 1 M KCl, 100 PL 0.5% phenol

red, H2O up to 50 mL. 11. 50 mL 40% Iodixanol: 33.4 mL 60% iodixanol, 5 mL 10× PBS,

50 PL 1 M MgCl2, 125 PL 1 M KCl, H2O up to 50 mL. 24 rAAV Vectors 361

12. 60% Iodixanol: 25 mL 60% iodixanol + 25 PL 1 M MgCl2, 62.5 PL 1 M KCl, 12.5 PL 0.5% phenol red. 13. Ti70 rotor (Beckman Coulter). 14. Two 20G needles and a 5-mL syringe.

2.2.2. Purification by 1. 5 mL Hitrap Q HP columns (GE Healthcare, Amersham, UK). Column Chromatography 2. Buffer A: 20 mM Tris, 15 mM NaCl, pH 8.5. For 500 mL, mix 10 mL 1 M Tris–HCl pH 8.4 and 1.5 mL 5 M NaCl, bring volume up to 500 mL, in between adjust pH to 8.5. 3. Buffer B: 20 mM Tris, 500 mM NaCl, pH 8.5. For 500 mL, mix 10 mL 1 M Tris–HCl pH 8.4 and 50 mL 5 M NaCl, bring volume up to 500 mL, in between adjust pH to 8.5. 4. Infusion pump or an Akta prime automated liquid chromatog- raphy system (GE Healthcare). 5. When using an infusion pump, twenty-five 15-mL tubes with a gradient from 100%A to 100%B have to be made, in each tube buffer A is decreased by 4% and buffer B is increased by 4%. Tube 1 contains 100%A; tube 2: 96%A + 4%B; tube 3: 92%A + 8%B, etc. until tube 25: 100%B. 6. Label 25 15-mL tubes (1 through 25), which are used to col- lect the eluate.

2.2.3. Screening Fractions 1. Dilution buffer containing 10 Pg/mL salmon sperm (Sigma for rAVV Vector Genomes Chemicals) in water. 2. 2 M NaOH. 3. 2 M HCl (from 37%, 12.1 M commercially available solution). 4. 1 M Tris–HCl (Tris obtained from Roche, Basel, Switzerland), pH 8.4. 5. PCR primers (available from Sigma Chemicals) (see Note 14), Taq enzyme, and 10× PCR mix (GE Healthcare).

2.2.4. Concentration 1. Amicon centricon Plus-20 Biomax-100 concentrator and Desalting of rAAV (Millipore, Billerica, MA). Vector Fractions

2.2.5. Titration by qPCR 1. Dilution buffer containing 10 Pg/mL salmon sperm (Sigma Chemicals) in water. 2. 2 M NaOH. 3. 2 M HCl (from 37%, 12.1 M commercially available solution). 4. LightCycler and capillaries (Roche). 5. Primers (available from Sigma Chemicals). 6. LightCycler FastStart DNA MasterPLUS Sybr Green I kit (Roche). 362 M.W.A. de Backer et al.

2.3. In Vivo Brain 1. Obtain approval for injection of rAAV in mouse or rat brain Injections and perform experiments according to guidelines and regula- and Determination tions of the relevant authorities. of Injection Site 2. 70% ethanol. 3. Anesthetics [0.1 mL/100 g Hypnorm (Janssen Pharmaceutica, Beerse, Belgium) intramuscular], [0.1 mL/100 g Rimadyl (Pfizer animal health, NewYork, NY) is diluted 1:10 resulting in 5 mg/mL before subcutaneous injections], and saline. 4. Lubricant eye ointment. 5. Surgical tools including small surgical scalpel and scissors, small dull forceps, fine sharp forceps, small bone scraper, and “surgical hooks.” 6. Laboratory scale. 7. Electric hair shaver. 8. Cotton swabs. 9. Hand-held drill. 10. 1- and 5-mL syringes. 11. Injection probes (make two probes for bilateral injections): insert a 30G needle (Kloehn, 9009-30) in a 22G needle (Kloehn, 9009-22) and make it to fit (see Fig. 1). Each probe is connected to a syringe via a PE10 tube. 12. Infusion pump. 13. Stereotaxic apparatus. 14. Digital angle meter.

Here a PE10 tube is inserted 4 mm

11 mm

Fig. 1. Injection needle to inject rAAV vectors into the brain. 24 rAAV Vectors 363

3. Methods

3.1. rAAV Production There are several protocols to produce and purify rAAV vectors (9, 45, 46). Some protocols can only be used only for AAV2 sero- typed rAAV vectors (e.g., heparin column and A20 affinity col- umn), while others can be used for all serotypes (e.g., CsCl gradient followed by dialysis). Here, we describe a protocol which is based upon the protocol by Zolothukin (45) and can be used for all sero- types and includes an Iodixanol gradient and an anion exchange column. One has to keep in mind that the purification scheme may interfere with the transduction pattern which is observed; Klein et al. showed that AAV8 purified on CsCl gradient transduced neu- rons and astroglia, while AAV8 purified on an Iodixanol gradient transduced only neurons, this difference may be caused by differ- ences in protein impurity in the rAAV preparations (23). During rAAV production and purification all operations with rAAV vectors have to be performed in a dedicated tissue culture hood (BL-2 level in the Netherlands) and an incubator separate from those used for maintaining cell lines. In addition, it is essen- tial to dispose virally contaminated materials properly. rAAV trans- fection and harvesting takes 5 days and the purification and titer determination of rAAV preparations takes another 2–3 days.

3.1.1. Cell Culture 1. 293T cells are passaged twice a week with trypsin (see Note 4). and Transfection For one rAAV preparation ten plates (of 15 cm) full with 239T cells are required (see Note 5). 2. On day 1, 18–24 h before transfection, plate 15 dishes of 15 cm with 293T cells, at 40–60% confluency. 3. On day 2, the cells should be 80–90% confluent. One to two hours before transfection replace the growth medium with prewarmed DMEM containing 2% FBS, n.e.a.a., glutamine and penicillin–streptomycin. 4. Perform transfections at a molar ratio of 1:1 of rAAV plasmid– helper plasmid. For each 15 cm dish 10 Pg of rAAV plasmid is necessary, (our rAAV plasmids are usually ~6 kb). The pDP helper plasmids are approximately 23.7 kb, thus for a 1:1 M ratio, 39.5 Pg of pDP helper plasmid is necessary (see Notes 6, 7 and Table 1). The transfection protocol is adapted from Reed et al. (47). 5. Thaw the plasmids and centrifuge the eppendorf tubes for 5 min at 14,000 × g in a table centrifuge prior to setting up the transfection (see Note 8). 6. Make master mix 1 containing the appropriate amounts of rAAV plasmid, helper plasmid, 1.5 M NaCl (end concentration 0.15 M NaCl), and water for 15 dishes (solution 1). For an example, see Table 1. 364 M.W.A. de Backer et al.

Table 1 Example transfection mixtures

Volume Concentration mg per 15 cm Volume per 15 cm 15 × 15 cm Solution 1 (mg/mL) dish dish (mL) dishes (mL)

rAAV plasmid 1 10 10 150 (~6 kb) pDP helper 1 39.5 39.5 592.3 (23.7 kb) 1.5 M NaCl 50 750

H2O 400.5 6,007.5 End volume 500 7,500

Solution 2 Volume per 15 cm dish (mL) Volume for 15 × 15 cm dishes (mL)

PEI (8 PL/Pg DNA) 396 5,940 1.5 M NaCl 50 750

H2O 54 810 End volume 500 7,500

7. Make master mix 2, for 15 dishes, in a separate tube. Solution 2 contains 8 PL of PEI per Pg of DNA together with 1.5 M NaCl (end concentration 0.15 M NaCl) and water; see Table 1. 8. Add solution 1 to 2, invert the tube several times and let it stand for 20 min at RT. During these 20 min occasionally invert the tube. This incubation is necessary to form PEI-DNA complexes. 9. After incubation the mix is added dropwise to the cells, add 1 mL per plate. Before returning the plates to the incubator, gently rock the plates back and forth and sideways to achieve uniform distribution of the PEI-DNA precipitates throughout the plate. 10. Three to six h after transfection once again gently rock the plates back and forth in the incubator to enhance the transfec- tion efficiency. 11. Approximately 20 h after transfection (day 3), replace the medium with fresh prewarmed DMEM + 2% FBS. The old medium is removed and placed in an empty old medium bottle. Before discarding the old medium, add 1% SDS and sodium hypochlorite to the bottle to inactivate possible rAAV contamination. 24 rAAV Vectors 365

3.1.2. Harvesting 1. At 60 h post transfection, scrape the cells from the plates with of Transfected 293T Cells a rubber policeman, resuspend them in their current media and collect the cells in prechilled 50-mL tubes (see Note 9). Store the tubes with the transfected cells on ice. 2. Centrifuge the 50-mL tubes at 1,000 × g for 10 min at 4°C. 3. Discard the supernatant. 4. Resuspend the cell pellets in a total of 40 mL cold 1× PBS with 5 mM EDTA (4°C) by pipetting up and down. Collect the pel- lets from the different tubes in one 50 mL tube. 5. Centrifuge at 1,000 × g for 10 min at 4°C. 6. Resuspend the pellet in 12 mL cold buffer (150 mM NaCl, 50 mM Tris, pH 8.4; 4°C) in a 50 mL tube. When fewer plates are used, resuspend the pellet of every 15-cm dish in 1 mL buf- fer (e.g., 10 plates are resuspended in 10 mL); however, do not exceed 12 mL because of size limitations of the Iodixanol gra- dient. Store the resuspended cells at −20°C until purification. We usually continue after 3 days.

3.2. rAAV Purification 1. Lyse cells by three freeze-thaw cycles between dry ice–ethanol (100%) and 37°C water bath, to release rAAV from cells. Every 3.2.1. Purification time the cells are thawed, vigorously vortex the tube to resus- by Ultracentrifugation pend the cells. in Iodixanol Gradient 2. Add benzonase to the cells to a final concentration of 50 units/mL and incubate at 37°C for 30 min. Benzonase is a nuclease and is used to remove much of the contaminating cellular DNA, unpackaged DNA and makes the solution less viscous. 3. Centrifuge the cells at 16,200 × g for 20 min at RT. 4. Discard the cell pellet. 5. Load 12 mL of supernatant into an OptiSeal tube and under- lay with iodixanol solutions using a glass Pasteur pipette and a perfusion pump at a setting of ~3 mL/min (Fig. 2). Prevent the layers from mixing. (a) 9 mL 15% iodixanol in PBS-MK + 1 M NaCl (b) 6 mL 25% iodixanol in PBS-MK (c) 5 mL 40% iodixanol in PBS-MK (d) 5 mL 60% iodixanol If necessary, top the gradient with PBS using a glass Pasteur’s pipette fitted with a rubber bulb (see Note 10). 6. Balance the tubes. 7. Seal tubes and centrifuge at 504,350 × g in Ti70 for 1.25 h at 18°C. 8. Insert a needle in the top of the tube to enable extraction of the 40% layer. Puncture the tube with a needle attached to a 366 M.W.A. de Backer et al.

Before After centrifugation centrifugation

Virus

15% 15%

25% 25%

40% 40% Virus

60% 60%

Fig. 2. Iodixanol gradient before and after centrifugation. rAAV is isolated from the 40 to 60% interface with a needle attached to a syringe; at the top of the tube another needle is inserted to enable extraction.

syringe just (~2 mm) below the 40–60% interface and remove ~4 mL from the 40% iodixanol layer (see Fig. 2 and Note 11). This solution may be stored at 4°C for use the next day or frozen at −20°C for longer storage.

3.2.2. Purification 1. Increase the volume of the extracted 40% layer to 20 mL with by Column Buffer A before loading onto Hitrap Q column. Chromatography 2. Attach the Hitrap Q column to the perfusion pump (see Note 12). 3. Perform a run as described in Table 2 and collect 2 mL frac- tions (see Note 13).

3.2.3. Screening Fractions 1. Mix the following solutions in eppendorf tubes to lyse the for rAAV Vector Genomes rAAV capsids and release single-stranded AAV DNA: 4 PL of every AAV fraction, 16 PL dilution buffer and 20 PL 2 M NaOH. Incubate at 56°C for 30 min. 2. Neutralize by adding: 20 PL 2 M HCl and 10 PL 1 M Tris– HCl, pH 8.4. 3. Use 2 PL for PCR reaction. 4. Make PCR master mix, which contains per reaction the follow-

ing: 17.8 PL H2O, 2.5 PL 10× Taq Buffer, 0.5 PL 10 mM dNTPs, 1 PL forward primer (10 pmol/PL) (see Note 14), 1 PL reverse primer (10 pmol/PL) (see Note 14), and 0.2 PL Taq. 24 rAAV Vectors 367

Table 2 Run Hitrap Q column on perfusion pump

Step Volume (mL) Buffer Task Flow rate (mL/min)

1 25 B Preequilibrate with buffer B 4 2 50 A Preequilibrate with buffer A 4 3 20 Sample Load virus onto Hitrap Q 2 4 50 A Wash away unbound material 3 5 2 per tube Gradient 100%A Gradient to elute virus from 2 to 100% B column 6 30 B Wash column 4

5. Add the mix to 2 PL of lysed rAAV fractions. Also run a negative and positive control. 6. Run the following PCR program: (a) 95°C for 5 min (b) 95°C for 30 s (c) 58°C for 30 s (d) 72°C for 1 min (e) 25 cycles (steps (b) to (d)) (f) 72°C for 10 min (g) 8°C forever. 7. Determine on agarose gel which fractions resulted in a PCR band and are thus positive for rAAV DNA. For AAV prepara- tions with AAV1 capsids and AAV2 ITRs, we usually find posi- tive fractions in tubes 9–20.

3.2.4. Concentration 1. Pool the fractions which were positive for rAAV vector and Desalting of rAAV genomes. Vector Fractions 2. Sterilize a Centricon Plus-20 Biomax-100 concentrator with 70% ethanol for a few minutes. 3. Aspirate the ethanol and rinse the column with 1× PBS. 4. Transfer the pooled fractions to the column and centrifuge at RT for 5 min, 200 × g. Discard the flow-through into a bottle with 1% SDS and sodium hypochlorite. 5. Add 15 mL 1× PBS to the column and pipette up and down carefully around the filter to make sure that the filter does not get blocked. However, be careful not to touch the filter with the end of the pipette. Centrifuge at RT for 5 min, 200 × g. 368 M.W.A. de Backer et al.

6. Discard the flow-through in 1% SDS-sodium hypochlorite and repeat steps 5 and 6. 7. At the end of the cycles of desalting and concentration, con- tinue to centrifuge until nearly all the liquid has passed through the filter. 8. Carefully pipette the last remainder (~250 PL) up and down along the filter and collect it into an eppendorf tube. 9. Aliquot the rAAV vector in batches of 10 PL and store them at −80°C (see Note 15).

3.2.5. Titration by qPCR We perform titration according to the paper of Veldwijk et al. (48). 1. Thaw one aliquot and lyse the AAV capsid to release the ssDNA by mixing 4 PL AAV stock, 16 PL dilution buffer, 20 PL 2 M NaOH. 2. Incubate at 56°C for 30 min. 3. Neutralize by adding 20 PL 2 M HCl. 4. Dilute the sample 1:100 in dilution buffer. 5. Use 2 PL for quantitative PCR in a LightCycler. 6. Make PCR master mix, which contains per reaction the following: 2 PL primer cocktail (25 pmol/PL F and 25 pmol/PL R), 2 PL water, 4 PL Sybr Green mix. 7. Load capillaries into the holder, pipette 8 PL master mix into capillaries and subsequently add 2 PL of the sample (lysed AAV stock), 2 PL DNA standards [1 × 104, 1 × 106 and 1 × 108 DNA

molecules/PL (preferably of an rAAV plasmid diluted in H2O, these standards also function as a positive control), or 2 PL water (negative control)]. 8. Push tops on the capillaries and centrifuge to transfer liquid to the bottom of the capillaries. Start LightCycler program and type sample name, standards and negative control with the corresponding capillary number. In addition, add the known values for the standards: 2 × 104, 2 × 106 and 2 × 108. 9. Run the following program: (a) 95°C for 10 min, temperature transition rate 20°C/s (b) 95°C for 10 s, 20°C/s (c) 60°C for 5 s, 20°C/s (d) 72°C for 20 s, 20°C/s (e) 35 cycles (from steps (b) to (d)). 10. Calculate titer as genomic copies/mL. The titer obtained with qPCR has to be multiplied by 0.75 × 106 to obtain the genomic copies/mL, since 4 PL of rAAV stock was diluted 15× to obtain 60 PL. This was diluted 100× before start qPCR. The titer 24 rAAV Vectors 369

value from qPCR is for 2 PL and has to be in milliliter there- fore the titer has to be increased with a factor 500. 11. To obtain an indication for the infectious particles perform a serial dilution of rAAV vector stock on cells. Usually, GFP- positive cells are counted after 72–96 h (see Note 16).

3.3. In Vivo When rAAV vectors are made they can be used in vitro or in vivo, Brain Injections to study gene function. Note that when rAAV vectors are used and Determination for in vivo research, the infected neurons retain their native of Injection Site properties in intact neuronal networks, and the effect on behaviors can be investigated. 1. Determine the coordinates of the brain area of interest with stereotaxic atlases such as The mouse brain in stereotaxic coordinates (G. Paxinos and K.B.J. Franklin, Academic Press) and The rat brain in stereotaxic coordinates (G. Paxinos and C. Watson, Academic Press) (see Note 17). 2. Before starting surgery, ensure that all tools and reagents are clean (sterilized) and ready to be used. The surgical area should be disinfected with 70% ethanol. 3. Fill the tubing via needles connected to the stereotact with PBS, air, and rAAV vector (see Note 18) and put a mark on the tubing where the virus is located. This to verify if the virus has moved through the tube during injection. 4. Weigh the animal and calculate the appropriate dose for anes- thesia. After administering anesthesia (Hypnorm), the animal should fall asleep and full anesthesia should be reached within 5–10 min (lack of response to foot pinch), after which Rimadyl is given. 5. Shave the fur of the skull, clean the skin with 70% ethanol, apply lubricant eye ointment (to prevent dry eyes), and place the animal in an aligned stereotaxic apparatus, fixing the animal with ear bars and incisor adapter. 6. Visualize the top of the skull and make a midline incision with a small surgical scalpel, separate the subcutaneous and muscle tissue and use small hooks to keep the area open. Gently clean bregma and lambda areas using a small bone scraper. 7. Measure the position of x and y coordinates of bregma and intra-aural line and calculate the coordinates of target injection area. The intra-aural line can be used as a control. 8. Verify the angle of the needles with a digital angle meter because the stereotact arms may not be exactly 0°. Afterward, determine the injection sites with the needles that are placed in the holder of the stereotaxic arms and label the area for drilling. 9. Drill holes in the skull. 370 M.W.A. de Backer et al.

10. Place needles back into position. Determine once again the angle of the needles with a digital angle meter. 11. Bring the needles to the correct x and y position and lower until they touch the exposed dura. Penetrate the dura with the tip and slowly lower the needles to the desired z coordinate. 12. At the desired coordinates turn on the infusion pump and infuse rAAV at a rate of 0.2 PL/min (see Note 19). When the appropriate amount of virus is injected, turn off the pump and wait for 10 min before withdrawing the needles. 13. Suture the skin. 14. Keep the animals warm under a blanket in a clean cage (single- housed). Inject analgesic (Rimadyl) on the first and second day after surgery. Monitor the recovery closely for at least 1 week (see Note 20). 15. rAAV expression in vivo depends on genomic context of the vector (ssAAV or dsAAV), serotype used and tissue transduced. Usually after 2–3 weeks, the expression of rAAV cassette has reached its plateau and the expression can remain this high for years (22, 49, 50). 16. At the end of the experiment, animals can be perfused or decapitated, depending on what you want to do with the brain tissue next. All our rAAV vectors contain the green fluorescent protein (GFP); therefore, we can use GFP to localize the cells transduced by rAAV. However, other parts of the vector, such as woodchuck posttranscriptional regulatory element (WPRE), can also be used for localization. After perfusion we perform free-floating immunostaining against GFP on 40-Pm thick vibratome or microtome cut brain slices (can be performed together with NeuN, to check if predominantly neurons are transduced). However, after decapitation an in situ hybridiza- tion against GFP, on 20-Pm thick cryostat slices, is most opti- mal to detect transduced cells.

