Super-resolution Microscopical Localization of Dopamine Receptors 1 and 2 in Rat Hippocampal Synaptosomes Master Thesis For the attainment of the academic degree Master of Science From the University of Applied Sciences FH Campus Wien Submitted by: András-Gábor Miklósi Personal identity code 1410544003 Supervisor: Ao. Univ.-Prof. Dr. Gert Lubec Department of Pharmaceutical Chemistry Faculty of Life Sciences University of Vienna Vienna Austria Submitted on: 20. 11. 2017 Abstract Although dopamine receptors D1 and D2 play key roles in hippocampal function, their synaptic localization within the hippocampus has not been fully elucidated. In order to understand precise functions of pre- or postsynaptic dopamine receptors (DRs), the development of protocols to differentiate pre- and postsynaptic DRs is essential. So far, most studies on determination and quantification of DRs did not discriminate between subsynaptic localization. Therefore, the aim of the study was to generate a robust workflow for the localization of DRs. This work provides the basis for future work on hippocampal DRs, in light that DRs may have different functions at pre- or postsynaptic sites. Synaptosomes from rat hippocampi isolated by a sucrose gradient protocol were prepared for super-resolution direct stochastic optical reconstruction microscopy (dSTORM) using Bassoon as a presynaptic zone and Homer1 as postsynaptic density marker. Direct labeling of primary validated antibodies against dopamine receptors D1 (D1R) and D2 (D2R) with Alexa Fluor 594 enabled unequivocal assignment of D1R and D2R to both, pre- and postsynaptic sites. D1R immunoreactivity clusters were observed within the presynaptic active zone as well as at perisynaptic sites at the edge of the presynaptic active zone. The results may be useful for the interpretation of previous studies and the design of future work on DRs in the hippocampus. Moreover, the reduction of the complexity of brain tissue by the use of synaptosomal preparations and dSTORM technology may represent a useful tool for synaptic localization of brain proteins. Abstract Die Dopamin Rezeptoren D1 und D2 spielen eine Schlüsselrolle in der Funktion des Hippocampus. Dennoch ist die synaptische Lokalisation dieser Rezeptoren innerhalb des Hippocampus unklar. Für ein umfassendes Verständnis von prä- wie auch postsynaptischen Dopamin Rezeptoren (DRs) ist die Entwicklung von Protokollen zur feineren Differenzierung von prä- und postsynaptischen Rezeptoren essentiell. In bisherigen Studien wurde die Detektion und Quantifizierung von DRs unabhängig von ihrer synaptischen Lokalisierung getestet. Das Ziel dieser Studie war die Erstellung eines Protokolls, welches die synaptische Lokalisation von DRs erlaubt. Damit ist die Basis für zukünftige Arbeiten an unterschiedlichen Funktionen zwischen prä- und postsynaptischen DRs geboten. Das Synaptosom wurde aus dem Hippocampus von Ratten mittels Saccharosegradient isoliert und für die Visualisierung im ”Super- resolution direct stochastic optical reconstruction microscope” (dSTORM) wurden präsynaptische Areale mit Bassoon und postsynaptische Areale mit Homer1 markiert. Durch die Verwendung von validierten, primären Antikörpern gegen Dopamin Rezeptor D1 (D1R) und Dopamin Rezeptor D2 (D2R) und der Visualisierung dieser mittels Alexa Fluor 594 konnten die Rezeptoren eindeutig ihrer synaptische Lokalisation zugeordnet werden. Eine Anhäufung von D1R Immunoreaktivität wurde innerhalb der aktiven präsynaptischen Zone sowie in perisynaptischen Bereichen am Rande der aktiven präsynaptischen Zone beobachtet. Diese Ergebnisse könnten zu einem verbesserten Verständnis vorheriger Studien beitragen sowie die Gestaltung zukünftiger Studien an DRs im Hippocampus beeinflussen. Darüber hinaus könnte die Verwendung von synaptosomalen Präparationen in Verbindung mit dSTORM, durch Reduktion an Komplexität gegenüber Hirngewebe, ein nützliches Werkzeug für die synaptische Lokalisation von Proteinen im Gehirn darstellen. iii 1. TABLE OF CONTENTS 1. TABLE OF CONTENTS ........................................................................... IV 2. INTRODUCTION .................................................................................... 10 2.1 The dopaminergic system ....................................................... 10 2.1.1 Dopamine Biosynthesis ........................................................................... 11 2.1.2 Dopamine metabolism ............................................................................. 12 2.1.3 The dopamine receptors .......................................................................... 13 2.1.3.1 D1 type receptors ................................................................................ 