A Dissertation

entitled

Monoamines Differentially Modulate Release from Distinct Sites Within A

Single Neuron Pair

by

Tobias Samuel Thomas Clark

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the

Doctor of Philosophy Degree in

Biology

______Dr. Richard Komuniecki, Committee Chair

______Dr. Bruce Bamber, Committee Member

______Dr. Patricia Komuniecki, Committee Member

______Dr. Robert Steven, Committee Member

______Dr. Ajith Karunarathne, Committee Member

______Dr. Amanda Bryant-Friedrich, Dean College of Graduate Studies

The University of Toledo August, 2018

Copyright 2018, Tobias Samuel Thomas Clark

This document is copyrighted material. Under copyright law, no parts of this document

may be reproduced without the expressed permission of the author. An Abstract of Monoamines Differentially Modulate Neuropeptide Release from Distinct Sites Within A Single Neuron Pair By Tobias Samuel Thomas Clark Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biology

The University of Toledo August 2018

Monoamines and modulate behavior, but monoaminergic-peptidergic cross talk remains poorly understood. In C. elegans, serotonin and the adrenergic-like ligands, tyramine (TA) and octopamine (OA) require distinct subsets of neuropeptides in the two

ASI sensory neurons to inhibit nociception. For example, the ASI neuropeptides required for TA inhibition, encoded by nlp-1, -14 and -18 are distinct from those required for OA inhibition, encoded by nlp-6, -7, and -9. In the present study, NLP-9, -14:: or -18::GFPs

co-localized with the synaptic marker, mCHERRY::RAB-3 in the distal portion of the

ASI axons, where synapses have been identified previously by electron microscopy.

Neuropeptide and RAB-3 positive axonal puncta were also consistently observed extra-

synaptically near ASI soma. ASI::NLP-14::GFP and ASI::NLP-9::mCHERRY co-

localized to both synaptic and extra-synaptic puncta, but the GFP/mCHERRY ratios in

extra-synaptic/synaptic puncta differed significantly, suggesting that TA- and OA-

dependent neuropeptides may be differentially trafficked and/or localized. TA, but not

iii OA, selectively increased the release of ASI neuropeptides encoded by nlp-14 or nlp-18 from either synaptic/perisynaptic regions of ASI axons or the ASI soma, respectively. In contrast, OA, but not TA, selectively increased the release of ASI neuropeptides encoded by nlp-9 asymmetrically, only from synaptic/perisynaptic regions of the right ASI axon.

The predicted preprosequences of the three TA-dependent neuropeptide-encoding genes

differed markedly from the three OA-dependent genes. However, these distinct

preprosequences were not sufficient to confer monoamine-specificity, as the release of

chimeric neuropeptides containing swapped pre-pro-sequences did not respond to the

predicted monoamine. In contrast, the release of a truncated NLP-14::GFP containing only the first 95 amino acids and only five of a predicted 12 neuropeptides responded to

TA but not OA, demonstrating that additional N-terminal sequence was required for monoamine-specificity. Collectively, our results demonstrate that TA and OA differently modulate the release of distinct subsets of neuropeptides from different subcellular sites within the ASIs and highlight the complexity of monoaminergic/peptidergic modulation,

even animals with a relatively simple nervous system containing only 302 neurons.

iv Acknowledgements

First and foremost, I would like to sincerely thank my advisor and mentor, Dr. Rick

Komuniecki. Rick has taught me so many lessons about science and life, which often echo daily, and have helped steer my development, as both a person and scientist. For these lessons and patience, I am eternally grateful.

My dear thanks go to all my friends on this side of the Atlantic for helping me adjust to life in America and making me feel at home. In particular, I offer my heartfelt appreciations to Steven Dajnowicz, Mitchell Oakes and Dr. Wen Jing Law for their engaging discussions, assistance inside and outside the lab, lifelong lessons taught and general friendship over the years. I am also forever thankful to have RaeLynne MacBeth by my side. She has been a constant motivation and support, spending countless nights and weekends working with me. I am a better person for having her in my life.

To my many family members and friends back home, especially my parents, sisters and brother, thank you for the ongoing support and love over the years. You have each helped in your own way to keep me moving along this path and know that although I am no longer close by, nothing has changed between us.

Last, and certainly not least, I thank Dr. Patricia Komuniecki, Dr. Bruce Bamber,

Dr. Robert Steven and Dr. Ajith Karunarathne for agreeing to be on my committee and their time.

v Table of Contents

Abstract ...... iii

Acknowledgements ...... v

Table of Contents ...... vi

List of Figures ...... ix

List of Abbreviations ...... x

List of Symbols ...... xv

1. Introduction ...... 1

1.1 Monoamines and Neuropeptides as ...... 4

1.2 The Role of TA, OA and Neuropeptides in Invertebrates ...... 9

1.3 Dense Core Vesicle Formation and Neuropeptide Packaging ...... 14

1.4 Significance...... 22

2. Materials and Methods ...... 26

2.1 Materials ...... 26

2.2 Nematode Strains ...... 27

2.3 Construction of C. elegans Transgenes ...... 27

2.4 Behavioral Assays ...... 28

vi 2.5 Confocal Microscopy ...... 29

2.6 Neuropeptide Fluorescence Assay ...... 30

2.7 RNA Isolation and cDNA Synthesis...... 31

2.8 Quantitative PCR ...... 31

2.9 Bioinformatic Analysis ...... 32

2.10 Statistics ...... 32

3. Results ...... 33

3.1 Distinct Subsets of ASI Neuropeptides Are Essential for the TA- and OA- dependent Inhibition of Aversive Responses to 1-octanol ...... 33

3.2 TA- and OA-dependent Neuropeptides Appear to Co-localize at Individual

ASI Synapses...... 37

3.3 TA and OA Stimulate the Release of Distinct Subsets of ASI

Neuropeptides...... 42

3.4 ASI Neuropeptide Proproteins Required for Either TA- or OA-dependent

Inhibition Contain Different Leader Sequences ...... 48

4. Discussion ...... 55

4.1 Monoaminergic/peptidergic Crosstalk is Extensive and Modulates Most

Key C. elegans Behaviors ...... 57

4.2 Monoaminergic/peptidergic Interactions Are Also Important in Other

Organisms ...... 62

4.3 Both TA- and OA-dependent ASI Neuropeptides Co-localize in the ASIs .....64

4.4 TA and OA Differentially Modulate Neuropeptide Release from the ASIs ....68

4.5 OA Asymmetrically Stimulates Neuropeptide Release from the ASIs ...... 71

vii 4.6 Monoamines and Neuropeptides Interact Directly to Modulate Release ...... 73

4.7 The TA- and OA-dependent ASI Neuropeptides Contain Different Predicted

Preprosequences ...... 77

4.8 Summary ...... 83

5. References ...... 86

viii List of Figures

3 – 1 – 1 The two ASI Sensory Neurons Integrate Both Monoaminergic and Peptidergic

Signaling to Modulate Nociceptive Behavior ...... 35

3 – 1 – 2 The TA- and OA-dependent Inhibition of Aversive Responses Requires

Different Subsets of ASI Neuropeptides...... 36

3 – 2 – 1 Position of C-terminal GFP does not Affect DCV Packaging or Localization ..38

3 – 2 – 2 TA- and OA-dependent Neuropeptides Co-localize at ASI Synapses ...... 41

3 – 3 – 1 TA Modulates ASI Neuropeptide Release ...... 45

3 – 3 – 2 OA Modulates ASI Neuropeptide Release ...... 47

3 – 4 – 1 Both the TA- and OA-dependent Preproprotein Sequences Contain Several

Predicted Sorting Sequences Involved in DCV Packaging ...... 51

3 – 4 – 2 Effect of TA and OA on ASI GFP Fluorescence in Animals Expressing NLP-

14/NLP-9 Chimeric Neuropeptides ...... 52

3 – 4 – 3 Truncation of the ASI::NLP-14 Preproprotein does not Alter TA and OA

Sensitivity ...... 54

4 – 1 Model of Monoamine-specific ASI Neuropeptide Release ...... 56

ix List of Abbreviations

5-HT ...... 5-hydroxytrptamine (Serotonin) 5-HT1A ...... G-protein-coupled serotonin subtype 5-HT1B ...... G-protein-coupled serotonin receptor subtype 5-HT2C ...... G-protein-coupled serotonin receptor subtype 8-OH-DPAT ...... 8-Hydroxy-n,n-dipropylaminotetralin (5-HT1A )