4. Notes

1. The size of the insert of interest plus inverted terminal repeats (ITRs) may not exceed 4.8 kb, since packaging of larger viral genomes is less efficient, thus resulting in low amounts of rAAV particles (27, 28). 2. The rAAV vectors are prone to recombination due to the inverted terminal repeats (ITRs). The ITRs are required for rAAV replication and packaging and need to be intact. To min- imize recombination rAAV plasmids are propagated in JC8111 at 30°C with 2× ampicillin (these cultures will grow slowly, 24 rAAV Vectors 371

20–22 h for minipreps). Other groups reported that Stbl2 (Invitrogen) bacteria may also be used for propagation of rAAV (51, 52). Since recombination may still occur, every maxiprep is checked with the restriction enzyme SmaI to ensure that there was no (or only little) recombination in the ITRs. The SmaI digestion should be optimal (26°C for 2 h or overnight at RT); when there is an intense band at a size, which occurs after partial digestion, a new maxiprep should be made, since this may indicate that one of the ITRs cannot be cleaved any- more and is recombined. In addition, several other restriction enzymes are used to check the plasmid. 3. We generally use helper plasmids constructed by (53), which we obtained from the Plasmid factory (Bielefield, Germany). These helper plasmids contain adeno-helper genes and AAV rep and cap genes on one plasmid. The helper plasmids are grown into gigapreps (Qiagen) at 37°C with 1× ampicillin, since large amounts of helper plasmids are necessary. In addi- tion, there is also a packaging system where the adenohelper genes and AAV genes are located on two different plasmids (4, 54). 4. All tissue incubations are performed in a humidified 37°C, 5%

CO2 incubator. All equipment and reagents that come into contact with tissue culture cells should be sterile as should be the purified virus. 5. We generally do not use cells with passage number higher than 25 for rAAV preparations. 6. The different pDP helper plasmids encode different capsid proteins; this makes it possible to pseudotype rAAV vectors with another capsid, which uses a different (often unknown) cell entry receptor, and thereby the tropism of the rAAV vector can be altered so that it may transduce other cell types. 7. When the adeno-helper genes and the AAV rep/cap genes are located on two plasmids the molar ratio for transfection is 1:1:1 for rAAV–adeno helper–rep/cap plasmids. 8. This spin is to remove column debris from maxi prep DNA (Qiagen maxi preps); the debris can reduce transfection efficiency. 9. If possible, check transfection efficiency under a microscope before collecting the cells; there are fluorescent markers in the pDP helper constructs. High transfection efficiency (at least 80%) is important to obtain high titers of rAAV preparations. 10. Preferably these large Beckman tubes in combination with 70Ti rotor have to be used. Previously, we used the smaller OptiSeal tubes (16 × 60 mm) in combination with 70.1Ti rotor and had to prepare several iodixanol gradients for one rAAV 372 M.W.A. de Backer et al.

preparation and subsequently pooled the different 40% layers; however, this may reduce the titer. 11. Sometimes, there is a “fluffy” band at the 25–40% interface; this is cell debris and should not be extracted. 12. We use perfusion pump; however, the column can also be attached to an Äktaprime automated liquid chromatography system (Amersham Biosciences). The program is shown in Table 3. 13. During the gradient run, the pump is set at a flow rate of 2 mL/min. Every minute, the pump inlet is moved one gradient tube further (from 1 to 25, thereby decreasing buffer A with 4% and increasing buffer B with 4%). Meanwhile, once per minute the tubes collecting the column eluate are changed, generating fractions of 2 mL. Usually, we change the pump inlet at 1.00 min, 2.00 min, 3.00 min etc. and the collection tubes are changed at 1.30 min, 2.30 min, 3.30 min, etc. 14. Depending on the rAAV vector used, we use bgh-polyA (F2 and R2) or GFP (F and R) primers. Their sequences are the following: (a) bgh-polyA F2: 5c CCTCGACTGTGCCTTCTAG (b) bgh-polyA R2: 5c CCCCAGAATAGAATGACACCTA (c) GFP-F: 5c CACATGAAGCAGCACGACTT (d) GFP-R: 5c GAAGTTCACCTTGATGCCGT

15. The rAAV vectors should not be thawed often, since every freeze–thaw step reduces the titer. 16. We primarily use HT1080 cells to verify if the ratio of genomic copies to infectious particles is constant over different rAAV vector preparations. However, depending on the serotype other cell lines may be more optimal, since different serotypes use different entry receptors (55). 17. These coordinates can be checked by anesthetizing rats of same age and gender, as the rats that will be used in the rAAV experi- ment, and inject them with methylene blue instead of rAAV. Directly after injection, the animals are decapitated and the brains are removed, frozen, sectioned, and inspected for methyl blue localization. 18. Use an appropriate titer of rAAV to transduce the area of interest. We generally inject 1 PL containing 1 × 109 g.c. of an AAV1 serotyped vector to transduce nuclei in the hypothala- mus, such as PVN, VMH, and LH. Increasing or decreasing the volume or the amount of rAAV genomes can increase or decrease the area that is transduced (26). There appears to be a minimum threshold of ~5 × 107 g.c. rAAV genomes to achieve consistent transduction of areas in the rat brain (49). Different 24 rAAV Vectors 373 superloop buffer A automated liquid chromatography system prime 1 0 100 40 0 1 Waste Prime buffer B 2 24.9 100 40 0 1 Waste End priming B 3 25 0 40 0 1 Waste Prime buffer A 4 50 0 5 0 1 Load Equilibrate 5 74.9 0 5 0 1 Load End equilibration 6 75 0 2 0 1 Inject Inject sample from 78 95 145 0 0 5 3 0 2 1 1 Load Load column with Wash Start gradient elution 9 195 100 3 2 1 Load End elution 10 195.1 100 3 2 1 Load Continue elution with B 11 225 100 3 2 1 Load End Table 3 Table Run Hitrap Q on Äkta Step Volume Concentration %B Flow rate Fraction Buffer valve Injection valve Comments 374 M.W.A. de Backer et al.

rAAV serotypes can have different transduction efficiencies in one brain area. In addition, the transduction efficiency of one rAAV serotype can be different between brain areas, develop- mental stages, and species. 19. We generally infuse 1 PL; thus, we inject for 5 min. Larger volumes can be injected by increasing the injection time, but keeping the injection speed at 0.2 PL/min. 20. Monitor for any signs of distress, such as piloerection, lack of grooming, reduced locomotor activity, and wound scratching. During the first days after surgery, food and water intake drop, but they usually return to normal levels ~7 days post operation.

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Deorphanizing G Protein-Coupled Receptors by a Calcium Mobilization Assay

Isabel Beets, Marleen Lindemans, Tom Janssen, and Peter Verleyen

Abstract

G protein-coupled receptors (GPCRs) comprise one of the largest families of transmembrane proteins involved in signal transduction of diverse external stimuli and represent the most successful target class in drug discovery. The availability of genome sequences in the postgenomic era has paved the way for in silico identification of novel GPCR family members based upon sequence similarity. Consequently, newly discov- ered receptors are by definition orphan GPCRs with no known ligand, and their functional characteriza- tion now poses a major challenge. Over the years, advances in understanding of GPCR biology have led to the development of cell-based assay systems that link orphan GPCRs to their activating ligand(s) in high- throughput format (reverse pharmacology). Many of these technologies monitor important steps in the GPCR activation cycle such as the accumulation of secondary messenger molecules (e.g., cAMP, calcium). In this chapter, we present a calcium mobilization assay in mammalian cells to detect changes in intracel- lular calcium concentration upon receptor activation by the use of a fluorescent probe. This is currently one of the most frequently used assay systems for GPCR deorphanization.

Key words: Receptor deorphanization, G protein-coupled receptor, Reverse pharmacology, Calcium mobilization, FLEXstation®

1. Introduction

G protein-coupled receptors (GPCRs) comprise one of the largest families of membrane-spanning proteins involved in signal trans- duction with representatives across animals, plants and fungi (1). By transmitting external stimuli as diverse as hormones, neurotrans- mitters, odorants, light, and bioactive peptides, they occupy a high hierarchical position in the regulation of physiological processes and represent the most successful target class in terms of drug dis- covery with therapeutic benefits across a broad spectrum of human

Adalberto Merighi (ed.), Neuropeptides: Methods and Protocols, Methods in Molecular Biology, vol. 789, DOI 10.1007/978-1-61779-310-3_25, © Springer Science+Business Media, LLC 2011 377 378 I. Beets et al.

diseases (2, 3). In general, GPCRs display a seven D-helical transmembrane topology with alternating extra- and intracellular loops. Signal transduction upon ligand binding primarily relies on the activation of intracellular heterotrimeric G proteins (3). Despite these similarities, individual receptors can have unique signaling outcomes and growing insights into non-G protein-linked signaling pathways, receptor dimerization, and the structural basis of GPCR function underscore that many additional elements of the GPCR signaling scheme are yet to be discovered (4, 5). In the postgenomic era, bioinformatic search programs (e.g., BLAST, phylogeny analysis, etc.) have complemented PCR-derived screening methods in the search for novel GPCRs, which has resulted in the large-scale identification of novel GPCR family members (6, 7). Consequently, newly discovered receptors identi- fied upon sequence similarity are by definition orphan GPCRs (oGPCR) with no known ligand. The fact that many of these receptors are conserved among different species suggests that they should be active and the search for their natural ligand(s) repre- sents a crucial step in functional characterization. For this purpose, the reverse pharmacology approach was adopted as a general and high-throughput “deorphanization” strategy (Fig. 1). The oGPCR is first expressed in a recombinant eukaryotic system by DNA transfection. Following expression, a compound set of candidate ligands is screened against the receptor and recep- tor activation is measured (7, 8). Various compound resources can be used in oGPCR screening, for example biological tissue extracts (classic orphan receptor strategy) that provide the oppor- tunity to isolate novel, yet uncharacterized ligand molecules (7, 9, 10). Over the years, many groups have also adopted the strat- egy of randomly screening large compound libraries, mainly con- sisting of synthetic replicates of known and predicted GPCR ligands (reverse pharmacology) (11–13). In contrast to tissue extracts, this strategy requires no further fractionation, purifica- tion and molecular characterization of the ligand once an activat- ing compound has been identified. More recently, in silico search methods have become of growing importance for predicting oGPCR ligands, such as analyses of sequence homology and/or phylogenetic relationships with known receptor–ligand pairs (14, 15). Specifically for the identification of peptide ligands, pattern/ motif-based search algorithms can be used as an alternative approach to detect consensus sequence motifs (15, 16). Even if the exact iden- tity of the ligand cannot be determined from bioinformatic approaches, its nature (e.g., subfamily) can often still be discrimi- nated to guide future functional assays. The concept of reverse pharmacology has presented a major driving force for the development of cell-based assay technologies that allow high-throughput GPCR characterization. Deorphanization success of these assay types depends on the appropriate expression 25 Receptor Deorphanization 379

Fig. 1. Principle of reverse pharmacology to identify ligands of oGPCRs. A compound test set, consisting of candidate GPCR ligands, is composed through one or more different strategies: by preparing fractionated tissue samples (tissue extract- based approach), by using large compound libraries consisting of all known and predicted GPCR ligands (library-based approach) or by synthesizing synthetic replicates of prospective ligands that were identified through in silico search methods (in silico-based approach). The resulting compounds are screened for their receptor-activating abilities. The oGPCR is, therefore, cloned into a given cellular expression system and one of the many messenger molecules is used to measure activation of the receptor. An activating ligand will be identified according to its ability to cause a dose-dependent increase in the receptor activity.

of the orphan receptor in a recombinant system. Over the years, a number of expression systems have been adopted for oGPCR screening including immortalized mammalian cell lines, yeast, and Xenopus melanophores (8). The majority of GPCR characteriza- tions have been accomplished using Human Embryonic Kidney (HEK) and Chinese Hamster (CHO) cells due to their ease of use and proven history of functional GPCR expression (17). However, no expression system is suitable for deorphanizing all vertebrate or invertebrate oGPCRs, and screening conditions might need optimization to guarantee that the receptor is expressed at the cell surface and can couple to the signal transduction machinery of the host cell. The selection of rationally designed expression vectors (e.g., pcDNA vector series for mammalian cell lines) that generate high recombinant protein levels, therefore, facilitates ligand identi- fication (3). Very often, a transient expression system is chosen, as the construction of stable cell lines can present a labor-intensive task. Epitope-tagging strategies, GPCR-specific antibodies or green fluorescent protein (GFP)-fusion proteins can be used to ensure 380 I. Beets et al.

that the receptor is localized to the cell membrane in case there is little or no interference with ligand binding and/or functional acti- vation of the receptor (8, 17). On top of an appropriate expression system, oGPCR screening experiments require a broad-based assay system, capable of detect- ing receptor activation mostly without prior knowledge on the activating compound or evoked signaling cascade. Advances in understanding GPCR biology have permitted the implementation of such functional assay technologies in high-throughput format. The resulting detection strategies were already the subject of many reviews and are generally based on different steps in the GPCR activation cycle such as the activation of G proteins, production and release of secondary messenger molecules (e.g., cAMP, cal- cium), translocation of arrestin, changes in membrane potential, altered gene transcription, etc. (17–21). Most of these cell-based assays employ fluorescence or bioluminescence detection and thus require a certain degree of engineering, manipulation, or labeling (such as loading of fluorescent molecules into cells or expression of a reporter gene). Recently, label-free cell-based assays have been introduced to monitor ligand-induced responses in living cells. The principle of these noninvasive detection systems is based on the use of optical or electrical biosensors that measure the dynamic redistribution of cellular contents caused by receptor activation (19). Different assay systems often complement one another and application of a certain technology mostly depends on the expres- sion system, the knowledge on the endogenous signaling cascade of the receptor of interest (which is often very limited), and the need for automated assay systems for high-throughput screening. Probably the most widely used oGPCR screening method is the calcium mobilization assay, which has already proven its valid- ity in a large number of GPCR characterizations in humans, as well as in invertebrates (21). The method is based on the measurement of intracellular Ca2+, which is released from storage sites upon

ligand binding of GDq-coupled GPCRs. General application of this assay has been advanced by the discovery that coexpression of

GPCRs with promiscuous G protein D-subunits (e.g., GD15 and

GD16) can mostly direct intracellular signaling to a calcium flux,

regardless of the endogenous G protein coupling (GDq, GDi, or

GDs) of the receptor of interest (22). An alternative approach is the use of chimeric G proteins, in which the C-terminal five/six amino acids of one G protein have been replaced with those of another, leading the signal cascade to a pathway of choice (23). This way, calcium assays provide an almost generic detector of GPCR activa- tion. On top, there is a large signal-to-noise ratio due to rapid amplification of the calcium signal, which often occurs within seconds of activation of a GPCR (17). Fully automated high- throughput systems allowing simultaneous compound addition and signal detection for every well in an assay plate (96-, 384- or 25 Receptor Deorphanization 381

1,536-well plates) have been developed to measure rapid calcium fluxes in real time. The two predominant assay systems adopted in oGPCR screening experiments are based on the use of biolumines- cent proteins, such as the calcium-sensitive photoprotein aequorin and fluorescent calcium indicators (e.g., Fluo-4) (21, 24). Mostly fluorescence-based assays, in which receptor-expressing cells are loaded with a fluorescent calcium indicator prior to ligand screen- ing, have become increasingly popular in high-throughput experi- ments due to their ease of use, short reading time, and the flexibility of screening multiple oGPCRs on a single plate. In this chapter, we describe a detailed methodology for identi- fying ligands of oGPCRs with a fluorescence-based calcium mobi- lization assay in mammalian cells, as up till now this is one of the most generally applied deorphanization methods. Prior to ligand binding, it is often difficult to predict the likely G protein-coupling of a receptor. In the presented example, signaling of the oGPCR is, therefore, directed to a calcium flux by transient coexpression of

the GPCR and the promiscuous GD16 protein. The protocol is described in detail for protein expression in HEK293T cells but other mammalian cell lines (such as CHO cells) have been used successfully. For best results, parameters including transfection and dye loading conditions should be optimized for each receptor–cell- line combination. Fluorescence measurements are performed by the FLEXStation® device (Molecular Devices, CA, USA), which is a benchtop sister device of the FLIPR® (Molecular Devices) and currently one of the main platforms available for the development of fluorescent plate-based assays in 96- or 384-well microtiter plate format. The instrument allows simultaneous compound addition and signal detection of every well in the microtiter plate, which is necessary to measure the fast kinetics of the calcium response. Other instruments, e.g., the NOVOstar® (BMG Labtechnologies, Offenburg, Germany), can be adopted for this assay as well. Along the protocol, we highlight the opportunities and challenges associ- ated with this deorphanization technique.

2. Materials

2.1. Cell Culture 1. Dulbecco’s phosphate-buffered saline (PBS), without calcium and Cell Transfection chloride and magnesium chloride (available from Sigma Chemicals, St. Louis, MO). 2. Solution of 0.25% trypsin and 1 mM ethylenediamine tetraa- cetic acid (EDTA). 3. DMEM complete medium: Dulbecco’s Modified Eagle’s Medium (DMEM, with 4,500 mg/L glucose, L-glutamine, sodium bicarbonate, without sodium pyruvate) supplemented 382 I. Beets et al.

with 10% fetal bovine serum and penicillin–streptomycin (100 units/mL–0.1 mg/mL). 4. FuGENETM 6 Transfection Reagent (Roche, Brussels, Belgium). 5. DMEM/HEPES (25 mM): DMEM supplemented with 25 mM HEPES.

6. Purified GPCR expression construct and GD16 plasmid DNA (see Note 1).

2.2. Calcium 1. Dulbecco’s PBS, without calcium chloride and magnesium Mobilization Assay chloride. 2. HBSS buffer: Hank’s Balanced Salt Solution (with sodium bicarbonate, without phenol red).

3. HEPES stock solution: 1 M HEPES in distilled water (dH2O), pH 7.4. Store at room temperature.

4. CaCl2 stock solution: 1 M CaCl2 in dH2O. Store at room temperature. 2+ 5. HBSS/HEPES/Ca /BSA buffer: Add 165 PL CaCl2 stock solution, 500 PL HEPES stock solution, and 0.05 g bovine serum albumin (BSA) to 50 mL HBSS buffer and heat to 37°C. Prepare 50 mL buffer per 96-well plate. Always use fresh buffer, as precipitate might form upon storage. 6. Probenecid solution (100×, 250 mM): Dissolve 0.71 g probenecid (Sigma Chemicals) in 5 mL NaOH (1 M). Add 50 PL HEPES stock solution and 5 mL of heated HBSS/ HEPES/Ca2+/BSA buffer. Use on the same day. 7. Wash buffer: Add 500 PL (100×) probenecid to 50 mL heated HBSS/HEPES/Ca2+/BSA buffer (see Note 2). Adjust pH to 7.4 and sterilize buffer solution by filtration. Prepare 50 mL buffer per 96-well plate. 8. 10% pluronic acid solution: Mix 50 PL pluronic acid (20% w/v in dimethyl-sulfoxide (DMSO), Invitrogen, Carlsbad, CA) with 50 PL DMSO. When crystallization occurs, heat to 37°C. Use on the same day. 9. Fluo-4 AM solution (1 mM): Add 44 PL 10% pluronic acid solution to a vial containing 50 Pg fluo-4 AM (Invitrogen) and vortex until dissolved (see Note 3). Fluo-4 AM solution should not be exposed to light to avoid bleaching of the fluorophore. 10. Loading buffer: Add 8.8 mL DMEM supplemented with 10 mM HEPES and 2.5 mM probenecid (pH 7.4) to the Fluo-4 AM solution and vortex. Loading buffer for one 96-well plate now contains a final concentration of 5 PM fluo-4 AM. This buffer should not be stored for later usage. 25 Receptor Deorphanization 383

2.3. Assay 1. FLEXStation® device (Molecular Devices, CA, USA). on FLEXstation® 2. Well plates: black-walled polystyrene plates (96 wells) with clear bottom and poly-D-lysine coated surface (Corning Incorporated, NY, USA) are used as cell plates for the FLEXstation assay. Polystyrene V-shaped 96-well plates (Greiner Bio-One, Frickenhausen, Germany) are used as compound plates.

2.4. Data Analysis 1. Soft Max Pro® software (Molecular devices).

3. Methods

The principle of the fluorescence-based calcium mobilization assay for GPCR deorphanization is depicted in Fig. 2a. Cells transfected

with the GPCR of interest and the promiscuous GD16 subunit (see Note 4) are screened with different compounds. During the assay, receptor activation is measured by the release of intracellular cal- cium from storage sites such as the endoplasmic reticulum upon agonist binding. Prior to ligand screening, receptor-expressing cells are, therefore, loaded with the fluorescent calcium indicator Fluo-4, which is one of the most commonly used dyes for this assay system (see Note 5). The fluorophore is administered to the cells as a cell membrane permeable derivative carrying acetoxymethyl (AM) ester groups. Once inside the cells, Fluo-4 AM is cleaved by intracellular esterases, thereby unmasking negative charges in the dye molecule and subsequently trapping the fluorophore in a cell- impermeant form, capable of binding Ca2+ ions. Hydrolyzed Fluo-4

binds calcium with a Kd of 345 nM and is, therefore, well suited for measuring physiological important calcium changes in a wide range of cells. In its purified form, the dye is essentially nonfluorescent but upon Ca2+ binding fluorescence enhancement can be increased to more than 100-fold depending on the free calcium concentration. The Fluo-4 signal is measured over time by a microplate reader. Activating ligands of the oGPCR are identified according to their ability to cause a dose-dependent increase in receptor activity, with

EC50 values typically corresponding up to the nM-range. A graphi- cal output for the calcium response of a ligand-activated receptor measured on the FLEXstation® device is depicted in Fig. 2b. Advanced software packages such as Soft Max Pro® (Molecular devices) can be used for further data analysis. During the calcium mobilization assay, several parameters such as transfection efficiency, cell quality, and cell adherence can influence the final calcium response. Therefore, it is recommended to validate the calcium assay with a “standard” ligand for which the receptor is already known. Recombinant cells expressing this standard receptor and its corresponding ligand should be included as positive control on every cell and compound plate respectively. Moreover, a positive 384 I. Beets et al.