13 2.1.3.2 D2 type receptors ................................................................................ 15 2.2 The Hippocampus .................................................................... 16 2.2.1 The anatomy of the hippocampus ............................................................ 17 2.2.2 Cell types of the hippocampal formation .................................................. 18 2.2.2.1 The CA1 region ................................................................................... 18 2.2.2.2 The CA2 and CA3 regions ................................................................... 19 2.2.2.3 The dentate gyrus ................................................................................ 20 2.3 The synapse .............................................................................. 22 2.3.1 Structure of the presynaptic active zone .................................................. 24 2.3.2 Structure of the post synaptic density ...................................................... 25 2.4 Basics of super resolution fluorescence microscopy .......... 27 2.4.1 Stimulated emission depletion (STED) microscopy .................................. 28 2.4.2 Stochastic optical reconstruction (STORM) microscopy .......................... 29 3. MATERIALS AND METHODS .................................................................. 33 3.1 Preparation of hippocampal synaptosomes .......................... 33 3.2 Primary neuronal cell cultures used for dSTORM and immunocytochemistry........................................................................... 33 3.3 Mass spectrometrical characterization of the synaptosomal fraction ....................................................................................................34 3.4 Liquid Chromatography Tandem Mass Spectrometry (LC- MS/MS) ....................................................................................................34 3.5 Processing of synaptosomal fraction for electron microscopical characterization ............................................................ 35 3.6 Fixation and staining of synaptosomes and primary neuron cultures for dSTORM ............................................................................. 36 3.7 Three channel dSTORM imaging ............................................ 36 3.8 dSTORM processing ................................................................ 37 iv 3.9 Measurement of the distance between pre-and postsynaptic markers ................................................................................................... 37 3.10 Antibodies used for dSTORM analysis .................................. 38 3.11 Staining for characterization of neuron culture morphology by confocal microscopy ........................................................................ 38 4. RESULTS ............................................................................................ 40 4.1 Mass spectrometrical characterization of the synaptosomal fraction ....................................................................................................40 4.2 Electron microscopical characterization of the synaptosomal fraction ....................................................................................................43 4.3 Characterization of the primary hippocampal neuronal cultures ................................................................................................... 45 4.4 Expression of D1R and D2R in rat hippocampal neuronal cultures ................................................................................................... 46 4.5 Measurement of the distance between pre- and postsynaptic markers for validation of the synaptosomal preparation .................. 48 4.6 Super resolution localization of dopamine receptors .......... 49 5. DISCUSSION ........................................................................................ 52 6. REFERENCES ...................................................................................... 56 7. ACKNOWLEDGEMENTS ........................................................................ 66 8. LIST OF FIGURES ................................................................................. 67 9. LIST OF TABLES .................................................................................. 69 10. APPENDIX .................................................................................... 70 v List of abbreviations TH tyrosine hydroxylase L-DOPA L-3,4-dihydroxyphenylalanine VMAT2 vesicular monoamine transporter 2 DAT dopamine transporter NET norepinephrine tranporter ADHD attention deficit hyperactivity disorder MAO monoamine oxidase DOPAL 3,4-Dihydroxyphenylacetaldehyde MPTP 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine MPP+ 1-methyl-4-phenylpyridinium DR dopamine receptor