α-MSH ...... Alpha-melanocyte stimulating hormone act-1 ...... Actin-encoding gene ACTH ...... Adrenocorticotropic hormone ADF...... Sensory neuron ADR ...... Adrenalin AEX-2 ...... G-protein-coupled AgRP ...... Agouti related AIA ...... Interneuron AIB ...... Interneuron AIY ...... Interneuron AMP ...... Adenosine monophosphate ARC ...... Arcuate nucleus of the ASE ...... Sensory neuron ASEL...... Left ASE sensory neuron ASELON ...... Left ASE sensory neuron response ASER ...... Right ASE sensory neuron ASEROFF ...... Right ASE sensory neuron response ASH...... Sensory neuron ASI ...... Sensory neuron ATGL-1...... Adipose triglyceride lipase AtT-20 ...... Mammalian neuronal cell line AUA ...... Interneuron AWC ...... Sensory neuron AWCOFF ...... AWC sensory neuron not expressing str-2 AWCON ...... AWC sensory neuron expressing str-2

BAG ...... Neuronal cell

CabTRP Ia ...... Cancer borealis tachykinin-related peptide Ia

x cAMP ...... Cyclic adenosine monophosphate CB1 ...... G-protein coupled CgA ...... Chromogranin A CgB ...... Chromogranin B CP ...... Male-specific neuron CPE ...... Carboxypeptidase E CRF ...... Corticotropin releasing factor CRH ...... Corticotropin releasing hormone

D2 ...... G-protein-coupled receptor D3 ...... G-protein-coupled DA ...... Dopamine DAF-7 ...... A member of the transforming growth factor beta superfamily (Caenorhabditis elegans) DAF-28 ...... Beta-type (Caenorhabditis elegans) DAG ...... Diacylglycerol DCV ...... Dense core vesicle DOP-1 / dop-1 ...... G-protein-coupled dopamine receptor / G-protein-coupled dopamine receptor-encoding gene DOP-3 / dop-3 ...... G-protein-coupled dopamine receptor / G-protein-coupled dopamine receptor-encoding gene DOP-4 ...... G-protein-coupled dopamine receptor DRN ...... Dorsal raphe nucleus DVA ...... Interneuron

EC50 ...... Half maximal effective concentration EGL-3 ...... Proprotein convertase 2 (Caenorhabditis elegans) EGL-6 ...... G-protein-coupled neuropeptide receptor EGL-21 ...... Carboxypeptidase E (Caenorhabditis elegans) ELH ...... Egg-laying hormone f25b3.3 ...... Ras nucleotide exchange factor-encoding gene flp ...... FMRFamide (Phe-Met-Arg-Phe-NH2)-related flp-1 ...... Neuropeptide-encoding gene flp-3 ...... Neuropeptide-encoding gene FLP-7 / flp-7 ...... Neuropeptide / Neuropeptide-encoding gene flp-8 ...... Neuropeptide-encoding gene flp-9 ...... Neuropeptide-encoding gene flp-10 ...... Neuropeptide-encoding gene flp-11 ...... Neuropeptide-encoding gene flp-12 ...... Neuropeptide-encoding gene flp-17 ...... Neuropeptide-encoding gene FLP-18 / flp-18 ...... Neuropeptide / Neuropeptide-encoding gene flp-19 ...... Neuropeptide-encoding gene flp-20 ...... Neuropeptide-encoding gene flp-21 ...... Neuropeptide-encoding gene xi

GABA ...... Gamma-Aminobutyric acid gcy-1 ...... Receptor-type guanylyl cyclase-encoding gene gcy-3 ...... Receptor-type guanylyl cyclase-encoding gene gcy-4 ...... Receptor-type guanylyl cyclase-encoding gene gcy-5 ...... Receptor-type guanylyl cyclase-encoding gene gcy-6 ...... Receptor-type guanylyl cyclase-encoding gene gcy-7 ...... Receptor-type guanylyl cyclase-encoding gene gcy-14 ...... Receptor-type guanylyl cyclase-encoding gene gcy-20 ...... Receptor-type guanylyl cyclase-encoding gene gcy-22 ...... Receptor-type guanylyl cyclase-encoding gene GEF ...... Guanine nucleotide exchange factor GFP ...... Green Fluorescent Protein GH4C1 ...... Mammalian neuronal cell line gpa-4 ...... G-protein alpha subunit-encoding gene GPCR ...... G-protein-coupled receptor GTP ...... Guanosine triphosphate

HEK293 ...... Human embryonic kidney cell line HPA...... Hypothalamic-pituitary-adrenal axis HSN...... Motor neuron ins-1...... Insulin-like neuropeptide-encoding gene INS ...... Insulin-like neuropeptide IP3 ...... trisphosphate ISV ...... Immature secretory vesicle

L1 ...... Caenorhabditis elegans larval stage 1 L4 ...... Caenorhabditis elegans larval stage 4 let-858 ...... Nucampholin-encoding gene LGC-55 ...... Tyramine-gated chloride channel LH ...... Lateral hypothalamus

MC ...... receptor MC3 ...... MC4 ...... Melanocortin receptor mCHERRY ...... A monomer fluorescent protein derived from DsRed MCN1 ...... Modulatory commissural neuron 1 mGluR2 ...... G-protein coupled µM ...... Micromolar mM ...... Millimolar MOD-1 ...... Serotonin-gated chloride channel MPN ...... Modulatory proctolin neuron MS ...... Mass spectrophotometry

N2 ...... Wild-type Caenorhabditis elegans NA ...... Noradrenaline xii NEP-2 ...... Neprilysin peptidase Neuro-2a ...... Mammalian neuronal cell line ng...... Nanogram NGM ...... Nematode growth medium nlp-1 ...... Neuropeptide-encoding gene nlp-3 ...... Neuropeptide-encoding gene nlp-6 ...... Neuropeptide-encoding gene nlp-7 ...... Neuropeptide-encoding gene nlp-9 ...... Neuropeptide-encoding gene NLP-12 / nlp-12 ...... Neuropeptide / Neuropeptide-encoding gene NLP-14 / nlp-14 ...... Neuropeptide / Neuropeptide-encoding gene nlp-15 ...... Neuropeptide-encoding gene NLP-18 / nlp-18 ...... Neuropeptide / Neuropeptide-encoding gene NLP-24 / nlp-24 ...... Neuropeptide / Neuropeptide-encoding gene NLP-40 / nlp-40 ...... Neuropeptide / Neuropeptide-encoding gene nM ...... Nanomolar NPR-1 ...... G-protein-coupled neuropeptide receptor NPR-2 ...... G-protein-coupled neuropeptide receptor npr-17 ...... G-protein-coupled neuropeptide receptor-encoding gene npr-19 ...... G-protein-coupled neuropeptide receptor-encoding gene NPS ...... NPSR...... G-protein-coupled NPY...... nrD ...... Dorsal segment of the RIA neuron nrV ...... Ventral segment of the RIA neuron ntc-1 ...... Neuropeptide-encoding gene ntr-1...... NTC-1 receptor-encoding gene ntr-2...... NTC-1 receptor-encoding gene (predicted to be G-protein-coupled)

OA ...... Octopamine OCTR-1...... G-protein-coupled octopamine receptor OP50 ...... Escherichia coli strain

PC1/3...... Proprotein convertase PC2 ...... Proprotein convertase PC12 ...... Pheochromocytoma cell line PDF-1 / pdf-1 ...... Neuropeptide / Neuropeptide-encoding gene PDFR-1 ...... G-protein-coupled PDF-1 receptor POMC ...... PPT ...... Preprotachykinin PVN...... Paraventricular nucleus PVP ...... Interneuons