Fig. 2. Overview of the fluorescence-based calcium mobilization assay for oGPCR screening in mammalian cells. (a) HEK293T cells transfected with the GPCR of interest and the promiscuous GD16 subunit are screened with different compounds.

Upon receptor activation, an exchange of GDP for GTP takes place at the GD16 subunit, which dissociates from the GEJ subunit and activates phospholipase CE (PLCE). PLCE in turn hydrolyzes phosphatidylinositol bisphosphate (PIP2) into diacylglycerol (DAG) and inositol trisphosphate (IP3). IP3 diffuses into the cytoplasm where it opens the IP3 dependent cal- cium channels, thereby releasing Ca2+ from the endoplasmic reticulum (ER). Subsequent binding of Ca2+ to Fluo-4 AM, which the cells are loaded with prior to compound addition, results in a fluorescent signal. (b) Typical time-fluorescence curve (representing raw “relative fluorescent unit” (RFU) values in time) for a ligand-activated receptor measured by the FLEXstation® device. A baseline read is taken prior to compound addition and fluorescence measurements continue for 2 min to ensure that the peak fluorescence is obtained. The graph demonstrates the rapid kinetics of the calcium response as a result from receptor activation. The same profile, displaying only lower peak values, can be found for decreasing compound concentrations up to the nM-range.

control with an endogenous agonist (see Note 6) and a negative control (e.g., wash buffer) should be incorporated into the plate design. To obtain good calcium response, parameters presented in the protocol below (cell density, dye concentration and incubation time/temperature, transfection conditions) should be optimized for different receptor–cell-line combinations.

3.1. Cell Culture For the read-out of one 96-well plate, HEK293T cells are grown and Cell Transfection in 150 cm2 culture flasks (see Note 7) and for transient expression,

transfected with GPCR and GD16 (if needed) expression constructs 2 days before assay. Several methods exist to introduce the con- structs into host cells. The example presented here uses the lipid transfection reagent FuGENETM 6. 1. Remove all media from cells and wash dead cells off with PBS. Harvest cells with trypsin-EDTA solution and collect them in DMEM complete medium. 25 Receptor Deorphanization 385

2. Count and seed cells in a culture flask (150 cm2) containing 25 mL DMEM complete medium at a density of 70,000 cells/cm2 (see Note 8).

3. Incubate for 24 h (37°C and 5% CO2) or until the cell culture approaches 50–70% confluence. 4. Transfer 1,406.4 PL serum-free DMEM/HEPES (25 mM) to a sterile polystyrene tube. Add dropwise 93.6 PL FuGENETM 6 Transfection Reagent (do not allow FuGENETM 6 to contact the walls of the tube). Gently tap the tube and incubate 5 min at room- temperature. Add a total amount of 15.6 Pg purified plasmid DNA

(e.g., 7.8 Pg oGPCR and 7.8 Pg GD16 expression construct) to the transfection mix without contacting the walls and gently tap the tube. Incubate for 30–45 min at room temperature (see Note 9). 5. Add the transfection mix dropwise to the cell culture, still con- taining 7 mL of DMEM complete medium. Pipette directly into the medium without contacting the walls of the culture flask.

6. Incubate the cells for 24 h (37°C and 5% CO2).

3.2. Calcium 1. Collect the cells as described in step 1 of Subheading 3.1. Mobilization Assay 2. Seed the cells in a 96-well black-walled, clear-bottom plate at 2 3.2.1. Cell Plating a density of 70,000 cells/cm (see Notes 8 and 10). Use a volume between 100 and 200 PL per well.

3. Incubate the cell plate for 16–24 h (37°C and 5% CO2).

3.2.2. Loading Cells 1. Prepare wash and loading buffer. with Dye 2. Remove media from the cells and wash with 200 PL PBS per well (see Note 10). 3. Add 55 PL loading buffer per well and incubate for 1 h at 37°C (see Note 11). Avoid exposure of the plate to light, for example by wrapping the plate in aluminum foil. 4. Prepare ligand solutions in wash buffer and place them into a 96-well plate (see Note 12). Remark that ligands will be diluted during the assay in 100 PL wash buffer per well. Thus, when transferring 50 PL of the compound plate to the cell plate, compounds must be prepared at 3× their final concentration. It is recommended to prepare a small excess of ligand solution to correct for death volume when transferring the ligand solution from the compound plate to the cell plate. Always use negative (e.g., wash buffer) and positive controls (e.g., endogenous agonist, deorphanized receptor–ligand pair) on each plate. 5. After 1 h incubation time, remove all loading buffer and replace with 100 PL wash buffer. Incubate the cell plate for 15 min at room temperature. Avoid light exposure. 6. Remove wash buffer and replace with 100 PL wash buffer. 7. Incubate the cell plate, together with the compound plate and tip rack, for 15 min at 37°C. 386 I. Beets et al.

3.3. Assay on 1. Prepare the FLEXstation® device for measurement. Load the FLEXstation® appropriate protocol file and activate the temperature control unit for the reading chamber (see Note 13). 2. Place the cell plate, compound plate, and pipette tips in the appropriate drawers. It is recommended to wait 5–10 min before starting the experiment to reduce fluorescent signal variability caused by disruption of cells during transfer to the microplate reader. 3. Initiate compound addition and data capture (see Note 14).

3.4. Data Analysis Advanced software packages such as SoftMax Pro® can be used to visualize and analyze data from the FLEXstation®. An example of the graphical output is depicted in Fig. 2b. Fluorescent measure- ments usually start prior to compound addition and continue at least until after the peak fluorescence upon receptor activation has been obtained (e.g., for 2 min). The kinetic profile of an agonist typically shows a rapid increase in fluorescence upon addition to the cells. Corresponding peak heights decrease when lower concentra- tions of the agonist are used. High concentrations ranging from 10 to 1 PM are normally used for initial screening of a compound set. Compounds that elicit a clear and fast calcium response, as com- pared to a positive control, should then be tested in dilution series

up to nM concentrations to obtain a dose–response curve and EC50 value (typically in the nM-range) as illustrated in Fig. 3 (25).

Fig. 3. Representative dose–response curve of the Caenorhabditis elegans pigment dis- persing factor (PDF) neuropeptide PDF-2 on the PDFR-1b receptor. Calcium fluorescence was measured at PDF-2 concentrations ranging from 10−4 to 10−12 M. Values are repre-

sented as relative (in %) to the highest value (100% activation). The resulting EC50 value typically corresponds to the nM-range (25). 25 Receptor Deorphanization 387

4. Notes

1. Several high-level eukaryotic expression vectors are commercially available for protein expression in mammalian cells, such as the pcDNATM vectors (Invitrogen) that generate high recombinant expression driven by the human cytomegalovirus (CMV) pro- moter. Using a PCR-based strategy, the open reading frame (ORF) of the GPCR of interest is cloned into the vector according to the manufacturer’s guidelines. Incorporation of a (partial) Kozak sequence immediately preceding the authentic initiation codon is recommended for optimized translational initiation in eukaryotic cells. When isolating the plasmid DNA, it is best to use an endotoxin-free plasmid preparation kit, as bacterial endotoxins can reduce transfection efficiency in mam- malian cells. Always verify the sequence of the constructs before use in transfection studies. 2. Probenecid is an inhibitor of inorganic anion transporters that are able to extrude the hydrolyzed form of Fluo-4 AM from the cytoplasm, thus reducing the fluorescent signal. Comparison of dye loading in the presence and absence of probenecid can determine whether inorganic anion transporters present a problem in the cell line under investigation, and thus whether probenecid should be added to the wash buffer or not. 3. Addition of a nonionic detergent, such as pluronic acid, can improve dispersion of apolar Fluo-4 AM in aqueous solution. Therefore, it should be mixed with Fluo-4 AM before adding the fluorophore to other buffer solutions.

4. The GD16 subunit is one of the most frequently used promiscuous G proteins due to its ability to link a wide range of GPCRs to

a calcium response. Nevertheless, it should be noted that GD16 binds to most, if not all, GPCRs and has been reported to have a dominant-negative effect on receptors that endogenously

couple via GDq. GD16 is most efficient on GDs-coupled recep- tors but provides suboptimal results for GPCRs that normally

couple via GDi (26). Therefore, it is recommended to test dif- ferent combinations of G proteins when optimizing a novel

calcium assay and to compare assay results for GDq-coupled

GPCRs in the absence or presence of GD16. Alternatively, func- tional assays have been developed to detect ligand-induced receptor activation independent of the interacting G protein. One of the best known examples is the translocation study of GFP-labeled arrestin, but so far it is still difficult to format this assay system in a high-throughput manner. The development of kits that allow fluorescent detection of changes in mem- brane potential (e.g., FLIPR Membrane Potential Assay Kit, Molecular Devices) might also present a promising method for 388 I. Beets et al.

studying GPCR activation in the future, as GPCR signaling often results in ion fluxes across membranes. Label-free assays based on optical or electrical biosensors can measure GPCR activity regardless of the receptor’s endogenous G-protein coupling, but still represent a significantly higher cost when compared to calcium mobilization assays. 5. Different types of calcium-sensitive fluorophores (e.g., Fluo-3, Rhod-2, Fluo-5, Calcium Green-1, etc.) with varying spectral and chemical properties are commercially available. A selection should be made according to the GPCR, cell type and/or available fluorescent plate reader, and verified experimentally. Recently, direct calcium assay kits have been developed that no longer require media removal or wash steps. This way, variabil- ity in fluorescent signal caused by disruption of cells is reduced and nonadherent cells can be used in calcium mobilization assays. Background fluorescence caused by the extracellular dye is reduced by the addition of a suppression dye. Kits utiliz- ing no-wash dyes (e.g., Fluo-4 NW, Invitrogen) share the advantage that additional washing steps after addition of the dye solution are eliminated. Nevertheless, it should be noted that all these assay kits are significantly more expensive than the use of standard Fluo-4 AM. 6. Carbachol is a known activator of the acetylcholine receptor and can be used as positive control for proper loading of the cells with Fluo-4. Alternatively, many cell types constitutively express receptors for ATP. 7. Cells in continuous culture allow 20–25 usable passages for screening. For best results, start the experiment with a cell cul- ture of maximum 3–4 days old when approaching 100% con- fluence. For some cell types, frozen stocks can be directly used to eliminate the workload of continuously culturing large amounts of cells, but this needs to be verified in advance (27). Cells stably expressing the receptor of interest have been used successfully in the calcium mobilization assay, but the genera- tion of these cell lines might be laborious. 8. It is our experience that the cell density for optimal calcium response varies according to the counting method and cell type and requires optimization at the start of a new assay. In general, cell densities fall in the range of 60,000–90,000 cells per/cm2. 9. The amount of transfected DNA and the DNA/FuGENETM 6 ratio should be optimized for each receptor–cell-line combina- tion. Transfection efficiency for a particular DNA/FuGENETM 6 ratio can be determined by using expression constructs encoding GFP. Epitope-tagging strategies, GPCR-specific antibodies, or GFP-fusion proteins can be used to ensure that the receptor is also localized to the cell membrane upon 25 Receptor Deorphanization 389

expression. Take into account that some of these detection strategies might interfere with receptor pharmacology. 10. HEK293T or other weakly adherent cell types can easily dis- lodge from the bottom of the cell plate during subsequent washing steps in the assay. For these cell types, the use of poly- D-lysine coated 96-well plates is recommended. Alternatively, direct calcium assay kits and no-wash dyes have been devel- oped to eliminate media removal and/or washing steps for dye loading (see Note 5). Cells can also dislodge when removing fluid from the cell plate. This can be avoided by simply inverting the plate instead of using a pipette. Afterward, check the cell layer under a microscope. 11. The optimal concentration of dye (normally in the range of 2–8 PM), incubation time, and temperature will vary depend- ing on the cell type and should be optimized for new receptor– cell-line combinations. In general, values described in the protocol above give to our experience good results. 12. The final concentrations used for ligand screening are com- monly 0.1 nM–10 PM. Care should be taken when compound stocks are maintained in solvents that are potentially harmful to living cells, such as peptides stored in acetonitrile. In this case, it is recommended to dry the compound stock before preparing ligand solutions or to use highly concentrated stocks. The final concentration of acetonitrile should not exceed 0.05% after addition of wash buffer. 13. The SoftMax Pro® Software can be used to control and analyze FLEXstation® measurements. Prior to running the assay, a pro- tocol file must be written containing the selected instrument settings. These include the excitation and emission wave- lengths, the type of plates being used, the wells to be read, the reading time for each well, and the frequency by which measure- ments are captured during the run. The speed, height and transferred volume for the integrated pipettor should be opti- mized to reduce fluorescent signal variability caused by disrup- tion of the cell layer upon compound addition. On top, temperature can influence measurements and should be con- trolled by the microplate reader. We generally measure calcium response at 37°C for 2 min (with a 1.52-s interval between successive readings) at 525 nm. Excitation of Fluo-4 is done at 488 nm. A total volume of 50 PL compound is added, 18 s after the reading start, with the dispense speed set at 26 PL/s and a height of 135 PL. 14. Prior to starting the measurements in FLEX mode, a single reading of the microplate can be taken at the same excitation and emission wavelength in the ENDPOINT mode. Values reported are relative fluorescence units (RFUs) and display the variability between different wells of the plate. It is best to 390 I. Beets et al.

optimize these values in advance by performing the assay with a standard receptor and known ligand. According to our expe- rience, values between 20,000 and 40,000 RFU represent good results.

Acknowledgments

ML, TJ and PV benefit a postdoctoral fellowship and IB a PhD fellowship of the Research Foundation – Flanders (FWO- Vlaanderen). We also thank J. Puttemans for elaborating the illustrations.

References

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INDEX

A Aequorin ...... 393 Affinity ...... 66, 70, 71, 144, 190, 211, 304, 308, A260:A280 ratio ...... 270 312, 313, 328, 330, 338, 340, 346, 364, 375 AAV2. See Adeno-associated viral type 2 (AAV2) Agar...... 199 ABC. See Avidin-biotin (ABC) Agarose ...... 102, 103, 106, 107, 118, Absolute concentration ...... 164, 165 145, 147, 256, 258–260, 262, 266, 267, 270, 379 Absorbance ...... 260, 304 Agonist ...... 395–398 Absorption...... 60, 67, 80, 162, 304, 316, 317, 328, 345 Air bubble ...... 131, 133, 168, 178, 270, 366 Absorption, distribution, metabolism, Albumin ...... 72, 87, 129, 133, 185, 208, and elimination (ADME)...... 328 212, 218, 226, 277, 286, 304, 394 Absorptive effect ...... 312 Alcohol ...... 54, 55, 57, 62, Accumulation time ...... 163 133, 135, 229, 255, 258, 269, 270, 287, 288, 330, Acetate buffer ...... 301, 303, 304 333, 345, 351 Acetic acid ...... 51, 62, 81, 207, 209, 246, Alcohol dehydrogenase ...... 218 301, 332, 338, 341 Aldehyde ...... 51, 74, 75, 80, 97, 130 Acetic anhydride ...... 87, 94, 104, 106, 130, 134, 237 Alexa Fluor ...... 71, 72, 75, 79, 80, 95 Acetone ...... 135, 238, 241, 245 Algorithm ...... 177, 240, 390 Acetonitrile (ACN) ...... 207, 209, 216–221, Alignment ...... 167, 170, 178 223, 225, 232, 239–242, 246, 332, 338, 341, 401 Alkaline phosphatase ...... 88, 90, 92–94, Acetylation ...... 18, 87, 94, 134, 246 98, 105, 124, 127–129, 132, 308, 309 Acetylcholinesterase ...... 85 Alternative processing ...... 19 Acidification ...... 363 Alternative RNA splicing ...... 126 ACN. See Acetonitrile (ACN) Alzheimer’s disease ...... 299, 330 Acquisition ...... 109, 155, 156, Amidation ...... 18, 246 198, 240, 241, 243, 338–340, 345 Amide...... 18, 23, 327, 335 ACSF. See Artificial cerebrospinal fluid (ACSF) Amine...... 18, 280, 334, 344 ACTH. See Adrenocorticotropic hormone (ACTH) Amino acid ...... 13, 14, 16–18, Activation ...... 145, 160, 186, 190, 245, 327, 50, 86, 150, 185, 205, 211, 243, 246, 273, 302, 317, 334, 363, 390–392, 395, 396, 398–400 334–336, 344, 353, 371, 372, 392 Activity-dependent secretion ...... 149 Aminoacyl-B-naphthylamide ...... 300 Adaptor ...... 250, 252, 253, 260–263, 265, 267, 270 Amino group ...... 237, 296, 334, 344 Adeno-associated viral type 2 (AAV2) ...... 370, Aminopeptidase ...... 299, 301–303 371, 375, 379 G-Aminopropyltriethoxysliane ...... 283 Adrenocorticotropic hormone (ACTH)...... 15, 19, 20, 203 Amino-terminal. See N-terminal Adenovirus ...... 370 Ammonia...... 218 Adhesion molecule ...... 184, 186 Ammonium acetate ...... 256, 332, 338 Adipose tissue ...... 19, 38 Ammonium citrate ...... 218, 221 ADME. See Absorption, distribution, metabolism, Amphibian ...... 51, 53 and elimination (ADME) Amphiphilic ...... 364 Administration ...... 199, 315–321, Ampicillin...... 382, 383 326, 329, 330, 343, 350, 353 Amplicon ...... 113, 116, 118, 124, 132, 142 B-Adrenergic receptor ...... 186 Amplifier ...... 194, 195, 197, 200 Adsorption ...... 206, 212, 225, 232 Amplitude ...... 161, 173–176, 179, 199

Adalberto Merighi (ed.), Neuropeptides: Methods and Protocols, Methods in Molecular Biology, vol. 789, DOI 10.1007/978-1-61779-310-3, © Springer Science+Business Media, LLC 2011 393 NEUROPEPTIDES 394 Index

AMV reverse transcriptase ...... 131 Arrestin ...... 392, 399 Anaesthetic, Arterial blood ...... 353 Analepsia ...... 330 Artificial cerebrospinal fluid (ACSF) ...... 194, 196, Analeptic ...... 330, 331, 333, 335, 341–343 198–200, 273, 274, 277, 279, 280, 294 Analgesic ...... 62, 299, 362, 374, 382 Artificial membrane ...... 328, 331–332, 337–339 Analysis ...... 78, 109, 110, 113, Arylamide ...... 300, 302, 305 114, 118, 120, 123, 139–147, 151–155, 161–163, [A–35S]-citosine 5’-triphosphate (35S CTP) ...... 103, 119 165, 167, 171–173, 176, 179, 183–191, 194, 196, Aseptic ...... 167, 277 197, 199, 206, 210, 216, 235–246, 253, 255, Astrocyte ...... 16 265–269, 273–276, 283, 287, 294–295, 299, 337, Astroglia ...... 375 338, 342, 352, 357, 360, 362, 390, 395, 398 [A–35S]-uridine 5’-triphosphate (35S UTP) ...... 103, 107, 119 Analytical technique ...... 215 Atmospheric-pressure chemical ionization (APCI) ...... 337, Anesthesia ...... 51, 53–54, 75, 339, 340, 346 280, 281, 318–321, 353, 361, 381 Atmospheric-pressure ionization (API) ...... 241, 338 Anesthetic ...... 51, 53, 61, 62, 199, ATP...... 186, 254, 350, 351, 400 276, 277, 279, 281, 300, 301, 321, 343, 353, 374 Atrium...... 305 Anesthetic mask ...... 53 Audio monitor ...... 196, 199 Angiotensin ...... 362, 365 Auditory meatus ...... 278 Anion exchange ...... 375 Autoantibodies (AutoAbs) ...... 307–313 Annealing ...... 106, 112, 136, 145, 250, 252, 359 Autoclave ...... 102, 127, 185, 190, 280 ANOVA ...... 110, 342, 343 Autocorrelation ...... 172–173 Anterograde transport ...... 149, 371 curve ...... 162–165, 171–176, 179 Antibiotic ...... 185, 276, 278, 279, 318, 362 Autocrine ...... 14, 43 Antibody...... 52, 55, 59–61, 63–67, Autoimmune disorder ...... 183 71, 72, 74, 75, 80, 88–90, 92–95, 97, 124, 129, 132, Autoimmunity ...... 307 190, 191, 195, 198, 204, 206, 210, 211, 269, Autolytic ...... 305 283–297, 307–313 Autoprime ...... 117 microprobes ...... 283–297 Autoradiographic ...... 91, 283, 284, 287, 294–296 penetration...... 65 Autoradiography ...... 104, 109, 119 replacement ...... 60, 312 Autosampler ...... 223, 225, 240, 244, 337 Antidepressant ...... 299 Avalanche photodiode detector (APD) ...... 160, 161, 166 Antifading ...... 88, 95 Avidin...... 55, 70, 88, 93, 195, 198 Antigen...... 50, 53, 55, 57, 59, 60, Avidin-biotin (ABC) ...... 55–57, 60, 64, 70, 74 63–67, 74, 191, 204, 205, 307, 308, 312, 313 Avidity...... 67 Antigenicity ...... 54, 63, 64, 70, 78, 80, 89 AxoGraph ...... 196, 199 Antihypertensive ...... 299 Axon(al) ...... 71, 75, 76, 77, 194 Antisense ...... 103, 107, 116, 117, 349–354, 370 Axo-Tape ...... 196, 199 Antisera ...... 50, 51, 57–60, 205, 207, 208, 210, 296 Azide...... 52, 195, 198, 208, 285, Antiserum ...... 51, 60, 67, 210 286, 290–293, 295, 308, 357, 360 Antrum ...... 57 Aorta...... 75 B APCI. See Atmospheric-pressure chemical ionization (APCI) Bacitracin...... 195, 198 APD imaging ...... 160–162, 166–176, 179 Background ...... 50, 52, 55, 57, 59, API. See Atmospheric-pressure ionization (API) 64, 65, 80, 97, 98, 106, 110, 120, 129, 136, 159–162, Aplysia californica ...... 238 171, 179, 253, 267, 400 Aqueous phase ...... 216, 258, 259, 328, 335, 363, 364 correction ...... 110 Ar. See Argon (Ar) normalization...... 110 Ara-C...... 184, 188 Balance ...... 63, 197, 200, 326, 331, 333, 377, 394 Araldite ...... 73 Barrier filter ...... 162, 195 Arcuate nucleus ...... 145 Basic motif...... 16, 17 Argon (Ar)...... 166, 168, 169, 191, 331, 337, 356, 359 Bath application ...... 195, 198 Ar-ion...... 166, 168, 169 Baytril...... 318, 319 Aromatic ring ...... 211 BBB. See Blood–brain barrier (BBB) Arrayer ...... 255, 268 BC4...... 190 NEUROPEPTIDES 395 Index

BCIP. See 5-Bromo–4-chloro–3-indolylphosphate (BCIP) Borate...... 102, 150, 152, 155, 237, 286 BDNF. See Brain-derived neurotrophic factor (BDNF) Borohydride ...... 286, 292 Beam...... 139, 162, 178 Borosilicate tube ...... 194, 196 path ...... 169 Bouin’s fixative ...... 51 Behavior ...... 15, 175, 176, Bovine serum albumin (BSA) ...... 72, 75, 179, 193, 204, 212, 236, 338, 350, 381 80, 87, 88, 92, 93, 95, 129, 136, 185, 191, 208, 210, Bell jar...... 61 218, 277, 280, 286, 292, 304, 394 Benzene ...... 51, 54, 62, 327 bp...... 102, 105, 113, 116, 134, 144, 145, 269, 366 Benzonase ...... 372, 377 Bradford concentrated solution ...... 301 Benzotriazole–1-yloxy-tris-pyrrolidinophosphonium Bradford stock solution ...... 301 hexafluoro-phosphate (PyBOP) ...... 331, 334–336 Bradykinin ...... 31, 239 Best fitting curve ...... 165 Brain...... 13, 15, 16, 19, 25, 37, 38, 40–46, B/F ratio ...... 204 64, 69, 106, 108, 110, 139, 140, 142–143, 145, 149, Bgh-polyA ...... 384 196, 197, 199, 200, 273–276, 279, 280, 299–302, Bicuculline ...... 197 316, 320, 325–347, 349, 350, 352–357, 361, 365, Bi-dimensional HPLC ...... 220–221 366, 370, 371, 374, 381, 382, 384, 386 Bioactivation ...... 329, 334 Brain-derived neurotrophic factor (BDNF) ...... 43, Bioavailability ...... 316, 363 45, 70–72, 77, 150–155 Bio-beads ...... 300, 302, 305 Brain injury ...... 330 Biocompatibility ...... 362 Brain uptake ...... 328 Biocytin ...... 195, 198, 200 Brain uptake index (BUI) ...... 328, 355 Biodegradable ...... 316 Bregma ...... 278, 279, 361, 381 Bioinformatics ...... 240, 243, 390 Bridge balance ...... 197 Biological activity ...... 14, 15, 18, 41, 43, Brightness ...... 172, 179, 180 326, 329, 355, 362 5-Bromo–4-chloro–3-indolylphosphate (BCIP) ...... 88, Biological fluid ...... 215, 308, 311, 362, 365 92–94, 98, 129, 132, 133 Bioluminescence ...... 392, 393 BSA. See Bovine serum albumin (BSA) Bioreversible ...... 326, 327 Bucket...... 285, 286, 288–290, 292, 296 Biotin...... 55, 64, 70–72, 75, 87, 88, 92–94, 96, 105 Buffered formalin ...... 51, 54 Bipolar...... 195, 197 Buffer RPE ...... 107, 119 Bipolar stimulating electrode ...... 195, 197 BUI. See Brain uptake index (BUI) Bird...... 51, 53, 61 Blade...... 199, 200, 300, 301, 339 C Blank...... 73, 79, 143, 144, 242, 304, 309, 310, 312 BLAST...... 116, 145, 269, 390 14C...... 205 Bleeding ...... 221, 278, 317, 321, 322 C18. See Octadecyl carbon chain (C18) Block...... 54, 55, 57, 59, 75, 93–95, 98, 204, 263, 270 C(t). See Intensity autocorrelation function (C(t)) Blood...... 15, 64, 187, 274, 278, 301, Ca2+...... 18, 186, 189, 191, 392, 394–396 317, 326–328, 339, 342, 352, 353, 365 CADM1 ...... 186 Blood–brain barrier (BBB) ...... 315, 326–328, Cage...... 274, 277, 279, 321, 332, 342, 343, 362, 382 338, 349–354 CALCA...... 24, 126 Blood-cerebrospinal fluid barrier (CSFB) ...... 326 CALCB ...... 24, 126

Blood-to-brain unidirectional influx rate (Ki) ...... 352–354 Calcitonin ...... 24, 37, 70, 126 Blunt end ...... 250, 259, 292 Calcitonin gene-related peptide (CGRP) ...... 24, cloning ...... 267 37, 70, 72, 77, 126–128, 131, 134 B27 medium ...... 15, 152 Calcium flux ...... 392, 393 BMMC. See Bone marrow-derived mast cell (BMMC) Calcium Green–1 ...... 400 BMP. See Bone morphogenic factors (BMP) Calcium imaging ...... 183 Body fluid ...... 204, 211, 329, 346 Calcium indicator ...... 393, 395 Body heat ...... 53 Calcium measurement ...... 185–186, 189–190 Body weight...... 301, 332, 341–343, 346, 347 Calcium mobilization assay ...... 389–402 Bone marrow ...... 188, 189 Calcium response ...... 393, 395, 396, 398–401 Bone marrow-derived mast cell (BMMC) ...... 188, 189 Calibration...... 165, 167, 171, 178, 246, 338, 341, 365 Bone morphogenic factors (BMP) ...... 43 Calibration sample...... 341 Booster dose ...... 204, 208 cAMP...... 392 NEUROPEPTIDES 396 Index

Cancer...... 36, 62, 86, 307 Charge...... 133, 206, Cannula ...... 199, 276–280, 284, 317–322 216, 239, 243, 244, 328, 330, 395 Cap gene ...... 369, 370, 383 CHCA. See A-Cyanohydroxycinnamic acid (CHCA) Capillary ...... 58, 217–221, 225, Chemical communication ...... 13, 16 236, 326, 328, 339, 365, 373, 380 Chemical signal ...... 13, 14, 46 Capillary electrophoresis...... 215 Chemokine ...... 19, 43–45 Capsaicin ...... 190 CHEMS. See Cholesteryl hemisuccinate (CHEMS) Capsid...... 370, 378–380, 383 Chicken ...... 53, 72, 75 Capsule ...... 73, 80 Chimeric G protein ...... 392 Capture ...... 139–141, 143–144, 146, 189, 253, 268, 398 Chloral hydrate ...... 195, 198, 300, 301 Carassius auratus ...... 57, 58 Chloramine T ...... 205, 207, 209 Carbachol ...... 400 Chloranil test ...... 344 Carboxypeptidase E (CPE) ...... 150–155 Chloroform ...... 73, 81, 87, 92, 93, Carcinogen(ic) ...... 61, 62, 65, 79, 305 255, 258, 269, 270, 356, 357, 364 Carotid artery ...... 187, 351, 352, 354 CHO cells ...... 391, 393 Carprofen ...... 381 CHOL. See Cholesterol (CHOL) Carrier...... 308, 312, 316, 326 Cholecystokinin (CCK) ...... 21, 22, 57 protein ...... 205, 208 Cholesterol (CHOL) ...... 356–358, 362, 365, 366 Catecholamine ...... 273 Cholesteryl hemisuccinate (CHEMS) ...... 363

Catestatin ...... 43 Chromatographic capacity factor (k’IAM) ...... 330, Caveolin ...... 160 335, 339, 340, 346, 355 Cavitation ...... 364 Chromatographic column ...... 216, 345 CCK. See Cholecystokinin (CCK) Chrome-gelatin ...... 133 cDNA. See Complementary DNA (cDNA) Chromogranin ...... 37, 41 cDNA amplification ...... 256–258, 269 Chromophore ...... 338 Cell density ...... 168, 396, 400 Chronic action ...... 355, 356 Cell line...... 16, 168, 188, 370, 375, Circulation ...... 317, 326, 328, 329 384, 391, 393, 396, 399–401 Citric acid ...... 52, 218, 346 Celloidin ...... 51, 54 Clathrin ...... 160 Cell-penetrating neuropeptide (CPnP) ...... 159–180 Clearing agent ...... 63, 347 Cell penetrating peptide (CPP) ...... 21, 160 Cleavage ...... 17, 45, 243, 246, 334, 344, 345 Cell plates(ing) ...... 395, 397, 398, 401 Clone...... 253, 267, 269 Cell-surface receptors ...... 159, 160 CLSM. See Confocal laser scanning microscopy (CLSM) Cell suspension ...... 167, 168, 188, 189 Cluster...... 74, 75, 168, 171, 176 Cellular membrane ...... 74, 299 CMV. See Cytomegalovirus (CMV) Cellular uptake ...... 160 Coagulation ...... 63

Centrifuge ...... 105, 107, 108, 111, CO2 asphyxiation ...... 346 112, 119, 152, 166, 167, 184, 185, 187, 188, Coating buffer ...... 308, 309 207–210, 239, 241, 242, 244, 257–261, 263, 266, Co-culture ...... 183–191 270, 300, 302, 333, 339, 341, 350, 352, 359, 375, Coding RNA ...... 86, 90 377, 379, 380 Codon...... 399 Cerebellin ...... 19, 37 Coefficient ...... 120, 160, Cerebral cortex ...... 275 161, 165, 171, 177, 210, 305, 328, 335, 347 Cerebral ventriculi ...... 199 Coelenterate ...... 50 Cerebrospinal fluid (CSF) ...... 194, 206, Co-elution ...... 220 209, 274, 277, 311, 315, 317, 326 Coil...... 285, 288 Cervical dislocation ...... 339, 346 Collagen ...... 38, 166 c-fos...... 113–115, 117, 118 Collection efficiency ...... 161 CGRP. See Calcitonin gene-related peptide (CGRP) Collection tube ...... 107, 108, 119, 274, 384 Chain...... 14, 216, 308, 309, 327, 330, 334, 344–346 Collimator ...... 170 Chain assembly ...... 334 Colloidal gold ...... 70, 74, 75 Chambered coverslip ...... 166–169, 171, 178 Colloidal stability ...... 362 Chaperone ...... 43 Colorimetric assay ...... 366 Charcoal ...... 206, 208, 210 Column ...... 102, 103, 107, 108, 110, 119, NEUROPEPTIDES 397 Index

141, 207, 209, 216–221, 223–225, 239–245, 254, cpm. See Counts per molecule (cpm) 259, 331–333, 338, 339, 345, 346, 373, 375, 378, CPnP. See Cell-penetrating neuropeptide (CPnP) 379, 383–385 CPP. See Cell penetrating peptide (CPP) Column chromatography...... 373, 378 Cranioplastic ...... 318, 319 Combustion analysis ...... 337, 346 Craniotomy ...... 361 Complementary DNA (cDNA) ...... 96, 124, Cresyl violet ...... 140, 141, 143, 145, 146 129, 131, 134, 140–142, 144, 146, 151, 250–270, CRH. See Corticotropin releasing hormone (CRH) 371, 391, 399 Cribriform plate ...... 317, 319, 320 Computational methods ...... 328 Cross-link(ing) ...... 63, 130, 191, 268 Concentration ...... 52, 75, 97, Cross-reactivity ...... 205, 206, 505 112, 133, 145, 160, 188, 189, 196, 204, 216, 236, 250, Cryocutting ...... 95, 141, 146 275, 285, 301, 312, 315, 325, 353, 358, 373, 394 Cryostat ...... 86, 96, Concentrator ...... 239, 242, 373, 379 97, 103, 108, 128, 129, 131, 140–143, 195, 198, 382 Concentric probe ...... 275, 277 Cryostatic sections ...... 63

Confluence ...... 397, 400 Crystal phase transition temperature (Tc)...... 359 Confocal ...... 153, 162 CSF. See Cerebrospinal fluid (CSF) Confocal laser scanning microscopy (CLSM) ...... 161, CSFB. See Blood-cerebrospinal fluid barrier (CSFB) 186, 189 2-$$Ct...... 114 Confocal microscopy ...... 150, 153, 185–186, 189–190 Ct. See Cycle threshold (Ct) Connectivity ...... 78, 284 C-terminal ...... 18, 21, 205, 246, 334, 336, 392 Consensus sequence motif ...... 390 M-Culture dish ...... 189 Constitutive secretory pathway ...... 16, 17 Culture flask ...... 166, 167, 396, 397 Contaminant ...... 220, 221 Current ...... 156, 161, 164, Contamination ...... 113, 167, 193, 196, 198, 200, 218, 224, 316, 334, 339, 377 169, 216, 217, 221, 222, 232, 370, 376 Curve...... 113, 114, 145, 147, Control ...... 14, 50, 162–165, 171–176, 179, 204, 210, 304, 352–354, 70, 97, 109, 126, 145, 152, 167, 210, 216, 236, 258, 396, 398 275, 288, 299, 308, 320, 333, 360, 379, 395 Cut-off...... 242, 244, 245, 274, 281 Control gene ...... 114 Cuvette ...... 88, 94, 151, 152, 156 Convection-enhanced delivery ...... 371 A-Cyanohydroxycinnamic acid (CHCA) ...... 218 Coomassie Brilliant Blue ...... 301, 304 Cycle...... 106, 112, 115, 120, Coordinates ...... 154, 170, 131, 136, 145, 168, 200, 250, 255–258, 267, 270, 199, 276, 278, 279, 301, 361, 381, 382, 384 311, 377, 379, 380, 392 Coplin jar ...... 128, 130, 135 Cycle threshold (Ct) ...... 112–115, 120 Copper ...... 81, 285 Cylinder ...... 285, 288–290, 292, 318, 338, 356 Cornea...... 167, 319, 320 Cytochrome C ...... 218, 230 Coronal suture ...... 278 Cytokine ...... 16, 18, 43, 44, 46 Correction collar ...... 166, 170, 177, 178 Cytomegalovirus (CMV) ...... 399 Correlation time ...... 161, 163, 164, 179 promoter ...... 399 Corrosive ...... 62, 343 Cytoplasm ...... 91, 127, 129, 161, 172–176, 179, 396, 399 Corticotropin releasing hormone (CRH) ...... 19, 28 Cytoplasmic tail ...... 150 Co-transfection ...... 370 Cytoskeleton ...... 175 Cotransmission ...... 85 Cytosol ...... 299, 326, 363 Counterstain ...... 79, 96, 130, 132 D Count rate ...... 169, 170, 179 Counts per molecule (cpm) ...... 108, 119, D–19, 104 169–171, 177, 178, 210, 286, 292 DAB. See 3,3’-Diaminobenzidine (DAB) Coupling ...... 89, 205, 219, Danio rerio ...... 63 220, 225, 334–338, 344, 392, 393, 400 Dark chamber ...... 57 Covalent binding ...... 79, 89, 335 Dark current ...... 161 Coverslip ...... 58, 95, Data analysis software ...... 194, 243 104, 108, 128, 131, 135, 153, 155, 166–169, 171, Database ...... 18, 43, 45, 238, 240, 241, 243, 269 178, 198 Data capture ...... 398 CPE. See Carboxypeptidase E (CPE) Dead volume ...... 225 NEUROPEPTIDES 398 Index

Death...... 54, 61, 156, 397 Dialysis membrane ...... 207, 274, 275, 356 Debris...... 111, 156, 241, 242, 283, 284 3, 3’-Diaminobenzidine (DAB) ...... 52, 57, 60–62, 65, 88 Decay time ...... 164, 165 Diaminobenzimide ...... 93 Declaration of Helsinki ...... 343, 346 Diamond knife ...... 73 Deconvolution ...... 243 DIC. See Differential interference contrast (DIC) Degradation ...... 13, 101, 140, Dichloromethane ...... 331, 337 212, 236, 241, 280, 311, 316, 326, 327, 330, 341, Dichroic mirror ...... 162, 169 349, 362, 363 Diethylpyrocarbonate (DEPC) ...... 127 Degradation rate ...... 101 Differential expression ...... 19, 249, 250, 252, Degranulation ...... 186 253, 267, 269 Dehydration ...... 143, 146, 357 Differential interference contrast (DIC)...... 151, 189, Dehydration-rehydration vesicle (DRV) ...... 357, 359 194, 196 Dektol...... 104 Differentially expressed sequences ...... 250, 252, 253 Delta TPG dish ...... 150, 152, 153, 156 Differentiation ...... 43, 153 Denaturation ...... 106, 145, 270 Diffraction ...... 161, 162 Dendrite(ic) ...... 75, 76, 77 Diffraction limited volume element...... 161, 162 Denhardt’s solution ...... 87, 104, 108 Diffuse double layer ...... 365, 366 Dental cement ...... 276, 278, 279, 318 Diffusion ...... 63, 91, 160–162, Deorphanization ...... 390, 393, 395 164, 165, 171, 173, 175–177, 179, 326, 330 DEPC. See Diethylpyrocarbonate (DEPC) Diffusion coefficient ...... 160, 165, 171, 177 DEPC-PB. See DEPC-phosphate buffer (DEPC-PB) DIG. See Digoxigenin (DIG) DEPC-PBS. See DEPC-phosphate-buffered saline Digestion ...... 127, 129, 130, 133, 260, 270, 383 (DEPC-PBS) Digitizer ...... 194 DEPC-phosphate buffer (DEPC-PB) ...... 127, 128 Digoxigenin (DIG) ...... 86, 87, 90–96, DEPC-phosphate-buffered saline 98, 104, 105, 124, 128, 129, 132, 136 (DEPC-PBS) ...... 128, 130, 131 Digoxigenin–11-dUTP ...... 87, 124, 128, 132, 136 Dependovirus ...... 370 DiI. See 1,1’-Dioctadecyl–3,3,3’,’3’- Depression ...... 316, 330 tetramethylindocarbocyanine perchlorate (DiI) Deprotection ...... 334, 335, 344 Dimethylformamide ...... 129, 331 Desalting ...... 216, 217, Dimethyl sulfoxide (DMSO) ...... 185, 240, 394 219, 221, 223, 236, 239, 242, 245, 373, 379–380 Dimyristoylphosphatidycholine (DMPC) ...... 362 Desiccator ...... 362 1,1’-Dioctadecyl–3,3,3’, 3’-tetramethylindocarbocyanine Detection ...... 50, 57, 60, 63, 67, perchlorate (DiI) ...... 357, 366 70, 86, 88–97, 116, 119, 123, 124, 126, 132, 134, Dioleoylphosphatidylethanolamine (DOPE) ...... 363 160, 161, 163–166, 169, 177, 178, 210, 216, 219, Dioleylphosphatidylcholine (DOPC)...... 362 221, 225, 236, 242, 284, 308–312, 333, 337, 338, Dipalmitoylphosphatidycholine (DPPC) ...... 362 340, 344–346, 392, 393, 399, 401 DIPEA. See N,N’-diisopropyl-ethylamine (DIPEA) buffer ...... 309, 310 Direct esterification ...... 344 efficiency ...... 161, 163 Direct in situ RT-PCR ...... 123–137 limit ...... 50, 210, 219 Disease...... 86, 183, 249, 299, 307, 316, system ...... 60, 67, 88, 89, 97, 309, 312, 392 325, 326, 330, 355, 390 volume ...... 164, 165, 169, 177 Displacement distance ...... 154 Detector ...... 160–162, 169, 274, 280, 345, 392 Disposable ...... 88, 132, 206, 268, 288–289, 296, 333 Detergent ...... 109, 302, 399 Disposal ...... 79, 351 Development ...... 15, 43, 70, 78, 79, 85, Dissection ...... 141, 188, 199, 241 124, 126, 132, 161, 203, 216, 236, 249, 299, 313, Dissociation ...... 75, 145, 147, 156, 165, 191, 311–313 325, 326, 330, 350, 370, 371, 386, 390, 393, 1184 buffer ...... 156 Dextran ...... 87, 104, 108, 206, 208, 210 curve ...... 145, 147 Dextran-coated charcoal ...... 206, 210 Dissociative condition ...... 308 Dextran sulfate ...... 87, 104, 108 1,2-Distearoyl-sn-glycero–3-phosphocholine Diabetes ...... 313 (DSPC ) ...... 356–359, 362–364, 366 Dialysate ...... 275, 276, 280 1,2-Distearoyl-sn-glycero–3-phosphoethanolamine-N- Dialysis ...... 200, 207, 208, 273–276, 280, [methoxy(polyethylene glycol)–2000] 356, 360, 365, 366, 375 (DSPE-PEG(2000)) ...... 356, 357, 362, 363, 366 NEUROPEPTIDES 399 Index