RAB-3 / rab-3 ...... Ras GTPase / Ras GTPase-encoding gene RAB-3a ...... Ras GTPase, isoform a RAB-27a ...... Ras GTPase, isoform a xiii RFP ...... Red fluorescent protein RIA ...... Interneuron RIC ...... Interneuron RIF ...... Interneuron RIM ...... Interneuron RNAi ...... RNA interference rol-6...... Cuticle -encoding gene

SCV ...... Small clear vesicle SE ...... Standard error SER-1 ...... G-protein-coupled serotonin receptor SER-2 ...... G-protein-coupled tyramine receptor SER-3 ...... G-protein-coupled octopamine receptor SER-4 ...... G-protein-coupled serotonin receptor SER-5 ...... G-protein-coupled serotonin receptor SER-6 ...... G-protein-coupled octopamine receptor SIA ...... Interneuron / Motor neuron SNARE ...... Soluble N-ethyl-maleimide-sensitive fusion protein attachment receptor SNB-1 ...... Synaptobrevin SNET-1 ...... Peptide involved in environmental signaling srg-47 ...... Serpentine receptor class G-encoding gene SSTR2 ...... G-protein-coupled receptor str-2 ...... Putative -encoding gene

TA ...... Tyramine TBH-1 / tbh-1 ...... Tyramine β-hydroxylase / Tyramine β-hydroxylase-encoding gene TDC-1 tdc-1 ...... Tyrosine decarboxylase / Tyrosine decarboxylase-encoding gene TGN ...... trans-Golgi network tph-1 ...... Tryptophan hydroxylase-encoding gene TRH...... Thyrotropin-releasing hormone TYRA-3 / tyra-3 ...... G-protein-coupled tyramine receptor / G-protein-coupled tyramine receptor-encoding gene unc-54 ...... Myosin class II heavy chain-encoding gene unc-122 ...... Type II transmembrane protein-encoding gene UTR...... Untranslated region UV1 ...... Neurosecretory cell

VP ......

Y2 ...... G-protein-coupled Y5 ...... G-protein-coupled neuropeptide Y receptor YFP ...... Yellow fluorescent protein

xiv List of Symbols

°C ...... Degrees Celsius α ...... Alpha β ...... Beta γ ...... Gamma µ ...... Micro m ...... Milli n...... Nano

xv Chapter 1

Introduction

Most, if not all behaviors, are modulated by both monoamines and neuropeptides and the dysregulation of the monoaminergic/peptidergic crosstalk has been implicated in a myriad of diseases, including depression, anxiety, obesity and chronic pain (Charbogne et al., 2014; Kohler et al., 2016; Murrough et al., 2015). However, the specifics of these monoaminergic/peptidergic interactions are difficult to analyze due to the complexity of the mammalian . Caenorhabditis elegans contains a small nervous system, with only 302 neurons, yet exhibits complex behaviors modulated by both monoamines and neuropeptides, with many similarities to mammalian systems. For example, many pharmaceuticals, including selective serotonin (5-HT) reuptake inhibitors, opioids and cannabinoids work through orthologous proteins and pathways in both nematodes and mammals. , a 5-HT reuptake inhibitor, modulates synaptic transmission by blocking 5-HT reuptake transporters, increasing the amount of synaptic

5-HT in both mammals and C. elegans (Kullyev et al., 2010; Perez-Caballero et al.,

2014). Similarly, opioids exhibit analgesic properties in mammals via activation of opioid receptors and C. elegans contains an endogenous opioid signaling system also involved in nociception that can be modulated by and salvinorin A (Mills et al., 2016). 1 Indeed, phenotypes in the C. elegans with null mutations in the gene encoding its opiate receptor orthologue, npr-17, can be rescued by the expression of the human kappa opiate receptor driven by the npr-17 promoter (Mills et al., 2016). In addition, both mammals and C. elegans contain the endogenous cannabinoid receptor , 2- arachidonoylglycerol and anandamide (Lehtonen et al., 2008; Lehtonen et al., 2011).

Endocannabinoids mediate nociception in mammals, and interestingly, nociceptive and feeding phenotypes in C. elegans with null mutations in the gene encoding its cannabinoid receptor orthologue, npr-19, can be rescued by the expression of the human

CB1 cannabinoid receptor driven by the npr-19 promoter (Aviram & Samuelly-Leichtag,

2017; Clapper et al., 2010; Oakes et al., 2017). Lastly, the noradrenergic inhibition of chronic ‘pain’ is also similar in both C. elegans and mammals, as both systems express

α2-like adrenergic receptors to inhibit primary nociceptors and α1-like receptors to stimulate the release of an array of inhibitory neuropeptides (Kirkpatrick et al., 2015;

Knezevic et al., 2017; Mills et al., 2012a; Stein, 2013).

Crosstalk between monoaminergic and peptidergic signaling modulates most, if not all behaviors in C. elegans. For example, extensive work details how monoamines and neuropeptides modulate the two ASH sensory neurons in mediating aversive responses.

As we have demonstrated previously, 5-HT stimulates the initiation of aversive responses to dilute 1-octanol and promotes forward locomotion after the initial reversal is complete.

This pathway requires three distinct 5-HT receptors at different levels within the locomotory circuit and the neuropeptide-encoding genes nlp-3 and -24 (Harris et al.,

2010; Harris et al., 2009; Mills et al., 2016). Conversely, 5-HT stimulation is antagonized by the adrenergic-like ligands, tyramine (TA) or octopamine (OA), that dramatically

2 increase the time taken to initiate the aversive response, and this TA- and OA-dependent inhibition requires a different subset of neuropeptides (Hapiak et al., 2013; Mills et al.,

2012b; Wragg et al., 2007). For example, neuropeptides encoded by nlp-1, -14 and -18 are required for the TA inhibition of this 5-HT stimulated response (Hapiak et al., 2013).

Interestingly, TA and OA act through different subsets of monoamine receptors and neuropeptide-encoding genes to inhibit aversive responses to higher 1-octanol concentrations (Hapiak et al., 2013; Mills et al., 2012b; Wragg et al., 2007). For example, neuropeptides encoded by nlp-6, -7 and -9 are required for OA inhibition of aversive responses to higher 1-octanol concentrations (Mills et al., 2012b). Additionally, nociception and avoidance behavior is modulated by inhibitory cross talk between the

ASHs and ASIs, via additional , octopaminergic and peptidergic signaling

(Guo et al., 2015). Monoaminergic and peptidergic signaling also modulate many additional behaviors, including feeding, nutritional status, mating, egg-laying and locomotion (Banerjee et al., 2017; Carnell et al., 2005; Garrison et al., 2012; Greer et al.,

2008; Hapiak et al., 2009; Srinivasan et al., 2008). Monoaminergic and peptidergic cross talk is extensive and complex, with modulation occurring at the level of individual neurons, entire circuits and, as described in the present study, interacting directly, with monoamines differentially regulating the release of specific neuropeptides from somal and synaptic sites within a single pair of neurons.

The present study was designed to define the roles of these modulatory monoamines on ASI neuropeptide release using fluorescently-tagged neuropeptides. We have demonstrated that individual monoamines selectively stimulate the release of distinct subsets of ASI neuropeptides from unique sites within the ASIs, with the TA-

3 dependent neuropeptides encoded by nlp-14 and nlp-18 released at synaptic/perisynaptic sites or the ASI soma, respectively, and OA-dependent neuropeptides encoded by nlp-9 released asymmetrically only from synaptic/perisynaptic sites on the right ASI axon. The

TA- and OA-dependent neuropeptide-encoding genes also differed markedly in their preprosequences, but these differences did not confer monoamine-specific neuropeptide release and additional N-terminal sequence within the neuropeptide coding sequence was required. For example, a truncated NLP-14 protein, NLP-141-95 that contained the preprosequence and the first five of 14 predicted neuropeptides exhibited specific TA- dependent release. Collectively these studies demonstrate the potential for TA and OA to selectively modulate specific neuropeptide release from distinct sites within the ASIs and highlight the complex interactions of monoaminergic/peptidergic signaling.

1.1. Monoamines and Neuropeptides as Neurotransmitters

Even though monoaminergic and peptidergic signaling modulates many of the same behaviors, little is known about potential interactions between these two complex signaling systems. Both monoamines and neuropeptides can act as neurotransmitters and/or hormones, and exert their actions by activating seven transmembrane metabotropic G-protein-coupled receptors (GPCRs) or ionotropic ligand-gated ion channels. For example, mammals express genes encoding 13 different 5-HT GPCRs that can be separated into seven distinct 5-HT receptor families, and one 5-HT-gated ion channel. In addition, 5-HT receptor number is increased extensively by the presence of multiple isoforms of the individual 5-HT receptors and additional post-translational processing by RNA editing (Burns et al., 1997; Schellekens et al., 2012). C. elegans contains four different 5-HT GPCRs, each expressed as multiple isoforms and each with 4 clear orthology to one of the mammalian 5-HT receptor families in mammals, and one unique 5-HT-gated Cl- channel (Carre-Pierrat et al., 2006; Hannon & Hoyer, 2008;

Hapiak et al., 2009; Ranganathan et al., 2000). Typically, ionotropic receptors mediate fast synaptic transmission, while metabotropic receptors are associated with slow, long- lasting changes in potential. Both can either excite or inhibit a cell. GPCRs activate intracellular signaling pathways and secondary messengers, depending on the receptor type; most simply Gαs increases cyclic AMP (cAMP) production, Gαi/o is antagonistic to

Gαs and decreases cAMP production, Gαq/11 stimulates diacylglycerol (DAG) and

2+ inositol-1,4,5-triphosphate (IP3) production to increase Ca and lastly, Gα12/13 activate

RhoGEF (Heng et al., 2013). However, recent analyses have demonstrated that each of the GPCRs also initiate a wide variety of additional G-protein dependent and G-protein independent signaling cascades, and even at the level of individual G-proteins, these receptors exhibit extensive synergistic and antagonistic interactions. For example, stimulation of Gαs activates local adenylyl cyclases, increasing the amount of cAMP within the cell. The increased cAMP functions in several ways depending on the local protein environment, activating 1) protein kinase A to induce phosphorylation of target proteins, 2) exchange proteins that modulate neuronal polarity, or 3) directly modulating ion channels (Bradley et al., 2005; Godinho et al., 2015; Munoz-Llancao et al., 2015).

Furthermore, G-protein independent signaling regulates a diverse array of cellular functions, including signaling and trafficking events, and often desensitization of GPCRs.

For example, β-arrestins are activated following GPCR agonist stimulation and modulate

GPCR signaling by sterically inhibiting G-protein interactions with the second and third intracellular loops (Marion et al., 2006; Smith & Rajagopal, 2016).

5 Importantly, the co-localization of neurotransmitters within a single neuron has been observed in both vertebrates and invertebrates. For example, rat medullary slices contain both 5-HT and the neuropeptide, , both of which required for the excitatory modulation of respiratory interneurons and motor neurons (Ptak et al., 2009).

Similarly, both dopamine-β-hydroxylase, the enzyme that catalyzes the conversion of dopamine (DA) to noradrenaline (NA), and neuropeptide Y (NPY) co-localize in neurons within the of rats, and their expression increases in a model for spontaneous hypertension (Kourtesis et al., 2015). In C. elegans, monoamines and neuropeptides co-localize in several neurons. For example, nlp-3 and ins-1 are expressed in the serotonergic ADFs, and these neuropeptides, along with 5-HT, modulate thermotaxic responses (Li et al., 2013b; Nathoo et al., 2001). Similarly, the tyraminergic

RIMs also express the neuropeptide-encoding gene, flp-18, and both TA and FLP-18 neuropeptides are involved in hunger-dependent decision-making (Ghosh et al., 2016).

Lastly, the octopaminergic RICs also express several neuropeptides, although the signaling interactions among these neuromodulators has not been studied extensively

(Nathoo et al., 2001). Collectively, these studies highlight the co-localization of different neurotransmitters across species, suggesting each has a role in configuring neuronal circuitry and thus behavior.

Importantly, monoamines and neuropeptides can modulate both neuronal excitability and/or release. For example, NA, adrenaline (ADR), DA or

5-HT all increase VP secretion from cultured rat neurohypophyseal tissue (Kis et al.,

2011; Radacs et al., 2008). In contrast, either -A or -B, two peptides located in the hypothalamus, decrease monoamine-stimulated VP release, highlighting interactions

6 between monoaminergic and peptidergic signaling (Kis et al., 2011). New technologies to measure neurotransmitter release are always being developed, contributing to our understanding of how neurotransmitter release is regulated. Recently, Banerjee et al.

(2016) demonstrated that although histamine induced the release of both DA and NPY from pheochromocytoma 12 (PC12) cells, their EC50s differed significantly, suggesting that higher concentrations of histamine were required for DA release (Banerjee et al.,

2016). This study suggests that the two neurotransmitters were packaged into separate vesicles, and that the concentration of the upstream histamine stimulus differentially modulated which vesicles were released.

Growing evidence suggests that monoamines and neuropeptides interact to release a host of additional modulators and initiate global signaling cascades. For example, stress responses involve multiple neurotransmitters and several sites of activity. Initially, the perceives danger and signals to the hypothalamus, stimulating ADR release that circulates throughout the body, causing a myriad of physiological changes, including elevated heart rate and glucose release. Once the initial increase in ADR subsides, the hypothalamic-pituitary-adrenal axis (HPA), the second component of the stress response, is activated. To begin, the hypothalamus secretes two peptides, corticotropin releasing hormone (CRH) and VP, which act synergistically, stimulating the anterior pituitary to release adrenocorticotropic hormone (ACTH). ACTH acts on the adrenal cortex to stimulate glucocorticoid release, that reduces CRH and ACTH levels via a negative feedback loop to the hypothalamus and anterior pituitary (Smith & Vale, 2006).

The link between dysregulated neurotransmission and disease state has been well documented over time and continues to be a topic of intense interest. For example, 7 obesity is a global disorder, affecting an estimated 107.7 million children and 603.7 million adults worldwide, and is associated with several disease states, including hypertension, heart disease and type 2 diabetes (Kushner & Kahan, 2018). Serotonergic signaling plays a prominent role in feeding and satiety. Generally speaking, in vertebrates, increased 5-HT availability induces satiety and hypophagia; in contrast, reduced 5-HT availability confers hyperphagia (Voigt & Fink, 2015). For example, after

5-HT is depleted by administration of p-chlorophenylalanine, an inhibitor of 5-HT synthesis, rats exhibit hyperphagia and gain weight. In contrast, increasing synaptic 5-HT levels by administering 5-HT reuptake inhibitors decreases feeding and body weight

(Magalhaes et al., 2010). At the neuronal level, 5-HT most notably effects food intake by regulating two neuronal populations within the arcuate nucleus of the hypothalamus

(ARC), the anorexigenic proopiomelanocortin (POMC) expressing neurons, and the orexigenic NPY/Agouti related peptide (AgRP) expressing neurons (Sohn et al., 2013).

Activation of 5-HT2C receptors stimulates the POMC expressing neurons and conversely, activation of 5-HT1B receptors inhibits the NPY/AgRP expressing neurons (Wyler et al.,

2017). Neurons downstream of the ARC, such as the paraventricular nucleus (PVN), express melanocortin receptors (MCs) that regulate and are activated by alpha- melanocyte stimulating hormone (α-MSH), a proteolytic product of POMC (Berglund et al., 2014; Garfield et al., 2015; Krashes et al., 2016). Conversely, NA can mediate feeding by inhibiting the activity of POMC neurons and stimulating the activity of the

NPY/AgRP neurons (Paeger et al., 2017). The NPY/AgRP expressing neurons are believed to increase food intake by, 1) releasing NPY to stimulate Y receptors, 2) releasing the inverse MC4R agonist, AgRP and 3) releasing the inhibitory

8 neurotransmitter Gamma-Aminobutyric acid (GABA) to suppress signaling from several neurons, including those expressing POMC (Sohn, 2015). These studies highlight the complex role of 5-HT, NA and neuropeptides in regulating food intake and how their interactions can initiate signaling cascades to modulate neuronal activity.