Distilled water ...... 51–53, 55, 72–74, 76, 78, Dulbecco’s modified eagle medium (DMEM) ...... 150, 79, 81, 87, 109, 128, 129, 132, 207, 208, 260, 279, 152, 153, 155, 156, 185, 188, 371, 372, 375, 376, 300, 301, 359, 366, 394 393, 394, 396, 397 Dithiothreitol (DTT) ...... 103, 255, 304 Dummy cannula ...... 276, 279, 280 DMEM. See Dulbecco’s modified eagle medium (DMEM) Duplex...... 96 DMPC. See Dimyristoylphosphatidycholine (DMPC) Dura...... 279, 382 DMSO. See Dimethyl sulfoxide (DMSO) dUTP...... 87, 92, 124, 128, 132, 136 DNA...... 86, 87, 96, 102–108, 113, 116, Dye...... 65, 120, 144, 145, 165, 170, 175, 124, 127, 129, 130, 133, 136, 140, 144, 151, 152, 177, 178, 200, 304 156, 252–255, 260, 265, 266, 268, 269, 369, 370, Dye saturation ...... 170 373, 376–380, 383, 390, 394, 397, 399, 400 Dyn A. See Dynorphin A (Dyn A) contamination ...... 113 Dynamic ...... 161, 186, 236, 243, 357, 360, 392 ladder ...... 102 Dynamic light scattering ...... 357, 360 library ...... 253, 255, 264–267 Dynorphin A (Dyn A) ...... 159, 160, 165, localization...... 124 166, 168, 171–176, 179, 180 polymerase ...... 102, 103, 106, 107, E 136, 252, 254, 265, 266 polymerase mix ...... 254, 265, 266 EagI...... 253, 267 transfection ...... 390 Ear bar...... 278, 318, 319, 381 vector ...... 152, 369, 391 E. coli...... 162 DNase...... 87, 103, 105, 112, 119, 127, 129, EDTA. See Ethylenediamine tetraacetic acid (EDTA) 130, 133, 140, 144 Efficiency...... 97, 114, 134, 145, 156, 161, dNTP...... 86, 96, 102, 103, 106, 107, 163, 221, 245, 261–262, 266–267, 270, 335, 336, 131, 134, 255–257, 262, 265, 266, 378 344, 357, 360, 364–366, 376, 383, 386, 395, 399, 400 Donkey ...... 61 Efflux pump ...... 326 Donor...... 18, 50, 59, 328 Egg albumen ...... 54 Dopamine ...... 273 EIA. See Enzyme immunoassay (EIA) DOPC. See Dioleylphosphatidylcholine (DOPC) EL...... 35, 43 DOPE. See Dioleoylphosphatidylethanolamine ( DOPE) Electrical ...... 162, 281, 365 DOPE-PEG ...... 363 Electrical activity ...... 193 Dorsal horn ...... 71, 76, 77, 90, 91, 126 Electrical biosensor ...... 392, 400 Dorsal root ganglia (DRG) ...... 126, 187 Electrical pulse ...... 162 Double bond ...... 97 Electrical response ...... 193 Double labeling ...... 51, 57, 60, 66, 89, 198 Electrochemical gradient ...... 175 DPPC. See Dipalmitoylphosphatidycholine (DPPC) Electrode ...... 193–197, 200, 219, 365 DPX...... 129, 132 Electron microscopy ...... 72–77, 86, 88, 90, 128, 129, 285 DRG. See Dorsal root ganglia (DRG) Electrophoresis ...... 102, 103, 118, 206, 212, Drill...... 195, 199, 278, 279, 319, 321, 322, 374, 381 215, 256–258, 266, 267, 270 Driver cDNA ...... 250, 252, 262–264, 270 Electrophysiological ...... 43, 193 Drug...... 61, 195, 280, 299, 315, 325, 389 Electrophysiological recording ...... 193 Drug delivery ...... 280, 321, 322, 326, 329 Electroporation ...... 151–153, 156 DRV. See Dehydration-rehydration vesicle (DRV) Electrospray ionization (ESI) ...... 217, 236, 337 Dry ice...... 142, 145, 300, 301, 351, 372, 377 ELISA. See Enzyme-linked immunosorbent assay (ELISA) dsDNA ...... 144 Eluate...... 209, 210, 259, 270, 373, 384 DSPC. See 1,2-Distearoyl-sn-glycero–3-phosphocholine Elution ...... 119, 209, 216, 220, 221, 223, (DSPC ) 232, 242, 244, 245, 260, 345, 385 DSPE-PEG(2000). See 1,2-Distearoyl-sn-glycero–3- Elution buffer ...... 144, 216 phosphoethanolamine-N-[methoxy(polyethylene EM...... 66, 41, 43 glycol)–2000] (DSPE-PEG(2000)) Embedding ...... 54, 63, 73, 76, 78–80, 86, DSPE-PEG(5000) ...... 363 88, 96, 124, 126, 129, 139–140, 199 ds tester ...... 250–252 Embryo...... 57, 63, 64 3D-structure ...... 16 Embryonic development ...... 43 DTT. See Dithiothreitol (DTT) Emission ...... 80, 162, 211 Duck...... 57, 58 Emission filter ...... 169, 195, 366 NEUROPEPTIDES 400 Index

Emission wavelength ...... 303, 401 Eukaryotic expression vector ...... 399 Emitted light ...... 162 Euthanasia ...... 51, 53–54, 346 Encapsulated peptide-to-lipid ratio ...... 360 Evaporation ...... 131, 134, 270, 359, 363, 364 Encapsulation ...... 357, 360, 364–366 Evoked EPSC ...... 197 Endocrine ...... 15, 19, 46, 49, 57, 204, 330 Evolution ...... 50, 124 Endocrine gland ...... 19, 36, 40 Excitation ...... 161, 169–171, 177, Endocrinology ...... 14, 15, 204 178, 180, 191, 195, 303, 366, 401 Endocytosis ...... 160, 362 wavelength ...... 177, 303 Endopeptidase ...... 327 Excitatory amino acid ...... 18 Endoplasmic reticulum (ER) ...... 17, 90, 395, 396 Excited state ...... 170 B-Endorphin ...... 19, 20, 294 Exciting radiation ...... 162 Endosome ...... 363 Exonic probe ...... 101, 102, 107, 116 Endothelial ...... 326, 328 Exopeptidase ...... 327 Endothelium ...... 326 Explosive ...... 61, 62 Endotoxin-free ...... 399 Exponential fitting ...... 341 Enkephalin ...... 15, 20, 50, 205, 294 Exposure Time (ExpT) ...... 352, 353 Enrichment ...... 219 Extension ...... 16, 106, 191, 270 Enzyme ...... 17, 18, 85, 124, 135, 145, 150, Extracellular ...... 43, 71, 195, 197–198, 273–375, 400 215, 254, 274, 299, 300, 304, 305, 308, 326, 327, Extracellular domain ...... 71 330, 349, 351, 363, 373, 383 Extracellular field potential recording ...... 195, 197–198 Enzyme assay ...... 215 Extraction ...... 102–104, 106, 107, 111–112, Enzyme immunoassay (EIA) ...... 274 140, 141, 144, 209, 211, 236, 238–243, 245, 246, Enzyme-linked immunosorbent assay 353, 377, 378 (ELISA) ...... 308, 309, 312 buffer ...... 144 Epidermal growth factor (EGF) ...... 43 Extruder ...... 356, 359 Epifluorescence ...... 166, 195 Eye...... 62, 318, 319, 374, 381 Epilepsy ...... 299, 316, 321, 330 lubricant...... 318, 319, 374, 381 Epithelial cell ...... 151 ointment ...... 374, 381 Epithelium ...... 317, 319, 320 F Epitope ...... 64, 391, 400 Epitope-tagging ...... 391, 400 F–12, 185, 187, 188, 190 Epon...... 88 F(ab). See Monovalent F(ab)-fluorochrome conjugate Eppendorf tube ...... 88, 94, 333, 341, 375, 378, 380 FA. See Formic acid (FA) EPSC...... 197 Fab’-Alexa Fluor ...... 72, 75 Equilibration ...... 220, 225, 232, 245, 345, 385 Fading. See Photobleaching Equilibrium fraction ...... 164 Falcon tube ...... 268 Equithensin ...... 300, 301 False negative ...... 124, 130 ER. See Endoplasmic reticulum (ER) False positive ...... 64, 129, 130 ESI-IT ...... 236, 238, 240, 241, 243, 246 Fasted rat ...... 132 ESI-Q-TOF ...... 237, 239, 241, 244 Fast neurotransmitter ...... 18 Esophagus ...... 317, 322 Fast red ...... 132 Ester...... 51, 85, 104, 327, 330, 331, 337, 339, 344 FBS. See Fetal bovine serum (FBS) Esterases ...... 334, 395 FCA. See Fluorescence cumulant analysis (FCA) Esterification ...... 337, 344 FCS. See Fluorescence correlation spectroscopy (FCS) Estrogen ...... 316–317 Feeding ...... 193, 235 Ethanol ...... 51, 54, 63, 73, 78, 81, 87, 92, 93, Feeding behavior ...... 193 104, 105, 107–109, 111, 119, 130, 140, 143, 146, fEPSP...... 198 167, 206, 256, 267, 270, 276, 278, 285, 300, 301, Fetal bovine serum (FBS) ...... 150, 152, 153, 156, 331, 334, 344, 350, 351, 356, 357, 366, 372, 374, 167, 168, 371, 372, 375, 376 377, 379, 381 FGF. See Fibroblast growth factors (FGF) Ether...... 281, 337 Fibers...... 15, 57, 71, 91, 183 Ethidium bromide ...... 102, 118 Fibroblast ...... 151, 188 Ethylenediamine tetraacetic acid (EDTA) ...... 87, 92, Fibroblast growth factors (FGF) ...... 43 102–104, 108, 208, 254–256, 258, 260, 261, 267, FIDA. See Fluorescence intensity distribution analysis 269, 304, 372, 377, 393, 396 (FIDA) NEUROPEPTIDES 401 Index

Filling solution ...... 196 Formamide ...... 87, 88, 98, 104, 108 Film...... 119, 135, 146, 186, 284, 287, 293–296, Formic acid (FA) ...... 218, 224, 239–242, 246 358, 359, 363, 364 Fraction ...... 72, 129, 164, 179, 206, cassette ...... 293, 295 219, 222, 223, 252, 259, 277, 286, 303, 378, 385 Filter...... 52, 74, 79–81, 141, 143, 145, 146, Fractionation ...... 216, 220, 236, 240, 242–243, 390 162, 169, 178, 184, 190, 191, 194–197, 239, 242, Free diffusion ...... 164, 165, 173, 175, 176 244, 245, 269, 286, 296, 301, 309, 310, 332, 337, Free-floating ...... 75, 76, 78, 87, 98, 382 338, 359, 366, 379, 380 Free-moving ...... 273–281 Fire...... 62 Freezer...... 52, 120, 140, 143, 242, 289, 333 Firing rate ...... 199 Freeze-thaw(ing) ...... 52, 372, 377 First strand cDNA ...... 142, 256, 257 Frequency facilitation ...... 198 Fish...... 53, 54, 63 Frog...... 53 Fisher’s protected least significant difference (PLSD) .....110 Frozen section ...... 94, 96, 103, 200 FITC. See Fluorescein isothiocyanate (FITC) FSH...... 37 Fitting...... 19, 61, 165, 173, 175, 176, 179, 341, 347 FuGENE™ ...... 6, 394, 396, 400 Fixation ...... 51, 54–55, 63, 64, 80, 89, 96, 97, 124, 126, 130 Full-length trkB (fl-trkB) ...... 71, 72, 75, 77 Fixative ...... 51, 54, 63, 66, 72, 75, 97, 128, 200 receptor ...... 71 Flammable ...... 62, 65, 281 Fume cupboard ...... 62 FLEX station ...... 393, 395, 396, 398, 401 Fume hood ...... 53, 61, 62, 65, 72, 79, 128, 143, FLIPR...... 393, 399 288, 289, 343 Floating section ...... 76, 78, 87, 94, 95, 98 Functional group ...... 236, 327 Flow rate ...... 220–225, 232, 242, 244, Fusogenic ...... 363 274, 275, 280, 345, 346, 379, 384, 385 G fl-trkB. See Full-length trkB (fl-trkB) Fluctuation ...... 161–165, 173–176, 179 G(T). See Normalized autocorrelation function (G(T)) Fluctuation correlation analysis ...... 161 GABAB receptor ...... 90

Fluo–3...... 400 GAi...... 392, 399 Fluo–4...... 393–396, 399–401 Gain...... 160, 161, 169, 249, 319, 322 Fluo–5...... 400 B-Galactosidase ...... 218 Fluo 3-AM ...... 185, 189 Galanin ...... 32, 90 Fluo–4-AM ...... 394 Gallus domesticus ...... 56, 57 Fluorescein. See Fluorescein isothiocyanate (FITC) Gamma counter(ing) ...... 208, 210, 294 Fluorescein isothiocyanate (FITC) ...... 58, 59, 65, Ganglion ...... 57, 187, 191 66, 88, 95, 195, 198 GAPDH. See Glyceraldehyde 3-phosphate dehydrogenase Fluorescence ...... 55, 58, 65, 72, 74, (GAPDH)

75, 78, 80, 105, 114, 120, 124–126, 129, 132, 144, GA16 plasmid ...... 394

155, 156, 159–180, 186, 189–191, 195, 198, 300, GAq...... 392, 399

304, 360, 366, 392, 393, 395, 396, 398, 400, 401 GAs...... 392, 399 fluctuation...... 161 Gaseous molecules ...... 13 photobleaching recovery ...... 161 Gastrin...... 21, 26, 57 Fluorescence correlation spectroscopy (FCS) ...... 159–180, GDNF. See Glial-derived neurotrophic factor (GDNF) 185, 190 Gel...... 102, 103, 106–108, 117–120, Fluorescence cumulant analysis (FCA) ...... 163 145, 147, 206, 209, 256, 258–260, 262, 265–267, Fluorescence intensity distribution analysis (FIDA) ...... 163 270, 359, 362, 379 Fluorescent ...... 65, 71, 75, 89, 91, 94–95, phase ...... 362 113, 124, 150, 151, 153, 161, 162, 165, 169, 171, Gelatin ...... 95, 133, 208, 210 175, 176, 178, 179, 191, 366, 382, 383, 391–393, GenBank ...... 116, 269 395, 396, 398–401 Gene...... 16, 50, 70, 86, 101, 126, 141, 150, 249, 369, 392 Fluorescent probe ...... 124 conversion ...... 50 Fluorochrome ...... 52, 59, 65, 66, 74, 79, 89 duplication ...... 50 Fluorometric ...... 300 expression ...... 16, 20–36, 43, 86, Fluoronanogold™...... 70–72, 74–80 101, 102, 111, 141, 249, 268, 370, 371 Fmoc-Pro-Rink-MBHA resin ...... 331 normalization...... 134, 269 Foot pinch ...... 381 Gene Jockey ...... 255, 269 Formalin ...... 51, 54, 61, 63, 126, 195 GeneSpring ...... 255, 268 NEUROPEPTIDES 402 Index

Genetic fusion ...... 161 Green fluorescent protein (GFP) ...... 382, 391 Genome ...... 37, 40, 42, 45, 46, Grid coating pen ...... 73 117, 243, 369, 370, 373, 378, 379, 382, 384 Ground state ...... 170 Genomic copies ...... 371, 380, 384 Growth factor ...... 16–19, 42, 45, 46, 184, 185, 316 Gentamicin ...... 279 Growth hormone ...... 28, 37, 204 GFP. See Green fluorescent protein (GFP) Guide cannula ...... 276–279 GFP-fusion protein ...... 400 Guillotine ...... 194, 196, 333, 339 GFRA1 receptor ...... 71, 78 Gut...... 15 Ghrelin ...... 31, 57 H Gigaohm ...... 196 Gigaprep ...... 383 3H...... 91, 205 Gill...... 53, 57, 58 Half-life ...... 211, 281, 316, 360 Glass beads ...... 363 Halothane ...... 194, 196 Glass knife ...... 73 Hamilton ...... 195, 199, 277, 279, 317, 319, Glass microelectrode ...... 197 321, 332, 339, 357, 361, 366 Glass micropipette ...... 196, 199, 284, 285, 287–288 Handling ...... 61, 62, 66, 124, 343, 344 Glassware ...... 132, 199 Hanks’ balanced salt solution (HBSS) ...... 184, 187, 394 Glia...... 69 Hapten ...... 89 Glial cell ...... 16 Hazardous area ...... 177 Glial-derived neurotrophic factor (GDNF) ...... 45, 70 HBS. See Hepes-based saline buffer (HBS) Glomerular terminal. See Glomerulus HBSS. See Hanks’ balanced salt solution (HBSS) Glomerulus ...... 77 Head...... 50, 53, 187, 200, 270, 278, Gloves...... 53, 62, 132, 141, 267, 287, 343 285, 289, 318, 319, 339 Glucagon ...... 19, 27 activator peptide ...... 50 Glucose ...... 28, 185, 190, 191, 194, 273, 393 stage ...... 200 Glue...... 58, 133, 199 Heart...... 54, 274, 305 Glutamine ...... 185, 190, 246, 371, 372, 375, 393 Heat...... 51–54, 62, 108, 135, 143, 166, Glutaraldehyde ...... 72, 80, 97, 207, 208, 285, 290 209, 250, 256, 261, 263, 264, 268, 270, 285, 288, [Glu2]TRH ...... 329–331, 334–336, 339, 340, 343–346 289, 333, 342, 351, 394 Glyceraldehyde 3-phosphate dehydrogenase Heating pad ...... 195, 198, 318–320 (GAPDH) ...... 142 HEK cells ...... 391 Glycerin ...... 58 A-Helical ...... 390 Glycerol ...... 53, 92, 96, 195 Helper gene ...... 370, 383 Glycine ...... 18, 72, 75, 80, 240, 244, 246, 308 Helper plasmid ...... 372, 375, 383 Glycosylation ...... 18 Hemorphirin–7 ...... 205 Gold...... 70, 71, 73–81, 86, 88, 90, 91, 95–96, 129 Hemostasis ...... 278 cluster ...... 74, 75 HeNe...... 166, 168, 169 enhancement ...... 71, 73–74, 76, 78–79 Heparin ...... 333, 339, 371, 375 intensification ...... 71 HEPES ...... 177, 185, 186, 189, particle ...... 71, 74–80, 88, 90, 91, 95–96 191, 194, 254, 394, 397 probe ...... 70 Hepes-based saline buffer (HBS) ...... 150, 152, 155 Golgi...... 17, 35, 149 Herpes simplex ...... 370 Gonadotropin ...... 23, 32, 204 Heteronuclear RNA ( hnRNA) ...... 119 GPCRs. See G-protein coupled receptor (GPCRs) High affinity IgE receptor ...... 190 G3PDH ...... 255, 261, 262, 266, 267, 270 High-performance liquid chromatography G-protein ...... 18, 70, 296, 389–402 (HPLC) ...... 236, 274, 331–332 G-protein coupled receptor (GPCRs) ...... 18, 70, 389–402 High-throughput ...... 390, 392, 393, 399 deorphanization ...... 395 Hill coefficient ...... 347 Gradient ...... 175, 209, 216, 217, 219–225, 242, 276, Hindrance ...... 89, 216 345, 346, 365, 372–373, 375, 377–379, 383–385 Hippocampal ...... 149, 150, 152–156 elution ...... 345, 385 Histofluorescence ...... 85 Granin...... 37, 41–43 Hitrap Q column ...... 378, 379 Granulation tissue ...... 321 hnRNA. See Heteronuclear RNA Green fluorescence ...... 156 HOBt. See 1-Hydroxybenzotriazole (HOBt) NEUROPEPTIDES 403 Index