1.2. The Role of TA, OA and Neuropeptides in Invertebrates

Several monoamines (e.g. 5-HT, DA, TA, histamine) are present in both vertebrates and invertebrates, but relatively little is known about the potential roles of TA and OA in vertebrates. Trace amounts of TA and OA are present in mammals, and although trace amine-associated receptors have been identified in 26 vertebrate genomes, much remains to be learned about their function and physiological significance

(Borowsky et al., 2001; Eyun et al., 2016). In contrast, in invertebrates, TA and OA are important signaling molecules in their own right and are considered the invertebrate counterparts of ADR/NA, based on the observation that their cognate receptors exhibit significant similarities to mammalian adrenergic receptors. TA is synthesized by tyrosine decarboxylation catalyzed by tyrosine decarboxylase (TDC-1). TA can act as a stand- alone neurotransmitter or as a substrate for the synthesis of OA, via the addition of a hydroxyl group catalyzed by tyramine β-hydroxylase (TBH-1). TA and OA often act in concert or antagonistically. For example, OA induces hyperactivity in starved flies, promotes rhythmicity of a neuronal feeding circuit in Aplysia, and TA plays a key role in behavioral differences between foraging and nursing honeybees (Martinez-Rubio et al.,

2010; Scheiner et al., 2017; Yang et al., 2015). In addition, in locusts, aggregative and solitary behaviors are mediated by TA and OA, respectively, through distinct TA and OA receptors (Ma et al., 2015). TA and OA ratios determine behavioral outcomes with TA

9 injection successfully switching gregarious olfactory preferences to solitary, and vice versa, demonstrating the delicate balance between TA and OA signaling (Ma et al.,

2015).

As in other invertebrates, TA and OA modulate multiple behaviors in C. elegans and this monoaminergic modulation often involves other neurotransmitters, including neuropeptides. In C. elegans, neuropeptides are classified into three families, insulin-like peptides (ins), FMRFamide (Phe-Met-Arg-Phe-NH2)-related peptides (flp) and a diverse neuropeptide-like proteins (nlp) (Li & Kim, 2008). Much remains to be learned about peptidergic signaling in general and its interaction with monoaminergic signaling specifically, despite the fact that peptide neurotransmitters are found across a variety of species. In C. elegans, neuropeptides modulate essentially all behaviors, including locomotion, social behavior, learning and memory, reproduction, chemosensation, mechanosensation (Avery, 2010; Campbell et al., 2015; Li & Kim, 2010; Li et al., 2013a;

Rogers et al., 2003). For example, when removed from food, C. elegans increase their turning rate, initiating a local search behavior that is dependent on the AWC neurons and neuropeptides encoded by nlp-1 (Chalasani et al., 2010; Gray et al., 2005). Interestingly, nlp-1 mutants exhibit prolonged Ca2+ signals in the AWCs after odor removal, with increased turning during local search and decreased Ca2+ signals in the downstream AIA neurons when presented with an odor, highlighting the involvement of neuropeptides in the dynamic regulation of neuronal signaling (Chalasani et al., 2010). Similarly, the

ASEL sensory neuron responds to salt at concentrations ≥10 mM, but larger increases in salt concentration reconfigure the salt-sensing circuitry and requires INS neuropeptides from ASEL to recruit AWCON (Leinwand & Chalasani, 2013; Suzuki et al., 2008).

10 The interplay between 5-HT, TA/OA and peptidergic signaling has been explored in the modulation of several behaviors, with TA/OA often acting antagonistically to 5-

HT. For example, both TA and OA inhibit aversive responses mediated primarily by the two ASHs and the levels of OA are important, as low OA concentrations activate the

Gαo-coupled adrenergic-like OA receptor, OCTR-1, in the ASHs to inhibit 5-HT- stimulated aversive responses, but higher OA concentrations also activate the Gαs- coupled OA receptor in the ASHs to antagonize OCTR-1 signaling and abolish OA inhibition (Mills et al., 2012b). Neuropeptides play keys roles in this monoaminergic modulation. For example, neuropeptides encoded by nlp-3 and -24 are required for the 5-

HT stimulation of aversive responses to dilute 1-octanol, neuropeptides encoded by nlp-

1, -14 and -18 are required for the TA inhibition of this 5-HT stimulated response and neuropeptides encoded by nlp-6, -7 and -9 are required for OA inhibition of aversive responses to higher 1-octanol concentrations (Hapiak et al., 2013; Harris et al., 2010;

Mills et al., 2016; Mills et al., 2012b). In fact, neuropeptides appear to be involved in the monoaminergic modulation of most key behaviors in C. elegans. For example, egg-laying is mediated by a complex neuronal circuit that switches between acute, active phases of egg-laying, and longer, inactive phases. The 5-HT stimulation of egg-laying requires both excitatory and inhibitory inputs, the direct activation of the 5-HTergic HSN neurons that stimulate egg-laying, and several 5-HT receptors (Carnell et al., 2005; Emtage et al.,

2012; Hapiak et al., 2009; Sze et al., 2000). In contrast, both TA and OA inhibit egg- laying and tdc-1 and tbh-1 null mutants that lack TA/OA and OA exhibit hyperactive egg-laying phenotypes (Alkema et al., 2005; Collins et al., 2016; Horvitz et al., 1982).

The neuroendocrine UV1 cells express tdc-1 and are suggested to play a role in this TA-

11 inhibition (Alkema et al., 2005). Briefly, when the uterus is not filled with eggs, the UV1 cells release TA and other neurotransmitters to inhibit egg-laying. However, upon filling, the uterus increases in size and deforms the UV1 cells, inhibiting neurotransmitter release

(Jose et al., 2007; Zhang et al., 2010a). TA inhibits the HSNs that release 5-HT to stimulate egg-laying via the TA-gated Cl- channel LGC-55 and importantly neuropeptides from the UV1 cells encoded by flp-11 and nlp-7 play an essential inhibitory role in this process (Banerjee et al., 2017; Collins et al., 2016).

The ability to respond to changing nutritional cues is critical for survival. The molecular mechanisms behind food-related behaviors are still being deciphered, but both monoamines and neuropeptides play key roles. As in mammals, 5-HT is involved in the definition of nutritional state in C. elegans and is often referred to as the ‘food-at-hand’ signal, with 5-HT incubation mimicking many behaviors seen on bacteria, such as increased feeding (pharyngeal pumping) and egg-laying (Avery & Horvitz, 1990; Horvitz et al., 1982; Niacaris & Avery, 2003). TA and OA have also been implicated in food- related behaviors and again, appear to antagonize 5-HT signaling. For example, both

TA/OA are believed to increase during starvation and inhibit pharyngeal pumping and feeding (Horvitz et al., 1982; Komuniecki et al., 2014; Rex et al., 2004; Suo et al., 2006).

In addition, C. elegans also exhibit several food-dependent locomotory behaviors that are modulated by both monoamines and neuropeptides. These include: roaming, characterized by rapid movement and full-body propagation, dwelling, involving less movement that is usually limited to the anterior portion of the worm, and both feeding- and fasting-quiescence, a complete cessation of movement that occur both on and off food. In the presence of food, worms typically exhibit dwelling and feeding-quiescence,

12 presumably to stay on the food source (Ben Arous et al., 2009). Exogenous 5-HT mimics this dwelling behavior and tph-1 nulls that lack 5-HT exhibit decreased dwelling and fasting-quiescence (Churgin et al., 2017; Flavell et al., 2013). In contrast, TA and OA sometimes, but not always, antagonize the effects of 5-HT on many food-dependent locomotory behaviors. For example, animals lacking TDC-1 cannot synthesize TA or OA and have increased dwelling, fasting quiescence, and decreased roaming behaviors, indicative of behaviors on food. Exogenous TA increases dwelling and reduces roaming, in contrast to exogenous OA which reduces dwelling, increases roaming and increases fasting quiescence, suggesting separate roles for TA and OA in this behavior (Churgin et al., 2017).