Holder...... 104, 112, 143, 169, 178, IHC. See Immunohistochemistry (IHC) 276, 278, 279, 332, 380, 381 IL–3. See Interleukin–3 (IL–3) Holopeptide...... 50 Image...... 104, 109, 110, Homogenate ...... 111, 241, 329, 330, 339–341 119, 143, 152–154, 162, 167, 169, 172, 176, 189, Homogenization ...... 236, 245, 359, 364 284, 287, 294–295 Homogenizer ...... 207, 239, 300 analysis...... 109, 152, 287, 294–295 Horizontal probe ...... 275 size...... 169 Hormone ...... 14–37, 45, 46, 49, ImageQuant ...... 255, 268 203, 204, 211, 236, 238, 241, 246, 273, 307, 321, IMF. See Immunofluorescence (IMF) 330, 334, 389 Immersion ...... 54, 55, 169, 178, 191, 194, 196 Horse-radish-peroxidase (HRP) ...... 55–57, 64, 88, 93–95 medium...... 166, 177 Horse serum ...... 166 Immobilized artificial membrane chromatography Hot-start PCR ...... 129 (IAMC) ...... 328, 332, 335, 338–340, 346 HPLC. See High-performance liquid chromatography Immune cell ...... 184–189, 191 (HPLC) Immune complex ...... 308, 311–313 HPRT–1...... 134 Immunization ...... 204, 208, 308 HRP. See Horse-radish-peroxidase (HRP) Immunocytochemistry (ICC) ...... 56, 70, HT1080 cells ...... 384 85, 86, 89, 91, 95, 97, 123, 124, 126, 132 Human ...... 37, 39, 40, 42, 45, 64, Immunofluorescence (IMF) ...... 55, 57, 59, 60, 65, 132 79, 86, 124, 203, 204, 255, 262, 268, 270, 307–311, Immunogen ...... 50, 205, 369 316, 317, 371, 389–392, 399 Immunogenicity ...... 369 Humidity ...... 177 Immunoglobulin ...... 66, 205, 296, 307–309 Hybrid(ization)...... 16, 37, 40, 42, 45, Immunogold ...... 70, 77 86, 87, 89, 92–94, 97, 101–110, 116, 117, 123, 126, Immunohistochemistry (IHC) ...... 50, 51, 55, 58, 63, 65 249–270, 382 Immunological precipitation ...... 206 Hydra...... 50 Immunoprecipitate ...... 210 Hydrodynamic diameter ...... 360, 365 Immunoprobe ...... 75 Hydrodynamic radius ...... 165 Immunoreaction ...... 64 Hydrogen peroxide ...... 52, 55, 89, 206, 278 Incisor bar ...... 278 Hydrolytic ...... 299, 300, 302 Incorporation ...... 14, 124, 126, 129, 133, 399 Hydrophobic ...... 135, 216, 242, 364 Incubation ...... 57–59, 64–66, 71, 80, 94, 98, 108, Hydrophobicity ...... 219 133, 136, 144, 152, 156, 189, 210, 212, 292, 294, 1-Hydroxybenzotriazole (HOBt) ...... 331, 334–336 303, 308, 311, 341, 359, 376, 383, 396, 397, 401 Hydroxylamine ...... 240, 244, 246 Incubator ...... 95, 104, 105, 112, Hypnorm ...... 374, 381 151, 152, 156, 166–168, 177, 185, 187–189, 256, Hypothalamic ...... 14, 19, 106, 141, 145, 146 270, 375, 376, 383 Hypothalamus ...... 15, 56, 102, 105, Indicator ...... 120, 168, 191, 328, 393, 395 106, 275, 278, 361, 370, 371, 384 dye...... 120 Hypothermia ...... 279 Indirect in situ PCR ...... 124 Hystar...... 240, 243 Infectivity ...... 370 I Inflammation ...... 183 Inflammatory ...... 39, 160 125I...... 205–208, 211, 286, 292, 294 Infrared (IR) ...... 139, 140, 144, 194, 196 131I...... 286 Infusion pump ...... 277, 371, 373, 374, 382 IAMC, Immobilized artificial membrane chromatography Ingestion ...... 62, 79 (IAMC) Inhalation ...... 62, 79, 281 ICC. See Immunocytochemistry (ICC) Inhibitor ...... 36, 131, 132, 152, 280, 399 Ice...... 94, 108, 112, 142, 143, 145, 168, 171, Injection pipette ...... 200 186, 196, 209, 253, 255, 293, 295, 300, 301, 337, Injection volume ...... 219, 342, 361 339, 341, 351, 372, 377 %Inj/g...... 352, 353 IgE...... 190, 191, 312 In silico ...... 328, 390, 391 IgG...... 59, 61, 308–312 In situ hybridization (ISH) ...... 16, 37, 40, 42, subclass ...... 312 45, 86, 92–93, 101–110, 123, 382 IgM...... 309, 312 In situ PCR. See In situ polymerase chain reaction (PCR) NEUROPEPTIDES 404 Index

In situ polymerase chain reaction Isocratic ...... 216, 217, 223–225, 242, 338, 345 (PCR) ...... 124, 125, 135, 136 Isoflurane ...... 51, 53, 61, 318–320 In situ RT-PCR. See Direct in situ RT-PCR Isoform ...... 29–36, 40, 44, 50, 70, 71, 126 Insulin...... 14, 19, 33, 204, 313, 316, 317, 333 Isopropanol (IPA) ...... 217, 232 Insulin-like growth factor ...... 316 Isotope...... 204, 205, 211, 236, 245 Intensity autocorrelation function (C(t)) ...... 163 Isotope labeled peptide ...... 204 Interassay coefficient of variation ...... 210 Isotopic labeling ...... 236 Interferon ...... 316 ITR. See Inverted terminal repeat (ITR) Interleukin–3 (IL–3) ...... 185, 188 J Internalization ...... 172–174 Internal solution ...... 194, 196 JC8111 ...... 382 Interpulse interval ...... 198 Jugular vein ...... 351 Interstitial fluid ...... 326 Intersystem crossing ...... 162, 164 K Intestine ...... 39, 57 Intra-aural line ...... 381 Kaiser test, 335, 344. See also Ninhydrin test Intracellular ...... 18, 71, 77, 80, 126, 151, 160, Kappa-opioid receptor (KOP) ...... 160 186, 194–195, 197, 200, 316, 390, 392, 395 KCl. See Potassium chloride (KCl) Intracellular fluid ...... 280 Kellog antibody ...... 269 Intracellular recording ...... 194–195, 197 Keratinocyte ...... 184 Intradermal injection ...... 204 Ketamine ...... 51, 53, 276, 277 K . See Blood-to-brain unidirectional influx rate (K ) Intranasal delivery ...... 315–322 i i k’ . See Chromatographic capacity factor (k’ ) Intraperitoneal ...... 277, 301, 342 IAM IAM Intravenous ...... 315, 316, 330, 353, 365 Kinase buffer ...... 350, 351 Intravenous administration ...... 330 Kindling ...... 321 Intron-specific probe ...... 101, 102 Knife...... 73, 144, 277, 278 Invasive ...... 284, 317, 326 Knockdown ...... 349 Inverted micelle ...... 160 KOP. See Kappa-opioid receptor (KOP) Inverted terminal repeat (ITR) ...... 253, 369, Kozak sequence ...... 399 370, 379, 382, 383 L In vitro ...... 107, 119, 165, 183–191, 194, 196–198, 286, 287, 290, 292–296, Lacerta viridis ...... 57 327, 329, 330, 332–333, 339–341, 343, 357, 360, Lactic acid ...... 273 371, 381 Lactoperoxidase ...... 206 In vivo 114, 154, 195–196, Lambda ...... 381 198–199, 274, 277, 279–280, 284, 287, 290, Lamina ...... 78 292–297, 327–330, 332, 334, 342, 343, 355, 365, Laminar flow ...... 167 371, 374, 381, 382 Large dense-cored vesicle ...... 16 Iodination ...... 205, 206 Large granular vesicle (LGV)...... 77 Iodine...... 205, 206, 211, 276, 278 Large unilamellar vesicle (LUV) ...... 359–360 Iodixanol gradient ...... 372–373, 375, 377–378 Laser...... 139–147, 161–163, 166, 169–171, Ion channel ...... 193 177–179, 186, 189, 191, 217, 360 Ion flux ...... 18, 400 Laser beam ...... 139, 162, 178 Ionic strength ...... 366 Laser capture microdissection (LCM) ...... 139–147 Iontophoretic ...... 199 Lauroylsarcosine ...... 87 Ion trap ...... 236, 337, 339 LC. See Liquid chromatography (LC) IPA. See Isopropanol (IPA) LCM. See Laser capture microdissection (LCM) IR. See Infrared (IR) Lead citrate (Reynold’s) ...... 74 Irgo...... 196, 199 Learning ...... 15, 193, 235, 296, 316 Irritation ...... 62 Lens...... 168, 177, 191 Ischemia ...... 2997 Lentiviral DNA ...... 124 ISH. See In situ hybridization (ISH) Leptin...... 19, 38 Islet cell ...... 58, 184 Levamisole ...... 98 Isoamyl alcohol ...... 255, 258, 269, 270 LGV. See Large granular vesicle (LGV) NEUROPEPTIDES 405 Index

LH...... 37, 384 MALDI. See Matrix-assisted laser desorption/ionization LiCl...... 87 (MALDI) Life time ...... 160, 211 Maleate buffer ...... 73, 78 Ligand...... 35, 44, 45, 70, 86, 160, 164, 205, Maleic acid ...... 73 390–393, 395–397, 399, 401, 402 Mammalian ...... 6, 46, 50, 65, 129, 330, 391, 393, 396, 399 Ligation ...... 258–263, 267, 270 Manifold ...... 286, 293, 295 Light microscopy ...... 54, 70, 200 Manipulator ...... 194, 195, 197 Linear regression ...... 341 Mannitol...... 371 Line scan ...... 169 Marker ...... 91, 134, 150, 152, 200, 256, 258, Linker...... 191, 258, 259 259, 262, 278, 292, 313, 339, 342, 346, 366, 383 Lipid...... 61, 92, 326, 328, 338, 340, 346, Mass...... 246, 339 357–360, 362–364, 366, 396 MassLynx ...... 241, 244, 245 Lipid bilayer ...... 326, 363 Mass spectrometer ...... 218, 219, 224, 332, 337–339 Lipid film ...... 358, 359, 363 Mass spectrometry (MS) ...... 5, 206, 212, 215, Lipophilicity ...... 328, 345 216, 221, 224, 225, 231, 235–246, 337, 338, 346 Liposome ...... 355–366 Mass tolerance ...... 243, 246 Liposome-encapsulated neuropeptide ...... 355–366 Mast cell ...... 183, 184, 186, 188, 189, 191 B-Lipotropin ...... 19 Mastermix ...... 104, 112, 128, 132, 140, Liquid chromatography (LC) ...... 219–225, 145, 375, 376, 378, 380 236–242, 245, 337, 346 Matrigel ...... 184, 186–189, 191 Liquid nitrogen ...... 145, 167, 359 Matrix...... 112, 184, 217–219, 221, 224, Lissamine rhodamine (LRSC) ...... 195, 198 240, 254, 259, 260, 267, 270 Live neuron ...... 150, 151, 153–156 Matrix-assisted laser desorption/ionization Liver...... 54, 274, 275 (MALDI) ...... 217–221, 224 Lizard...... 51, 53 Maxiprep ...... 383 lmRNA ...... 71, 86, 89–93, 95, 97, 101, 102, 105, Measurement ...... 101, 118, 120, 161, 162, 165, 106, 110, 111, 113–115, 117, 118, 123, 124, 126, 167–178, 185–186, 189–190, 204, 206, 236, 269, 127, 129, 133, 134, 142, 250, 258, 371 280, 303–305, 328, 333, 349–354, 360, 366, 392, LNA probe. See Locked nucleic acid probe (LNA probe) 393, 396, 398, 401 Loading buffer ...... 394, 397 Median eminence ...... 56 Locked nucleic acid probe (LNA probe) ...... 86–89, 91, Medium ...... 57, 58, 65, 88, 89, 92, 94, 98, 126, 94–95, 98 127, 129, 132, 150, 152, 153, 156, 161, 164–168, Locus coeruleus ...... 90 171–177, 185, 187–190, 195, 198, 304, 325–347, Log...... 120, 352 363, 366, 371, 372, 375, 376, 393, 396, 397 LogP...... 328 Melanocortin ...... 19, 317 Long-term expression...... 369 Melanophore ...... 391 Loop...... 220, 242, 339, 346 Melting curve ...... 113, 145 LRSC. See Lissamine rhodamine (LRSC) Melting temperature (Tm) ...... 89, 96, 97, 113, 145, 147 Lumen...... 17, 347 MEM...... 185 Lung...... 317, 322 Membrane ...... 17, 70, 71, 75–77, 90, 107, 108, LUV. See Large unilamellar vesicle (LUV) 119, 139, 140, 149, 160, 161, 164, 172–175, 196, Lymnaea stagnalis ...... 50 198, 207, 239, 253, 268, 270, 274–281, 299, Lymph node ...... 317 302–304, 328, 330–332, 335, 337–339, 346, 356, Lymphoid organ ...... 183 359, 360, 362–366, 392, 395, 400 Lyophilizer ...... 332 fusion ...... 362 Lysis buffer ...... 103, 104, 111 potential ...... 196–198, 360, 392, 399 Lysosomal enzyme...... 363 Membrane-bound fraction ...... 303 Lysosome ...... 91 Membrane-spanning protein ...... 389 Lysozyme ...... 218, 230 Memory ...... 15, 193, 316 M mEPSC ...... 197 Mercaptoethanol ...... 104, 111 Macropinocytosis ...... 160 Mercuric formalin ...... 63 Magnetic shaker ...... 300, 301 Mercury lamp ...... 168 Magnetic stirrer ...... 293, 295 Metabolism ...... 86, 315, 327, 328, 346, 355 NEUROPEPTIDES 406 Index

Metamorph ...... 151, 152, 154 Monolayer ...... 328, 335 Met-enkephalin-Arg-Phe ...... 205 Monovalent F(ab)-fluorochrome conjugate ...... 59–61, 66 Methanol ...... 207, 208, 210, 216, 217, 232, Morphology ...... 63, 80, 97, 145, 146 237, 245, 246, 285, 331, 334, 356, 357, 360, 364 Motorneuron ...... 126, 127, 330 Methylbenzoate ...... 51, 54, 62 Motor protein ...... 175 2-Methylbutane ...... 145 Mouse...... 32, 33, 37–42, 44, 45, 59, 64, 71, 76–78, 88, 126, Methylene blue ...... 384 127, 134, 156, 185–187, 196, 255, 262, 311, 330,

MgCl2...... 88, 102, 131, 134, 185, 191, 341–343, 346, 352, 354, 374, 381. See also Mice 274, 277, 309, 372, 373 MS. See Mass spectrometry (MS) Mice...... 184, 185, 187, 188, 194, 245, 296, 301, MS222...... 51, 53, 61 317, 330, 332, 341, 343, 350, 354. See also Mouse MSH...... 15 Micro 1041 ...... 196, 199 A-MSH ...... 19, 20 Microarray ...... 250, 253, 255, 267–269 MS/MS ...... 219, 238, 240, 241, 243, 246 Microcap ...... 285, 286, 290–295 Mucosa ...... 57, 184, 316, 317, 321, 322 Microdialysis ...... 273–281 Mucous membranes ...... 62 probe ...... 273–281 Multidimensional analysis ...... 215 Microfluidcs-based platform ...... 141 Multilamellar vesicle (MLV) ...... 358, 359, 363, 364 Microfuge tube ...... 256, 259 Multiphoton FCS ...... 161 Microhyb ...... 255, 268 Multiphoton microscopy ...... 161 Microinjection ...... 355–366 Multiple labeling ...... 66, 70, 75, 89 Micromanipulator ...... 198, 284 Murine stem cell factor (SCF)...... 185, 188 Micropipette ...... 168, 169, 171, 195, 196, Muscle...... 64, 188, 305, 381 198, 199, 284, 285, 287–289, 291, 295, 309 Mutagenesis...... 50 Micropipettor ...... 264 Mutation ...... 50, 370 Microplate reader ...... 309, 310, 395, 398, 401 Myenteric plexus ...... 57 Micropump...... 220, 222–224 Mytilus edulis ...... 50 Micro RNA (miRNA) ...... 86, 89, 91, 94–96, 98 m/z value ...... 221 Microscope slide ...... 104, 128, 130, 131, 133, 140, 142, 268 N Microtome ...... 54, 72, 79, 86, 128, 194, 382 Microtubule ...... 150 N-A-Fmoc-N-G-allyl-glutamic acid ...... 331 Microwave ...... 55, 57, 64, 97, 133, 241 Nanocolumn ...... 219, 225, 244 Midbrain ...... 370 Nano-HPLC ...... 216, 217, 223 Minigene cDNA ...... 371 Nanoparticle ...... 316, 360 Miniprep ...... 104, 119, 383 Nanosizer ...... 360 miR–134 ...... 91 Nanotechnology ...... 356 miRNA. See Micro RNA (miRNA) NAP...... 317 Mirror...... 162, 169, 178 B-Naphthylamine ...... 300, 302–304 Mismatch ...... 60, 98 ...... 346 Mispriming ...... 129 Nasal cannula ...... 318–322 MLV. See Multilamellar vesicle (MLV) Nasal cavity ...... 316, 317, 319–322 MMLV reverse transcriptase ...... 253 Nasofrontal suture ...... 319–321 Mobile phase ...... 216, 224, 225, 240–242, Nasopalatine duct ...... 317, 322 244, 246, 332, 338, 339, 345, 346 NBT. See Nitro-blue tetrazolium (NBT) Modulator ...... 14, 193, 283 n-Butanol ...... 256, 258 Mold...... 54 N-cadherin ...... 186 Molecular mass ...... 339 Neck...... 187, 296, 343 Molecular movement ...... 160 Needle...... 185, 187, 188, 221, 225, 279, 289, 319, 321, Molecular size ...... 206, 241, 327, 328 322, 342, 347, 357, 361, 373, 374, 377, 378, 381, 382 Molecular weight ...... 74, 80, 175, 205, 206, 218, Negative reaction ...... 60, 66 239, 256, 262, 270, 273, 274, 281, 339, 346, 360 Negative staining ...... 60, 67 Mollusk ...... 50 Nembutal ...... 300, 301 Monoamine ...... 85, 280 Nerve growth factor (NGF) ...... 42, 45, 185, 186, 190, 316 Monodimensional ...... 217, 219 Nested PCR ...... 255, 264–266 Monodimensional RP-HPLC ...... 217, 219 Nested primer ...... 252, 253, 267 NEUROPEPTIDES 407 Index

NeuN...... 382 Normal serum ...... 55, 59, 60, 72, 129 Neurexophilin ...... 13 Northern blotting ...... 123 Neurite ...... 151, 153, 156, 186, 189–191 Nose...... 316 Neuroanatomy ...... 85 Nose clamp ...... 278 Neurobasal ...... 150, 152, 153, 156 Nothobranchius furzeri ...... 63 Neuro-immune interaction ...... 183–191 NotI...... 253 Neuromodulation ...... 149 NPY. See Neuropeptide Y (NPY) Neuron ...... 16, 19, 37, 43, 45, 46, 49, 56, NT–3...... 45 57, 69–81, 85, 90, 102, 124, 126, 127, 140, 149–156, NT–4/5 ...... 45 184, 186, 188, 193, 195–198, 200, 316, 330, 355, N-terminal ...... 16, 17, 150, 205, 246 371, 375, 381, 382 NTS. See Sodium thiosulfate (NTS) Neuronal connection ...... 69 Nuclear staining ...... 127, 130 Neuronal loss ...... 160 Nuclease-free water ...... 103, 104, 131, 132 Neuronal network ...... 69 Nucleic acid ...... 86, 91, 97, 104, 124, 126, 136 Neuropeptidase ...... 299–305 Nucleotide ...... 86, 96, 105, 108, 116, Neuropeptide Y (NPY) ...... 18, 23 119, 124, 126, 129, 133 Neurophysin ...... 15, 21 Nucleus ...... 105, 110, 118, 129, 145, 146, NeuroPred ...... 241 161, 172–174, 176, 361, 371 Neurosecretion ...... 15 Nucleus magnocellularis preopticus ...... 56 Neurotensin ...... 31, 57 Nylon...... 253, 255, 267 Neurotransmission ...... 14, 85, 149, 159, 193 O Neurotrophin ...... 43, 45, 149 Neutralization ...... 308, 312 Obesity ...... 316 NGF. See Nerve growth factor (NGF) Object...... 110 NGS. See Normal goat serum (NGS) Objective ...... 143, 144, 151, 156, 162, 166, Nick...... 129 168, 169, 171, 177, 178, 191, 194, 196, 218 Nickel grid ...... 73, 78, 81 Octadecyl carbon chain (C18)...... 216, 217, 223, Ninhydrin test ...... 335, 336, 344. See also Kaiser test 239, 240, 242, 244, 245, 331, 332 Nitric acid ...... 285, 288, 296 OD. See Optical density (OD) Nitric oxide ...... 273 oGPCR. See Orphan GPCR (oGPCR) Nitro-blue tetrazolium (NBT) ...... 88, 92–94, 98, 7...... 190, 194, 196, 197, 200, 223, 285, 362 129, 132, 133 Oil...... 151, 156 Nitrogen ...... 145, 167, 198, 217, 331, 337, 339, 359 Olfactory ...... 303, 315–317, 319, 320 4-Nitrophenyl phosphate disodium salt hexahydrate Olfactory cortex ...... 315 (pNPP) ...... 309, 310 Olfactory neuroepithelium ...... 316, 317, 321 NMR...... 337, 346 Oligonucleotide ...... 105, 253, 269 N,N’-diisopropyl-ethylamine (DIPEA) ...... 331, 334–336 Oligo(dT)15 primer ...... 131 N,N’-dimethylformamide...... 331 Oligo(dT) primers ...... 142, 144