Neuropeptides also modulate locomotion extensively, as dysregulated peptidergic signaling can either increase or decrease movement both on a solid surface and in liquid medium. For example, the neuropeptide encoding genes, flp-1 and flp-18 are both expressed in command interneurons, and the corresponding neuropeptide-null mutants exhibit decreases or increases, respectively, in swimming rate in liquid (Chang et al.,

2015). Interestingly, neuropeptides that are not expressed in the command interneurons also modulate swimming rate, as hyperactive locomotion is observed in the neuropeptide- encoding flp-3, -10 and -21 null mutants. In addition, 5-HT-dependent decreases in swimming rate are dependent on peptidergic signaling, as flp-1 and -19 null mutants are resistance to 5-HT inhibition (Chang et al., 2015). The role of neuropeptides in the modulation of locomotory state is only now beginning to be examined, but it is clear that many neuropeptide null animals exhibit extensive defects in locomotory behavior

(turning, reversal, etc.), in addition to defects in locomotory rate, suggesting that, as

13 observed for pharyngeal pumping and aversive behaviors, monoaminergic/peptidergic interactions will also be important in the modulation of these more complex locomotory behaviors (Campbell et al., 2015; Flavell et al., 2013; Harris et al., 2010; McCloskey et al., 2017). For example, the neuropeptides encoded by pdf-1 interact with serotonergic signaling to promote roaming behavior, as pdf-1 animals exhibit enhanced dwelling/quiescence that can be rescued with native pdf-1 expression (Flavell et al.,

2013). Neuropeptides are also involved in aging and reproduction. For example, neuropeptides encoded by nlp-7 play a role in aging, as nlp-7 RNAi reduces oxidative- stress resistance and lifespan, and the 5-HT stimulation of egg-laying requires neuropeptides encoded by flp-9, -12 and -20 mutants, as the corresponding null mutants exhibit decreased egg-laying and retained more eggs in the uterus (Chang et al., 2015;

Park et al., 2010; Park et al., 2009).

To summarize, the studies outlined above highlight the role of monoaminergic and peptidergic interactions in modulating a variety of complex behaviors, but also demonstrate that further examination of this extensive and important cross-talk between these signaling systems is required.

1.3. Dense Core Vesicle Formation and Neuropeptide Packaging

Neurotransmitter release from synaptic vesicles and neuroprotein secretion from dense-core vesicles (DCVs), either synaptically, perisynaptically and extra-synaptically, play key roles in neuronal plasticity. Protein/peptide secretion can be either constitutive or regulated. Several groups have proposed that the constitutive pathway is the default route for secretion and a specific signal or domain must be present to direct proteins to

14 the regulated pathway (Blazquez & Shennan, 2000; Dikeakos & Reudelhuber, 2007). The regulated secretory pathway requires the aggregation of secreted material into immature secretory vesicles (ISVs) that eventually mature into DCVs, so named because DCVs are electron-dense when viewed under an electron microscope (Blazquez & Shennan, 2000;

Tooze et al., 2001; Zhang et al., 2010b). The biogenesis of ISVs is a multi-step process and a model has been proposed based on morphology. Firstly, regulated secretory proteins begin to aggregate within a specialized region of the trans-Golgi Network

(TGN), possibly via membrane-associated secretory proteins, as proprotein convertase 2

(PC2), chromogranin A (CgA) and chromogranin B (CgB) associate with TGN membranes and interact with other proteins in the secretory pathway (Glombik et al.,

1999; Mahapatra et al., 2008; Seidah & Prat, 2002). In addition, the Golgi has a predicted pH 5.9 and contains 10 mM Ca2+, conditions that induce aggregation of several DCV proteins, including CgA, CgB, secretogranin II and PC2 (Blazquez & Shennan, 2000;

Demaurex et al., 1998; Park et al., 2002). Following aggregation, proteins secreted through the regulated pathway assemble into oligomers and additional components, such as soluble N-ethyl-maleimide-sensitive fusion protein attachment receptor (SNARE) proteins, associate and begin to bud from the TGN, forming ISVs. After formation,

SNAREs found on the ISVs initiate the homotypic fusion of several ISVs. Lastly, during homotypic fusion, membrane remodeling and protein sorting occur, with soluble peptides, membrane proteins and unprocessed proproteins removed from the maturing vesicle and targeted back to the TGN or endosomes (Kim et al., 2006). For example, in

PC12 cells, mannose-6-phosphate receptors and furin are present in ISVs, but are undetectable after maturation (Morvan & Tooze, 2008). In contrast, this pathway is not

15 associated with any of the proteins found in the constitutive pathway, in which smaller vesicles undergo continuous fusion and do not store their cargo for long, meaning that release is usually representative of protein biosynthesis. As such, the constitutive pathway are primarily involved in delivering essential proteins to the plasma membrane at constant, relatively low levels.

Two models have been proposed to explain aggregation as a mechanism of sorting: sorting-for-entry and sorting-by-retention. Sorting-for-entry stipulates that any cargo destined for the regulated secretory pathway is selectively recruited from the TGN, a model that includes aggregation and/or receptor-mediated packaging (Dikeakos &

Reudelhuber, 2007; Zhang et al., 2010b). As such, proteins excluded from this pathway would then reach the constitutive pathway by default (Sobota et al., 2006). Indeed, the condensation of secreted proteins starts at the TGN and in AtT-20 cells, no co- localization of regulated and constitutive proteins was observed after entry into the TGN or at sites from which ISVs were budding, supporting the idea that cargo separation occurs before ISV formation (Blazquez & Shennan, 2000; Kim et al., 2006). However, contrasting results have been reported in non-neuronal pancreatic β-cells, where aggregation appeared to occur within the ISVs (Blazquez & Shennan, 2000). In contrast, the sorting-by-retention model suggests that the bulk packaging of cargos into the ISVs and aggregation/condensation facilitates retention. ISVs would then undergo remodeling, and any proteins not required in the final DCVs would be selectively removed during maturation (Sobota et al., 2006). Evidence supporting both sorting models has been reported and a clear consensus has yet to be reached, possibly due to variations between cell lines, especially neuronal vs non-neuronal, and the specific proteins being sorted

16 (Dikeakos & Reudelhuber, 2007; Zhang et al., 2010b). Indeed, one could envision a mechanism involving aspects of both models. Initially, membrane-bound proteins, such as carboxypeptidase E (CPE), localize to distinct regions of the TGN, capturing soluble proteins destined for an ISV. These membrane associations could lead to the aggregation and recruitment of other proteins required for ISV formation, creating a specific lipid/protein microdomain (Kienzle & von Blume, 2014). ISVs then would form from these lipid microdomains and begin to bud from the TGN. The homotypic fusion of several ISVs is mediated by SNARE proteins and the differential sorting of cargo would send unnecessary, untargeted proteins back to the endoplasmic reticulum or endosomes, leaving a mature DCV containing specific cargo. However, the specifics of molecular mechanism involved in distinguishing necessary from unnecessary proteins/peptides remains to be elucidated. Any additional insight into neuropeptide packaging and DCV biogenesis will help us understand the mechanism of neuropeptide-specific DCV release and thus potential interactions between monoaminergic/peptidergic signaling.

Neuropeptides are synthesized as one, large preproprotein and packaged into DCVs often with other proteins, including their processing enzymes, such as PC1/3 and CPE

(EGL-21 in C. elegans) (Dikeakos et al., 2009; Hook et al., 2008; Lou et al., 2010; Salio et al., 2007). DCVs are often stored until release and require changes in cytoplasmic secondary messengers, such as Ca2+ for secretion in response to specific stimuli (de Wit et al., 2009; Farina et al., 2015). Stimulus-specific secretion allows for an acute increase in the release of the stored cargo, while keeping secretion in the absence of a stimulus low. As many neuropeptide precursors contain multiple neuropeptides, each with their own specific function, understanding how neuropeptides can be differentially packaged

17 and/or secreted is of biological importance and has been studied extensively. For example, Aplysia california bag cell neurons express egg-laying hormone (ELH), a prohormone that contains several functionally different peptides that localize to different types of DCVs. Smaller DCVs in the axon contain either N- or C-terminal products, in contrast to larger, somal-bound DCVs that only contain N-terminal peptides (Sossin et al., 1990). Pulse-chase experiments suggest that the initial ELH cleavage does not occur early during Golgi transit and the differential localization of N- and C-terminal products occurs after a 30-min pulse and 2-h chase, indicating that differential sorting occurs after the initial proELH cleavage (Sossin et al., 1990). Similarly, the differential sorting of prothyrotropin-releasing hormone (proTRH) has been studied in AtT-20 cells (Perello et al., 2008). ProTRH is initially cleaved by PC1/3 and further processing generates multiple products. The proTRH-derived peptides are differentially sorted to different populations of DCVs, as DCVs stain positive for either N-, C- or both N- and C-terminal products (Perello et al., 2008). NA is a natural secretagogue of TRH and stimulates the release of both N- and C-terminal proTRH-derived products. However, higher NA concentrations stimulates the more abundant release of C-terminal proTRH-derived peptides and the differential packaging of these proTRH peptides is disrupted when the

PC1/3 cleavage site is mutated (Perello et al., 2008). These data highlight the importance of the initial precursor cleavage in DCV sorting and demonstrate how both the differential packaging and release of peptides can occur from a single precursor.