NO2...... 13 Oligoprobe ...... 86, 87, 89, 90, 92, 95–97 Nociceptive ...... 70, 91 Open reading frame (ORF) ...... 399 Noise...... 343 Opioid...... 15, 19, 20, 160, 205 Nonamer primers ...... 144 Optical configuration ...... 169, 178 Non-coding RNA ...... 86 Optical density (OD) ...... 284, 287, 293–295, 310–312 Non radioactive ISH ...... 90 Optimization ...... 144, 206, 216, 217, 257, 312, 391, 400 Non radioactive probe ...... 124 Optimization reaction ...... 256, 258 Non-specific binding ...... 211, 312 Oral...... 58, 317 Non specific DNA ...... 127, 129 Orbital rotor ...... 300, 302 Non specific staining ...... 98 Orexin...... 34, 57 Nordarenaline ...... 273 ORF. See Open reading frame (ORF) Norepinephrine ...... 280 Organelle ...... 74, 299 Normal goat serum (NGS) ...... 95 Organic fluorophore ...... 161, 171 Normalization ...... 110, 134, 268, 269 Organic phase ...... 259 Normalization gene ...... 134, 269 Organic solvent ...... 62, 216, 236, 357–359, 364 Normalized autocorrelation function (G(T)) ...... 163, 164 Organotypic culture ...... 134 NEUROPEPTIDES 408 Index

Orphan GPCR (oGPCR) ...... 390–393, 395–397 PBS. See Phosphate buffered saline (PBS) Orthophosphoric acid ...... 301 PC12 cells ...... 166–168, 171, 172, 174, 176 Oryzias latipes ...... 63 pcDNA vector ...... 391 Oscilloscope ...... 196, 199 PCH. See Photon-counting histogram (PCH) Osmium ferrocyanide ...... 73, 78, 79 pClamp ...... 196, 199 Osmium tetroxide ...... 73, 79, 88 PCR. See Polymerase chain reaction (PCR) Osmolarity ...... 194, 280, 364 PCR-Select cDNA Subtraction ...... 250, 251 Osmotic shock ...... 364 PCs. See Prohormone convertase (PCs) Osmotic stimulation ...... 115 PdI. See Polydispersity index (PdI) Osteoblastic cell ...... 184 pDP...... 375, 376, 383 OT. See Oxytocin (OT) pDsRed2 ...... 151 Oven...... 55, 57, 63, 64, 86, 128, 130–133, pDsRed-Express-N1, 151 223, 225, 285, 288–290, 344 Peak...... 147, 221, 224, 225, 237, 240, Overamplification...... 98 243–245, 341, 345, 396, 398 Overdigestion ...... 97, 130 capacity ...... 216, 220, 224 Oxidation ...... 81, 206, 246, 269, 359 PEG. See Polyethylene glycol (PEG) Oxidizing agent ...... 62, 205 pEGFP-C1 ...... 151 Oxygen ...... 170, 318 pEGFP-N1 ...... 151 Oxytocin (OT) ...... 14, 15, 21, 50, 102, 103, PEG MS pattern ...... 221 105–107, 111, 113–119 PEI. See Polyethyleneimine (PEI) P PEI-DNA ...... 376 Pellet...... 152, 156, 167, 239, 260, 267, 302, 351, 377 32P...... 350, 351 Penetration ...... 65, 74, 97, 133, 328 PACAP ...... 28, 58 Penicillin ...... 166, 185, 372, 375, 394 Pain...... 62, 160, 193, 284, 316 Pentobarbital ...... 72, 75, 276, 277, 330, 333, 342, 343, 353 Paintbrush ...... 54, 200 Peptidase...... 302, 303, 327 Paired-pulse facilitation ...... 198 Peptide encapsulation efficiency ...... 360, 365, 366 PAM. See Peptidyl-aminotransferase (PAM) Peptide HI (PHI) ...... 18, 27 Pancreastatin ...... 43 Peptidergic vesicle ...... 149, 150, 153, 154 Pancreatic ...... 23, 58 Peptide YY (PYY) ...... 18, 23 PAP. See Peroxidase anti-peroxidase (PAP) Peptidomics ...... 215–232 Pap jar...... 140, 146 Peptidyl-aminotransferase (PAM) ...... 18 PAP pen ...... 128, 131, 135 Percentile ...... 269 Paracrine ...... 14, 43 Perfusate ...... 273–275, 280 Paraffin ...... 51, 54, 55, 62–64, 86, 96, 124, 126, 128–130 Perfusion ...... 72, 75, 196, 284, 300, 301, 377–379, 382, 384 Parafilm ...... 74, 79, 286, 292 Peripheral administration ...... 350 Paraformaldehyde ...... 51, 54, 61, 72, 87, Permeability ...... 326, 335, 362, 365 92, 95–97, 128, 130, 131, 153, 198, 200 Peroxidase anti-peroxidase (PAP) ...... 55–57, 60, Paralogous genes ...... 19 64, 65, 70, 74, 128, 131, 135 Paralysis ...... 160 Pestle...... 239, 340 Para-phenylenediamine ...... 195 Petri dish ...... 52, 74, 79, 167, 199, 286–288, 292 Pargyline ...... 280 pGEM–3 ...... 105, 107, 116 Particle...... 71, 74–81, 88, 90, 91, Phage T4 ...... 250 95–96, 152, 240, 316, 331, 332, 335, 359, 360, 365, Phagocytosis ...... 362 369, 370, 381, 382, 384 Pharmacokinetics ...... 353, 365 Particle size analyzer ...... 357, 360 Phase...... 216, 223, 313, 335, 340, 346, 359, 362, 363 Partition coefficient ...... 328, 335 Phenol...... 269, 344 Passive transport ...... 326–328 Phenolic group ...... 206 Pasteur pipette ...... 185–188, 377 Phenol red ...... 150, 155, 166, 168, 372, 373, 394 Patch pipette ...... 196, 200 Pheochromocytoma ...... 166 Pathogen ...... 307 PHI. See Peptide HI (PHI) Paw...... 365 pH indicator ...... 168 PB. See Phosphate buffer (PB) Phosphate buffer (PB) ...... 51, 52, 72, 87, 92, 95, 96, pBlueScript II SK ...... 103–107 98, 127, 128, 207, 208, 210, 240, 243, 300, 341, 351 NEUROPEPTIDES 409 Index

Phosphate buffered saline (PBS) ...... 52, 72, 87, Poly-D-lysine ...... 395, 401 128, 195, 308, 356, 372, 393 Polyethylene ...... 73, 80, 196, 206, 211, solution ...... 333 342, 356, 357, 361, 366 Phosphoimager ...... 104, 119 Polyethylene glycol (PEG) ...... 206, 221, 254, Phospholipid ...... 335, 359, 362–364, 366 356–358, 362, 363, 366 Phosphorothioate oligodeoxynucleotide Polyethyleneimine (PEI) ...... 372, 376 (PODN) ...... 349–354 Poly-L-lysine ...... 103, 108, 133, 150, 152, 153, 155 Phosphorscreen ...... 104 Polymer ...... 55, 64, 296 Phosphorylation ...... 18, 246 Polymerase chain reaction (PCR) ...... 102, 103, 105–107, Photobleaching ...... 65, 161, 170, 175, 179, 180 111, 113, 114, 116–120, 124, 128, 129, 131–136, Photodamage ...... 171 144, 145, 147, 151, 250–258, 260–262, 265–267, Photodestruction ...... 161, 179 269, 270, 373, 378–380, 390, 399 Photon ...... 162, 163 amplification ...... 111, 129, 135, 136, 144, 250, 253 Photon correlation spectroscopy ...... 360 buffer ...... 102, 103, 106, 107, 133, Photon-counting histogram (PCH) ...... 163 254, 257, 262, 265, 266 pH-sensitive liposome ...... 363 fragment ...... 103, 106 Phylogenetic ...... 390 mix ...... 257, 262, 265, 266, 373 Physiology ...... 14, 186, 203, 355 Polymer horseradish peroxidase (polymer HRP) ...... 55, 64 Picospritzer ...... 195, 198 polymer HRP. See Polymer horseradish peroxidase Picric acid ...... 51, 61, 63, 97, 195, 198, 200 (polymer HRP) Piloerection ...... 386 Polymeric beads ...... 216 Pinhole ...... 162, 169, 170, 178 Polypropylene ...... 211, 341, 342, 350, 352 Piperidine ...... 331, 334–336 Polystyrene ...... 211, 300, 303, 344, 395, 397 Pipette (Pipet) ...... 107, 108, 152, 186–188, 198, 200, Polystyrene beads ...... 327 264, 288, 305, 333, 352, 377, 379, 380, 397, 398, 401 Polyvinylpyrrolidone ...... 87 puller ...... 194, 196, 285, 288 POMC. See Pro-opiomelanocortin (POMC) Pipettor ...... 401 Pontamine sky blue ...... 196, 199, 200 Pituitary gland ...... 15, 19 Pore...... 74, 160, 240, 280, 286, 331, Pixel...... 109, 154, 169, 172 332, 356, 359, 360, 364, 365 Plasma...... 203, 206, 209, 212, 308, 311, 329, Positive control ...... 50, 60, 66, 97, 379, 333, 339–341, 362, 364 380, 395, 397, 398, 400 Plasma membrane ...... 70, 71, 75–77, 160, Positive reaction ...... 67 161, 164, 172–175, 363 Positive staining ...... 60, 65, 66, 80 Plasmid ...... 107, 370, 372, 375, 376, 380, Post-embedding ...... 70, 77 382, 383, 394, 397, 399 Post hoc Tukey test ...... 342, 343 Plastic...... 62, 88, 94, 139, 140, 151, 152, 178, 184, Post-translational modification 189, 207, 209, 216, 225, 268, 285, 288, 290–292 (PTM) ...... 18, 236, 240, 241, 243 Plasticity ...... 50 Potassium chloride (KCl) ...... 72, 102, 103, 106, 107, Plating...... 168, 397 185, 191, 194, 197, 274, 277, 285, 308, 372, 373 Platinum Taq polymerase ...... 102 Potassium ferrocyanide ...... 73 Pleiotropic ...... 18, 328 Povidone iodine ...... 276, 278 PLSD. See Fisher’s protected least significant difference Precipitate ...... 14, 54, 136, 241, 242, 260, (PLSD) 337, 364, 376, 394 Pluronic acid ...... 394, 399 Precipitating fixative ...... 97 pNPP. See 4-Nitrophenyl phosphate disodium salt Precolumn ...... 217, 219–222 hexahydrate (pNPP) Pre-concentration ...... 216, 217, 219, 236, PODN. See Phosphorothioate oligodeoxynucleotide 239, 240, 242, 245 (PODN) Precursor ...... 16, 17, 19–45, 50, 205, Point mutation ...... 50 241, 243, 246, 326, 371 Polar group ...... 327 Pre-embedding ...... 70, 72, 74, 75, 77, 78, 91 Poly(A) ...... 134 Pre-emptive analgesia ...... 62 Polycarbonate ...... 286, 364 Prehybridization ...... 92, 268, 270 Polycarbonate membrane...... 356, 359, 360, 364, 365 Pre-mRNA ...... 101, 111, 118 Polydispersity index (PdI)...... 360, 365 splicing ...... 86 NEUROPEPTIDES 410 Index

Preparative reaction ...... 256, 258 Purification cartridge ...... 220, 222, 223 Pre-pro-peptide ...... 16, 37, 40, 42, 45 P value...... 110, 269 Pre-separation ...... 206, 211 PyBOP. See Benzotriazole–1-yloxy-tris- Preservation ...... 63, 70, 78, 89, 96, 97, 126 pyrrolidinophosphonium hexafluoro-phosphate Pressure ...... 198, 215, 219, 225, 232, 290, 347, 359 (PyBOP) Primary amine ...... 344 Pyrex glass ...... 285, 286 Primary antiserum ...... 60, 67 PYY. See Peptide YY (PYY) Primary transcript ...... 101, 102 Primate ...... 126 Q Primer...... 102–104, 106, 107, 111–113, qISH. See Quantitative in situ hybridization (qISH) 116–118, 120, 128, 129, 131, 132, 134–136, 140, qPCR. See Real time PCR (qRT-PCR) 142, 144–147, 250, 252–257, 261, 262, 265, 266, QPCR/qPCR. See Quantitative PCR (QPCR/qPCR) 268, 269, 373, 378, 380, 384 qRT-PCR. See Real time reverse transcription PCR (Real Primer3 ...... 117 time RT-PCR) Primer dimers ...... 136, 145 Q-TOF. See Quadrupole time-of-flight (Q-TOF) Primer-independent DNA synthesis ...... 129 Quadrupole ion trap ...... 337 Probe...... 86–87, 89, 91–94, 96–98, 105–108, Quadrupole time-of-flight (Q-TOF) ...... 237, 239, 116, 119, 144, 206, 219, 225, 250, 253, 268, 241, 244 273–281, 284, 287, 289–297, 356, 364, 374 Quantitation ...... 104, 109, 110, 236, 237, Probenecid ...... 394, 399 240–241, 243–245 Process...... 50, 54, 58, 60, 63, 75, 141, 143, 173, Quantitative...... 105, 142, 162, 171, 175, 264, 283, 296, 305, 356, 357, 359, 364–366 179, 206, 210, 235–246, 350 Processivity ...... 149 Quantitative in situ hybridization Prodrug ...... 325–347 (qISH) ...... 101–111, 120 Prohormone ...... 150, 235, 238, 241, 246 Quantitative PCR (QPCR/qPCR) ...... 102, 104–105, Prohormone convertase (PCs) ...... 17, 45 111–115, 117, 120, 139–147, 373, 380–381. See also Prolactin ...... 24, 35, 37 Real time PCR Promoter ...... 151, 369, 370, 399 Quantum yield...... 161, 163, 171, 177 Pro-opiomelanocortin (POMC) ...... 19, 20, 102 Quasi-elastic laser scanning, Propane ...... 285, 288 Propylene oxide ...... 73, 78, 79 R Propylenglycol ...... 300, 301 Prostate ...... 330 rAAV. See Recombinant adeno-associated viral (rAAV) Protease ...... 17, 97, 129, 130, 133, 156, 241, 294 vector Protein...... 16, 17, 35, 38, 39, 42, 43, 53, 80, 86, Rabbit...... 60, 61, 72, 204, 207, 208, 210, 296, 330 130, 133, 150, 161, 204–206, 212, 218, 226–232, Radiation ...... 162, 167, 293 236, 241, 285, 290, 296, 301, 302, 304–305, 349, Radioimmunoassay (RIA) ...... 203–212, 274, 280 350, 362, 363, 371, 375, 391, 393, 399 Radiolabeled peptide ...... 284, 287, 292, 294 kinase ...... 43 Radioligand ...... 15, 205, 210 synthesis ...... 16 Radiommunoassay (RIA) ...... 203, 274 Protein A ...... 285, 290–291, 296 Random primers ...... 134, 135 Proteinase K ...... 87, 92, 127, 128, 130, 133 Random sampling ...... 140 Proteolytic ...... 17, 225, 231, 299 Rat...... 15, 72, 90, 91, 102, 105, 106, 114–116, Proteolytic processing ...... 16 118, 134, 142, 146, 150–153, 155, 156, 166, 184, Proteomics ...... 215, 220 185, 188, 196, 255, 262, 275, 279, 311, 316, Proventriculus ...... 57 318–321, 361, 370, 371, 374, 381, 384 Pseudo-first-order degradation ...... 341 Rat basophilic leukemia cell (RBL–2H3) ...... 184, 185, 188 PstI...... 151 RBL–2H3. See Rat basophilic leukemia cell (RBL–2H3) PTM. See Post-translational modification (PTM) Real time ...... 102, 104–105, 111–114, Puller...... 194, 196, 197, 199, 285, 288 117, 150–152, 162, 393 Pulmonary arteries...... 353, 354 Real-time live cell visualization ...... 149 Purification ...... 14, 86–87, 103, 107–108, 141, Real-time movement ...... 150 209, 220, 221, 254, 258–260, 334, 337, 350, 351, Real time PCR (qRT-PCR) ...... 101, 104, 105, 372–373, 375, 377–378, 390 117, 120, 140–142, 144, 145, 147, 373, 380–381 NEUROPEPTIDES 411 Index

Real time reverse transcription PCR Rh6. See Rhodamine 6 G (Rh6) (Real time RT-PCR) ...... 104, 111–115, 117, 119 Rhod–2 ...... 400 Receptor ...... 18, 43, 45, 70, 71, 75, 90, 160, 191, 316, Rhodamine 6 G (Rh6) ...... 167, 169–171, 177, 178 327, 370, 371, 383, 390–393, 395–398, 400–402 RIA. See Radioimmunoassay (RIA) activation ...... 160, 390, 391, 395, 396, 398, 399 Ribbon...... 54 binding ...... 327 Ribonuclease A (RNase A)...... 87, 89, 92, 93, dimerization ...... 390 98, 104, 105, 108, 112, 131–133, 146, 253, 256 signaling ...... 371 Riboprobe ...... 86, 87, 89, 92–94, 96, 98, Receptor-independent mechanism ...... 160 102, 103, 105–108, 111, 116, 117, 119 Receptor-ligand pair ...... 397 Ribosomal RNA ...... 97 Recessed end ...... 250 Righting reflex ...... 319, 320, 342 Recombinant ...... 131, 185, 369–386, 390, 391, 395, 399 Rimadyl ...... 374 Recombinant adeno-associated viral Ringer solution ...... 72, 75, 280 (rAAV) vector ...... 369–386 RNA...... 85–98, 102–105, 107, 108, 111–114, Recombination ...... 382, 383 116–119, 124, 126, 130, 132, 134, 140–142, 144, Recording ...... 172, 193–200 146, 250, 256, 257, 268, 270, 349 Recording chamber...... 194, 196 concentration ...... 112, 134 Red fluorescence ...... 155, 156 degradation ...... 140 Red fluorescent protein (RFP) ...... 150–156 localization...... 85–98 Reflected light ...... 169 polymerase ...... 86, 87, 92, 103, 107, 116 Reflex...... 319, 320, 342 purification ...... 103, 141 Refractive index ...... 166, 177 RNA-binding protein ...... 97 Regression curve ...... 354 RNase A. See Ribonuclease A (RNase A) Regulated secretory pathway ...... 16, 17, 43 RNase free water ...... 92, 133, 146. See also Relaxation ...... 165 Nuclease free water Release ...... 13, 15–18, 37, 45, 46, 61, 135, RNeasy column ...... 107, 108, 119 236, 275, 280, 283–297, 360, 363–365, 371, 377, Rodent...... 333, 342 378, 380, 392, 395 Rostral ventrolateral medulla ...... 352, 362, 365, 366 Releasing factors ...... 14 ROX reference dye ...... 145 Rep gene ...... 370 RP-HPLC. See Reversed phase-HPLC (RP-HPLC) Replication ...... 369, 370, 382 RPMI 1640 medium ...... 166, 177 Reporter molecule ...... 89–92, 96, 124, 136, 160, 161 Rsa I...... 253, 254, 259, 260, 263, 267, 270 Reptile...... 51, 53, 61 Rsq. See R-square (Rsq) Resin...... 73, 78, 88, 96, 327, 331, 334–337, 344, 345 R-square (Rsq) ...... 145 Resistance ...... 194, 200, 330, 341 RT. See Reverse transcription (RT) Resistivity ...... 190, 223, 362 S Respiratory epithelium ...... 317 Respiratory system...... 62 Saccharose ...... 96 Resting membrane potential ...... 197 Safety...... 61, 62, 167, 293, 343 Restriction digest ...... 254, 259–260 glasses ...... 62, 343 Restriction enzyme ...... 383 Sagittal ...... 278, 320 RET...... 70 Sagittal suture ...... 278 Retention ...... 63, 78, 216, 221, 225, 231, 328, 339, 346 Saline...... 52, 87, 128, 150, 195, 300, 301, factor ...... 231 305, 308, 317, 318, 333, 341–343, 346, 356, 364,

time (tR) ...... 71, 221, 225, 228, 230, 231, 239, 339 372, 374, 393 Retrograde transport ...... 149 Saliva...... 308 Retrospective studies ...... 126 Salmon testis DNA ...... 87 Reversed phase-HPLC (RP-HPLC) ...... 215–232, Salt loading ...... 114 331, 337, 341, 345, 346 Sample...... 54, 70, 86, 107, 124, 140, 162, Reverse pharmacology ...... 390, 391 204, 216, 236, 250, 274, 293, 308, 332, 380 Reverse transcriptase ...... 104, 112, 113, dissociating buffer ...... 308, 309 130, 131, 135, 253 enrichment ...... 219 Reverse transcription (RT) ...... 111, 117, 124, loop ...... 220 128, 131, 134 normal buffer ...... 308–310 RFP. See Red fluorescent protein (RFP) preparation ...... 97, 207, 209–210, 218, 236, 241, 341 NEUROPEPTIDES 412 Index

Saturable system ...... 349 Serial Analysis of Gene Expression (SAGE)...... 249 Saturation ...... 61, 136, 170, 257, 258, 270, 311 Serotonin ...... 86 Saturation point ...... 257, 258 Serotransferrin ...... 218, 227 scAAV. See Self-complementary rAAV (scAAV) Serotype ...... 370, 371, 375, 382, 384, 386 Scanner(ing) ...... 109, 119, 161, 166, 169, 172, 268 Serum...... 55, 59, 60, 67, 72, 80, 87, 95, 126, Scan speed ...... 169 150, 166, 185, 208, 212, 218, 225, 277, 286, 304, Scattered light ...... 162 308–312, 330, 352–354, 371, 394, 397 Scavenger ...... 345 Shaft...... 288, 290, 291, 296, 340 S37 cell ...... 188 Shaker...... 300, 301, 342 SCF. See Murine stem cell factor (SCF) Sharp electrode ...... 194, 197, 200 SCG. See Superior cervical ganglia (SCG) Short hairpin RNA ...... 370 Schizophrenia ...... 330 Shrinkage ...... 63 Scintillation counter ...... 103, 108, 350–352 Side effect ...... 316 Scintillation vial ...... 108 Signaling cascade ...... 392 SCN. See Suprachiasmatic nucleus (SCN) Signal peptide ...... 16, 17, 45, 238 Scorpion venom ...... 186, 191 Signal-to-noise ratio (SNR) ...90, 96, 98, 161, 170, 180, 392 Scrambled sequence ...... 98 Signal transduction ...... 71, 389–391 Screen(ing) ...... 109, 110, 204, 253, 266, 338, 373, 378, Silane...... 133, 284, 285, 288–291, 296, 297 390–393, 395, 396, 398, 400, 401 Silane coating ...... 285, 288–290, 297 35S CTP. See [A–35S]-citosine 5’-triphosphate (35S CTP) Silica...... 107, 108, 119, 216, 218, 219, 221, SCX. See Strong cation exchange (SCX) 223, 225, 254, 260, 270, 335 SDS. See Sodium dodecyl sulfate (SDS) matrix ...... 254, 260, 270 Seal...... 83, 135, 196, 285, 288, 289, 372, 377 particles ...... 335 Secondary amine ...... 334, 344 Siloxane ...... 295, 296 Secondary antibodies ...... 52, 57, 58, 60, 66 Silver...... 74, 88, 90, 91, 95, 98 Secondary antibody ...... 55, 59–61, 65, 66, 72, enhancement ...... 88, 90, 91, 95, 98 75, 80, 88, 195, 198 SIM. See Selected-ion monitoring (SIM) chromatogram Secretin ...... 14, 19, 27 Single-barrel ...... 196, 199 Secretogranin ...... 37, 41, 42 Single cell ...... 139, 140 Secretoneurin ...... 41, 43 Single-ion channel ...... 194 Secretory vesicle ...... 17, 149–157 Single labeling ...... 55–57, 92–93 Section ...... 55, 57–59, 63, 66, 73, 79, 80, 93, Single-molecule detection ...... 160, 161 97, 108, 110, 131–135, 144, 187, 361 Single-step PCR ...... 111 Seizure 321 Site-specific ...... 253, 267, 355–366 Selected-ion monitoring (SIM) Site-specific cloning ...... 253 chromatogram...... 331, 339, 340 Skin...... 53, 62, 64, 79, 274, 278, 279, 305, Self-complementary rAAV (scAAV)...... 370 319, 343, 362, 381, 382 Self-seal reagent ...... 135 Skull...... 195, 199, 278, 279, 319, 339, 381 SEM...... 115, 155, 342, 343 35S labeled riboprobe ...... 105 Semipermeable membrane ...... 274, 276 Sleeping ...... 235 Sense...... 92, 103, 107, 116, 117, 370 time ...... 330, 333, 342, 343, 347 Sensitivity ...... 55, 64, 65, 89, 96–98, 111, Slice preparation ...... 197 116, 124, 159–180, 204, 205, 211, 216, 219, 221, Slide...... 86, 98, 103, 104, 108, 133, 143, 236, 253, 294, 303, 363 144, 153, 268, 342 Sensitizer ...... 61 chamber ...... 128, 131, 135 Sensory system ...... 126 Slope...... 145, 225, 352 Separation ...... 65, 206–211, 215–217, 220–222, SmaI...... 253, 383 224, 225, 236, 241–243, 245, 363 Small unilamellar vesicle (SUV) ...... 359 Sephadex ...... 207, 209 SMART-PCR ...... 256–258, 269 Sequence ...... 16, 64, 97, 98, 101, 105, 113, 116, SNR. See Signal-to-noise ratio (SNR) 117, 119, 124, 134, 142, 145, 150, 189, 205, 211, Sodium azide ...... 52, 195, 198, 208, 308, 357, 360 218, 226–228, 231, 236, 238, 240, 243, 250, Sodium citrate ...... 52, 55, 57, 64, 104 252–255, 261, 267, 269, 281, 345, 369, 384, 390, 399 Sodium dodecyl sulfate (SDS) ...... 87, 104 homology ...... 390 Sodium hypochlorite ...... 376, 379, 380 NEUROPEPTIDES 413 Index

Sodium thiosulfate (NTS) ...... 31, 104, 108 Stationary phase ...... 216, 223 Soft Max Pro® ...... 395, 398, 401 Statistical ...... 110, 114, 120, 155, 161, Solid-phase peptide synthesis (SPPS) ...... 334, 342 163, 165, 199, 245, 287, 342, 354 Solid support ...... 327, 334, 337, 345 Stbl2...... 383 Solution-phase peptide synthesis ...... 327 Steam...... 289 Solvent ...... 62, 216, 218–225, 232, 236–242, Stereological sampling ...... 140, 142, 145, 146 244, 334, 335, 345, 346, 357, 358, 363, 364, 401 Stereo microscope ...... 185 Somatostatin (SST) ...... 22, 58, 70, 75, 76 Stereotaxic ...... 195, 198, 199, 275, 276, 278, SON. See Supraoptic nucleus (SON) 301, 318, 319, 357, 361, 374, 381 Sonication ...... 359, 363 Sterile...... 94, 132, 167, 184, 185, 256, Sonicator ...... 81, 356, 359 263, 274, 277, 280, 321, 359, 360, 383, 397 Sorting ...... 16, 17, 43 Sterilization ...... 167, 296 Southern blotting ...... 124 Sternum ...... 305 SP. See Substance P (SP) Stimulated release ...... 45 SP6...... 87, 92, 102, 103, 107, 116 Stimulation ...... 18, 115, 186, 190, 321, 365 Species...... 13, 50, 51, 53, 55, 57–60, 62, 65, frequency ...... 198 66, 72, 75, 79, 80, 95, 97, 117, 118, 129, 162, 165, Stimulator ...... 191, 195 205, 216, 217, 224, 225, 262, 308, 309, 311, 353, Stimulus ...... 16, 17 371, 386, 390 Stir bar ...... 293, 295, 337 Specificity ...... 60, 66, 78, 97, 204, 205, 211, Stirrer...... 333, 340 216, 294, 296, 299, 311 Stochastic ...... 163, 165 Spectrometer ...... 218, 219, 224, 332, 337–339 Stock solution ...... 51, 61, 72, 73, 87, Spectrophotometer ...... 103, 105, 112, 120 127–130, 132, 133, 136, 168, 198, 285, 286, 291, Spectrum ...... 161, 356, 389 300, 301, 309, 337, 341, 394 Spike 2 ...... 196, 199 Stomach...... 57, 316, 317 Spinal cord...... 64, 69–71, 78, 91, 126, Storage ...... 16, 18, 96, 104, 109, 141, 142, 127, 134, 187, 196, 330 146, 156, 225, 232, 242, 258, 296, 333, 344, 378, Spin cup...... 105, 111, 112 392, 394, 395 Spleen...... 64 Strand...... 142, 250, 253, 256, 257, 370 Splicing ...... 19, 37, 71, 86, 126 Streptavidin ...... 71, 72, 75, 79, 80, 88, 93, 94 Splitter ...... 169 Streptomycin ...... 166, 185, 372, 375, 394 SPPS. See Solid-phase peptide synthesis (SPPS) Striatum...... 370 SP6/T7 RNA polymerase ...... 87 Stroke...... 316, 350 Square-wave ...... 197 Strong cation exchange (SCX) ...... 216–218, 220–223, 225 SSC. See Standard saline citrate (SSC) Structure parameter ...... 171 SSH. See Suppression subtractive hybridization (SSH) Student’s test ...... 110 SSH-PCR ...... 249 Student’s t-test ...... 199, 245 SST. See Somatostatin (SST) Subcellular ...... 74, 90, 97, 194, 299 ss tester ...... 250 Subcellular localization ...... 70, 74, 97 SSTR1...... 70 Subcloning ...... 103 SSTR5...... 70 Subcutaneous ...... 374, 381 SSTR2a receptor ...... 76 Substance P (SP) ...... 70, 72, 77, 207, 209 Stable isotope labeling ...... 236 Subtilisin ...... 17 Stage...... 62, 66, 126, 135, Subtracted cDNA ...... 253, 255, 264–267 153, 167, 171, 173, 186, 189, 196, 199, 285, 291, Subtracted DNA ...... 250, 251, 253, 255, 264–267 292, 296, 371, 386 Subtraction ...... 250–253, 262–264, 267 Standard ...... 51, 64, 87, 119, 120, 124, 145, Subtraction efficiency ...... 266–267 167, 169, 171, 178, 190, 204, 210, 217, 224, 226, Subtractive hybridization ...... 249–270 239, 302, 304, 310, 312, 318, 334, 335, 343, 362, Succinic anhydride...... 237, 239–241, 243–245 380, 395, 400, 402 Sucrose ...... 195, 198, 200 curve ...... 145, 204, 210, 304 Suction ...... 334, 335, 337 solution ...... 167, 169, 171 Sulfation ...... 18, 246 Standard saline citrate (SSC)...... 87, 88, 92, 93, Sulfinpyrazone ...... 185, 191 98, 104, 108, 109, 136, 268, 270 Superior cervical ganglia (SCG) ...... 41, 186, 187, 189 NEUROPEPTIDES 414 Index

Supernatant ...... 152, 167, 168, 187, 188, 209, TEM. See Transmission electron microscope (TEM) 210, 242, 245, 260, 302, 303, 305, 341, 377 Temperature ...... 54, 55, 57, 60, 61, 63–65, 72, Superscript II RT ...... 255, 268 75, 80, 87, 89, 91, 92, 96, 108, 109, 112, 113, 127, Suppression subtractive hybridization (SSH) ...... 249, 250, 128, 130–132, 135, 136, 142, 143, 146, 151, 153, 253, 255, 267–269 166, 167, 177, 196, 198, 208, 210, 223, 225, 241, Suppressive PCR ...... 265 242, 244, 254, 255, 259, 260, 275, 285, 287, 289, Suprachiasmatic nucleus (SCN) ...... 105, 106, 245 290, 292, 310, 311, 319, 320, 333, 339, 340, 344, Supraoptic nucleus (SON) ...... 105, 106, 110, 114, 115, 118 345, 359, 362, 363, 365, 380, 394, 396–398, 401 Surgery ...... 277, 279, 281, 319, 321, Template ...... 96, 120, 124, 134, 145, 268 322, 350, 351, 381, 382, 386 RNA ...... 112, 113, 118 Surgical ...... 53, 72, 185, 277, 300, 317, 318, Temporal autocorrelation analysis ...... 163, 179 326, 333, 339, 350, 374, 381 Terminal ...... 16–18, 21, 71, 76, 77, 205, 246, Surgical glove...... 53 253, 288, 302, 334, 336, 344, 369, 382, 392 Suspension ...... 157, 167, 168, 188, 189, transferase ...... 86 207, 310, 358–360, 363–366 Tester cDNA ...... 250, 251, 263, 268 Sustained release ...... 316, 356 Tetramethyl rhodamine isothiocyanate 35S UTP. See [A–35S]-uridine 5’-triphosphate (35S UTP) (TRITC) ...... 58, 59, 65, 169, 171–176, 179, 180 Suture...... 278, 318–321, 382 Tetrodotoxin (TTX) ...... 197 SUV. See Small unilamellar vesicle (SUV) Texas Red ...... 65, 81 SYBR Green ...... 112, 120, 140, 141, 144, TFA. See Trifluoroacetic acid (TFA) 145, 147, 373, 380 TGF. See Transforming growth factors (TGF) Sybr Green ...... 112, 120, 140, 141, 144, Therapeutic ...... 315, 316, 325, 326, 328, 349, 389 145, 147, 373, 380 Thermal cycler ...... 106, 128, 256–258, 263––267, 270 Sympathetic nerve ...... 183 Thermo cycler ...... 102, 103, 128 Sympathetic neuron ...... 184 Thermodynamics ...... 313 Synapse ...... 18, 69, 70, 77 Thermoplastic membrane ...... 139 Synaptic button ...... 18 Thickness ...... 54, 75, 96, 146, 166, 169, 177, 178, 322 Synaptic vesicle...... 75 Thick section ...... 142 Synaptoporin ...... 88, 91 Thighbone ...... 188 Synthesis ...... 13, 16, 46, 49, 85, 116, 127, Thin section ...... 54 129, 140–142, 144, 205, 253, 256, 257, 269, 327, Third ventricle ...... 56 331, 334–337 Three-dimensional diffusion ...... 162, 184 Syringe ...... 53, 74, 146, 184, 185, 188, 195, Three-way valve ...... 195, 198 199, 220, 272, 274, 277, 279, 289, 305, 317–319, Thyroglobulin ...... 205, 207, 208 321, 322, 332, 333, 339, 342, 346, 347, 356, 357, Thyrotropin-releasing hormone (TRH) ...... 34, 236, 321, 359–361, 366, 372–374378 329–331, 334–336, 339, 340, 343–346 Syringe pump ...... 273, 274, 357, 361 Time-lapse ...... 172 Systemic administration ...... 326, 329 Tip...... 80, 168, 171, 195, 198, 218, 264, 277, 278, 288, T 291–293, 295, 296, 318, 332, 339, 356, 359, 382, 397 diameter ...... 80, 218 T7...... 87, 92, 102, 103, 107, 116, 117 Tissue...... 18, 50, 72, 86, 103, 123, 139, 166, 184, 199, 206, Tachikynin ...... 37, 205, 206 220, 235, 250, 275, 284, 299, 328, 349, 365, 375, 390 T/A cloning vector ...... 253 culture plate ...... 84, 88 Tail...... 150, 151, 172, 175, 179, 199, 330, 343, 346, 347 grinder ...... 333, 339 vein ...... 199, 330, 346 processing ...... 51, 54–55, 73–74, 140 Taq polymerase ...... 102, 104, 112, 269 section ...... 66, 85–98, 105, 123, 124, Taste bud ...... 57 126, 132, 133, 135, 329

Tc. See Crystal phase transition temperature (Tc) Tissue-Tek ...... 96, 140, 142 T cell...... 186 Titanium ...... 359 293T cells ...... 371, 372, 375, 377, 393, 396 Titer...... 67, 95, 208, 371, 375, 380, 381, 383, 384 T4 DNA ligase ...... 254, 260 Titration ...... 80, 373, 380–381 TEA buffer ...... 104 Tm. See Melting temperature (Tm) TE buffer ...... 102, 103, 256 TNE buffer ...... 104, 108, 254, 259 Teflon...... 225, 274, 277, 279, 340 Toluene ...... 285, 288–290, 296 NEUROPEPTIDES 415 Index

Tool...... 75, 85, 86, 110, 117, 123, 144, 186, 199, Tyrosine kinase ...... 70, 71 203, 204, 269, 291, 299, 318, 329, 369, 374, 381 Tyrosine kinase receptor (trkB) ...... 70, 71, 77 Toxic...... 62, 79 T4 polynucleotide kinase ...... 350, 351 U t . See Retention time (t ) R R Ultracentrifuge ...... 300, 302 Trafficking ...... 150, 151 Ultrafiltrate(ion) ...... 360, 366 Transcription (rate) ...... 101, 102, 107, 111, 117, Ultramicrotome ...... 79 119, 124, 128, 131, 134, 392 Ultra performance liquid chromatography Transfection ...... 150–152, 156, 370–372, (UPLC) ...... 237, 240, 241, 244 375–377, 383, 390, 393–397, 399, 400 Ultrastructural ...... 69, 70, 74, 76, 77 efficiency ...... 152, 156, 376, 383, 395, 399, 400 Ultrastructure ...... 70, 80 Transforming growth factors (TGF) ...... 43 Ultrathin section ...... 73–74, 78–79, 96 Transgene (expression) ...... 370 Ultraviolet (UV) ...... 92, 139, 144, 167, 242, Transient expression ...... 391, 396 268, 274, 333, 338, 345, 346 Transient molecular complex ...... 160 Unsubtracted DNA ...... 260, 261, 264–267 Translation ...... 86, 165, 349 UPLC. See Ultra performance liquid chromatography Translocation ...... 160, 174, 392, 399 (UPLC) Transmission electron microscope (TEM) ...... 54, 70, Uranyl acetate ...... 73, 74, 78, 79, 88, 96 73–75, 78–80, 93, 164 Uridine ...... 103, 119 Transport(er) ...... 19, 35, 43, 49, 142, 149, 150, 161, U shape probe ...... 275 175, 316, 326–328, 330, 334, 349–354, 371, 399 UV. See Ultraviolet (UV) Transporting vesicles ...... 175 Trap...... 219, 221, 223, 225, V 236, 240, 242, 337, 339 Trauma ...... 317, 321, 322 Vacuum evaporator ...... 63 Tray...... 55, 143, 288–295 Vacuum pump ...... 293, 295, 296, TRH. See Thyrotropin-releasing hormone (TRH) 332, 338, 356, 359 Tricaine. See MS222 Valve...... 195, 198–223, 232, Triethanolamine ...... 87, 104, 108, 128, 130 338, 339, 346, 385 Trifluoroacetic acid (TFA)...... 207, 218, 224, Vapor(izer) ...... 61, 318–320, 339 239–242, 331–333, 336, 337, 343–346 Vasoactive intestinal peptide (VIP) ...... 27, 57, 58 Trimethylammoniumbutyrate ...... 237 Vasopressin (VP) ...... 21, 102–103, 105–107, Triplet correlation time ...... 164, 179 111, 113, 115–119 Triplet state ...... 162, 164, 170, 179 Vasostatin ...... 41, 43 TRITC. See Tetramethyl rhodamine isothiocyanate V bottom plate...... 267 (TRITC) Vector...... 71, 72, 88, 105, 107, 116, 129, Triton X–100 (TX) ...... 72 152, 195, 253, 267, 369–386 trkB. See Tyrosine kinase receptor (trkB) Vein...... 199, 330, 342, 346, 347, 351 Trophic ...... 193 Velocity ...... 150, 154, 175 tr-trkB. See Truncated trkB (tr-trkB) Venous blood ...... 353 Truncated trkB (tr-trkB) ...... 71 Ventral horn ...... 91, 127 Trypsin ...... 184, 187, 190, 372, 375, 393, 396 Ventricle ...... 56, 305 Tryptic...... 217, 218, 224–226 Vertebrate ...... 13, 49–67, 391, 392 TSA. See Tyramide system amplification kit (TSA) Veterinary ...... 62 TTX. See Tetrodotoxin (TTX) Vial...... 51, 54, 108, 167, 168, 210, 212, Tubing...... 196, 211, 218, 225, 277, 279, 285, 287, 288, 225, 232, 238, 277, 279, 394 317–319, 360, 381 Vibrating microtome ...... 72, 75, 194, 196, Turbinates ...... 317 199, 382. See also Vibratome Tween...... 88, 286, 292, 293, 295, 308 Vibratome...... 75, 196, 199, 382 Two-dimensional diffusion ...... 164 VIP. See Vasoactive intestinal peptide (VIP) Two-step PCR ...... 111 Viral particle ...... 370 TX. See Triton X–100 (TX) VNO. See Vomeronasal organ (VNO) Tyramide system amplification kit (TSA) ...... 88–95, 98 Voltage ...... 193, 197, 200, 218, 225, 288, 365 Tyrosine ...... 70, 71, 89, 205, 206, 211, 246 Voltammetry ...... 275 NEUROPEPTIDES 416 Index

Volume element ...... 161, 162, 164, 165, 171, 179 Woodchuck posttranscriptional regulatory element Volume transmission ...... 85 (WPRE) ...... 382 Vomeronasal organ (VNO) ...... 317 WPRE. See Woodchuck posttranscriptional regulatory Vortex...... 105, 111, 242, 244, 256, 258, 333, element (WPRE) 341, 350, 351, 356, 359, 377, 394 X VP. See Vasopressin (VP) x-direction ...... 170 W Xenopus ...... 57 Wash buffer ...... 103, 105, 111, 112, 308, XhoI...... 151 310, 394, 396, 397, 399, 401 Xylene...... 55, 57, 130, 135, 146, 285, 287 Water bath ...... 86, 128, 130, 166, 167, 333, XmaI...... 253 340, 341, 350, 351, 356, 359, 363, 377 X-ray cassette ...... 104 Water immersion objective ...... 156, 169, 194, 196 Xylazine ...... 276, 277 Waveform ...... 199 Y Wavelength ...... 162, 177, 191, 242, 303, 304, 401 y-direction ...... 170 Wax...... 54, 55, 57, 62–64 Yeast...... 87, 391 WE14...... 43 total RNA (tRNA) ...... 104, 108 Welsh t-test ...... 269 Z White glue ...... 133 Whole-cell ...... 194, 196–197, 200 z-direction ...... 170 patch clamp ...... 194, 196–197 Zebrafish ...... 53 recording ...... 196 Zeta potential ...... 360, 366 Whole organ...... 63 Zetasizer ...... 357, 360 Wnt...... 43 Zoom...... 169, 172