However, more information is needed to understand the mechanisms that separate proteins/peptides between the constitutive or regulated secreted pathways and then into specific DCVs.

18 The idea of a signal sequence/domain that efficiently traffics cargo to the regulated secretory pathway is supported by early transfection studies, in which heterologous cell lines successfully sort, process and eventually secrete non-native proteins destined for regulated secretion, if they are designed to contain the appropriate signal sequence. For example, when human proinsulin was expressed in a mouse anterior pituitary AtT-20 cell line, insulin-like products were secreted in response to secretagogues (Irminger et al.,

1994). Several possible signal sequences/domains have been proposed for cargo trafficking to DCVs. For example, the fusion of the 26 amino acids found at the N- terminus of POMC are sufficient to direct a reporter protein to the regulated secretory pathway (Cool & Loh, 1994; Loh et al., 2002). The sorting signal within the N-terminal of POMC is specific, i.e., 13 amino acids containing an amphipathic hairpin loop stabilized by a disulphide bridge (Cool et al., 1995). Interestingly, a similar hairpin loop that was essential for efficient sorting into the regulated secretory pathway is also found in both CgA and CgB. In contrast, a truncated CgB lacking this N-terminal loop expressed in PC12 cells was released constitutively and was not properly packaged and sorted into the regulatory secretory pathway. However, this truncated CgB was efficiently packaged in the presence of other proteins destined for the regulated secretory pathway, demonstrating that the N-terminal loop was needed for correct packaging, but the C- terminal sequence facilitated packaging that was dependent on the local protein environment (Kromer et al., 1998; Tooze et al., 2001). In addition, a chimeric fusion protein containing the CgB N-terminal loop on both the N- and C-terminus of the constitutively secreted α1-antitrypsin, switched sorting to the regulated secretory pathway in PC12 cells (Glombik et al., 1999). In contrast, not all proteins directed to the regulated

19 secretory pathway contain a disulphide-bonded loop. For example, PC1/3 and PC2 (EGL-

3 in C. elegans) are efficiently targeted to the regulated secretory pathway, but do not contain a disulphide-bonded loop (Dikeakos et al., 2009; Hook et al., 2008). Importantly, chimeric proteins and specific deletions may interrupt the natural folding of a protein or block potential sites involved in retention or sorting, and observations that must be considered when interpreting such data.

In addition to hairpin loops, internal dibasic residues have been postulated to be efficient sorting signals in the regulated secretory pathway. Dibasic residues are proteolytic cleavage sites for PC1/3 and PC2, and are also important for the correct sorting/secretion, as mutations at N-terminal dibasic sites in prorenin abolished regulated secretion in AtT-20 cells (Brechler et al., 1996). Similarly, chimeric proteins containing a prosequence with two dibasic cleavage sites increased vesicle sorting efficiency in

GH4C1 cells (Lacombe et al., 2005). Interestingly, when the initial processing sites of the rat proTRH were mutated to glycine, peptides derived from this mutant precursor were secreted in a constitutive fashion, suggesting that correct cleavage is required for efficient sorting to the regulated pathway (Mulcahy et al., 2005). In addition, α-helices direct secretory proteins to vesicles and interestingly, sorting was most efficient when N- terminal dibasic residues were combined with a C-terminal α-helix (Dikeakos et al.,

2007; Lacombe et al., 2005). Charge distribution along neuropeptide proproteins also plays a role in sorting. For example, protachykinin and procalcitonin gene-related peptide propeptide are negatively charged, but the mature peptide-containing sequence is positively charged. Mutations disrupting this charge distribution disturbs the overall electrostatic charge and causes inefficient DCV packaging, suggesting that electrostatic

20 interactions likely contribute to molecular aggregation/interactions that act as a sorting mechanism (Ma et al., 2008; Zhang et al., 2010b). These observations suggest that no single sorting domain appears to account for targeting into the regulated secretory pathway. In fact, a more specific receptor-mediated sorting may also play a role.

Preliminary evidence suggests the specific receptors are involved in sorting cargo into the regulated secretory pathway, but the specifics and identity of these receptors remains poorly understood. For example, CPE appears to function as a sorting receptor.

CPE removes basic residues after the original proprotein has been cleaved by PC1/3 and/or PC2 and is in both soluble and membrane-bound forms (Cawley et al., 2012). CPE binds to the loop structure in the putative POMC N-terminal sorting motif and CPE is required for the correct sorting of POMC to the regulated secretory pathway, both in vitro and in vivo (Cool et al., 1997; Loh et al., 2002). In fact, molecular modeling predicts the binding of CPE to POMC occurs between CPE254-273 and acidic residues in POMC1-26, based on site-directed mutagenesis (Loh et al., 2002). POMC and secretion in CPE-deficient mice was also misrouted to the constitutive pathway, resulting in elevated levels in circulation (Shen & Loh, 1997). In addition, proinsulin and secretion from mouse neuroendocrine cells was similarly misdirected after

CPE RNAi (Normant & Loh, 1998). In contrast, CPE RNAi did not affect the packaging of CgA, even though CgA also contains a putative N-terminal loop, suggesting that additional sorting receptors are present or that CgA contains sufficient signal/structure to be independently directed to DCVs (Normant & Loh, 1998). Similarly, proinsulin is efficiently targeted and released from pancreatic islets derived from CPE-deficient mice, suggesting that protein sorting and processing may be dependent on both cell type and

21 cargo (Irminger et al., 1997). Indeed, CPE appears to recognize additional sorting motifs, in addition to loop structures, as and insulin that do not contain loop structures but also form aggregates with CPE (Rindler, 1998). In fact, removal for the last 4 C- terminal amino acids from CPE impairs the correct sorting and lipid raft formation of both CPE and POMC, suggesting that the sorting signaling may be as little as four amino acids (Zhang et al., 2003). To summarize, several mechanisms have been proposed to explain the differential sorting of cargo to DCVs, although no definitive conclusions have been reached. However, it is clear that proteins/peptides can be selectively sorted to different DCV populations creating a framework for stimulus-specific release.

1.4. Significance

As stated above, monoamines and neuropeptides interact to modulate most, if not all, key behaviors in both vertebrate and invertebrates. These neurotransmitters/neuromodulators modify signaling from individual neurons, coordinately modulate neuronal circuits and ultimately define behavioral state. For example, in mammals olfactory sensitivity is downregulated during satiety through pathways involving an array of signaling pathways that include both monoamines and several peptides, such as insulin. In contrast, fasting increases olfactory sensitivity and is accompanied by increases in an array different peptides, including NPY and AgRP

(Palouzier-Paulignan et al., 2012; Tong et al., 2011). Similarly, in C. elegans, extensive work defining the coordinate nutritional modulation of locomotion (roaming, dwelling and quiescence), feeding (pharyngeal pumping) and egg-laying has revealed that 5-

HTergic, TAergic, DAergic (food perception) and peptidergic signaling all play key roles

(Banerjee et al., 2017; Collins et al., 2016; McCloskey et al., 2017; Zang et al., 2017).

22 Monoamines and neuropeptides often modulate the same behavior, with the knockout and overexpression of either a modulator or its cognate receptor yielding similar phenotypes.

For example, the enhanced-aversive response to dilute 1-octanol on food is phenotypically identical in animals on 5-HT or in animals that overexpress the neuropeptides encoded by nlp-24 or npr-17, the proposed NLP-24 receptor (Cheong et al., 2015; Harris et al., 2010; Mills et al., 2016). However, relatively little is known about how the release of one neurotransmitter regulates the release/activity of another, but a few studies have provided insight into these interactions and suggest that monoaminergic/peptidergic signaling can interact at many levels. For example, in

Drosophila melanogaster, OA directly stimulates the presynaptic release of GFP-tagged atrial natriuretic factor (Shakiryanova et al., 2011). In C. elegans both 5-HT and OA are required for the release of neuropeptides encoded by flp-7 through a process that is both

5-HT- and OA-receptor dependent (Palamiuc et al., 2017). In mammals, 5-HT differentially regulates the expression of vasoactive intestinal peptide and - releasing peptide mRNA expression in the suprachiasmatic nucleus (Hayashi et al.,

2001). Similarly, gut-derived peptide-YY3-36 increases ARC POMC mRNA expression, while simultaneously decreasing NPY expression (Challis et al., 2003). However, much still remains to be learned about the specifics of these complex monoaminergic and peptidergic interactions.

In the present study, we have built upon previous work that demonstrates the role of neuropeptides in mediating the effects of monoamines on a sensory-mediated locomotory behavior, avoidance of the volatile repellent 1-octanol. In these studies, the

TA and OA inhibition of 1-octanol avoidance required the expression of the Gαq-coupled

23 TA receptor, TYRA-3 and the Gαq-coupled OA receptor, SER-6, respectively, in the two

ASI sensory neurons, in addition to an array of neuropeptide-encoding genes, i.e., nlp-1, -

14 and -18 are required for TA inhibition and nlp-6, -7 and -9 for OA inhibition (Hapiak et al., 2013; Mills et al., 2012b). Interestingly, all of these neuropeptides are functionally expressed in the ASIs, suggesting that a complex regulatory network is involved in specific monoamine-dependent ASI neuropeptide release. The ASIs also express several other neuropeptide-encoding genes involved in the modulation of a host of additional behaviors (Mills et al., 2016; Nathoo et al., 2001; Palamiuc et al., 2017; White &

Jorgensen, 2012). The aim of this study is to better understand the cross-talk between monoamines and neuropeptides, using fluorescently-tagged neuropeptides, and to determine how OA and TA differentially regulate the release of neuropeptides encoded by nlp-9, -14 and -18 from the ASIs, as measured by monoamine-stimulated deceases in peptide::GFP ASI fluorescence. Specifically, we propose that these monoamine receptors generate local changes in Ca2+ dynamics and compartmentalized Ca2+signals that drive the monoamine-dependent release of specific populations of neuropeptide-containing

DCVs.

Importantly, compartmentalized neuronal Ca2+ dynamics have been observed in both vertebrates and invertebrates. For example, in C. elegans, the two RIA interneurons receive input from multiple sensory networks and form synaptic connections with several motor neurons. Anatomically, the axons of the RIAs can be divided into a hairpin ‘loop’ located below the axon, containing nrD and nrV (the dorsal and ventral segments that reach the nerve ring, respectively). When presented with the olfactory stimulant, nrD and nrV exhibit differential Ca2+ changes that correlate with head movements (Hendricks et

24 al., 2012). Similarly, in D. melanogaster, the Kenyon Cells extend a single axon that reaches the mushroom body, passing through several mushroom body γ lobes and forming en passant boutons with mushroom body output neurons. DAergic neurons project axons into the individual γ lobes 2-5, forming discrete, anatomic compartments, and interestingly, stimulant-dependent compartmentalized Ca2+ dynamics were observed along the Kenyon Cell axon, suggesting functionally distinct units (Cohn et al., 2015).

Compartmentalized Ca2+ dynamics have also been observed in mammalian cells. The starburst amacrine cell is a retinal interneuron with a symmetric, dendritic morphology.

These cells contain a soma, but lack a clear distinction between dendrite and axon, having synaptic outputs and inputs along the process, in many ways similar to the multifunctional processes associated with many C. elegans neurons. Processes from amacrine cells are divided into proximal, intermediate and distal portions, relative to the soma, and these sections have differential changes in Ca2+ in response to light (Euler et al., 2002). These section-specific changes in dendritic Ca2+ are mediated by metabotropic glutamate receptor 2 that inhibits voltage-gated Ca2+ channels to restrict cross- compartmental signaling (Koren et al., 2017). In summary, monoaminergic/peptidergic crosstalk dramatically expands the signaling capacity of the nervous system, a property especially important for organisms such as C. elegans that contain only 302 neurons.

Understanding this cross-talk will significantly increase our understanding of this complex phenomenon and provide additional insight into neuronal communication.

25 Chapter 2

Materials and Methods

2.1. Materials

Restriction enzymes were purchased from either New England Biolabs (Beverly,

MA) and/or Promega (Madison, WI). Serotonin creatinine sulfate monohydrate (H7752-

25G), tyramine hydrochloride (T2879-5G) octopamine hydrochloride (O0250-5G) and 1- octanol (112615-1L) were all purchased from Sigma Life Sciences (St. Louis, MO). cDNA pools were generated from either mixed stage (for PCR fusion) or synchronized

(for qPCR studies) C. elegans using standard techniques (Portman, 2006) and

SuperScript™ III First-Strand Synthesis system from ThermoFisher Scientific (Waltham,

MA). Nematode growth media (NGM) was made using potassium phosphate monobasic

(P285-3), sodium chloride (S271-3), Ca2+ chloride dehydrate (C79-500), magnesium sulfate heptahydrate (BP213-1), tryptone (BP1421-2), agar (DF0812071) (purchased from ThermoFisher Scientific (Waltham, MA)) and cholesterol (C3045-5G) purchased from Sigma Life Sciences (St. Louis, MO). Green fluorescent protein (GFP) reporter constructs were a kind gift from Andy Fire (Carnegie Institute of Washington,

Washington, DC).

26 2.2. Nematode Strains

General culture and handling of strains were maintained as previously outlined

(Brenner, 1974). The following alleles were used: wild-type N2 (Bristol strain), nlp-18

(ok1557) and tyra-3 (ok325) which were obtained from the Caenorhabditis Genetics

Center (University of Minnesota, Minneapolis, MN), and nlp-9 (tm3572) and nlp-14

(tm1880), which was received from the National Bio-Resources Project (Tokyo Women’s

Medical University, Tokyo, Japan). Animals containing double mutations were generated using standard genetic techniques and confirmed by PCR. All mutants were backcrossed with wild-type N2 strain at least four times.

2.3. Construction of C. elegans Transgenes

The gpa-4::nlp-9::gfp, gpa-4::nlp-14::gfp and gpa-4::nlp-18::gfp transgenes contain 2.6 kb gpa-4 promoter, full-length cDNA of the respective neuropeptide- encoding gene, and gfp::unc-54 3’ UTR fused in-frame to the C-terminal peptide.

Constructs were co-injected with the selectable marker unc-122::rfp (to 50 ng). The srg-

47::nlp-9::mCherry transgene contains the 750 bp srg-47 promoter, full-length nlp-9 cDNA, mCherry::let-858 3’ UTR and was co-injected with the selectable marker rol-6

(su1106) (to 50 ng). The srg-47::mCherry::rab-3 transgene contains the 750 bp srg-47 promoter, mCherry, full-length rab-3 cDNA and unc-54 3’ UTR. srg-47::mCherry::rab-

3 was co-injected with gpa-4::nlp-9::gfp and rol-6 (su1106) or the selectable marker f25b3.3::gfp (to 50 ng). The gpa-4::nlp-14[leader sequence]::nlp-9::gfp transgene contains 2.6 kb gpa-4 promoter, nlp-14 leader sequence (the first 66 bp), full-length nlp-9 cDNA (minus the first 69 bp) and gfp::unc-54 3’ UTR and was co-injected with rol-6

(su1106) (to 50 ng). The gpa-4::nlp-9[leader sequence]::nlp-14::gfp transgene contains

27 2.6 kb gpa-4 promoter, nlp-9 leader sequence (the first 69 bp), full-length nlp-14 cDNA

(minus the first 66 bp) and gfp::unc-54 3’ UTR and was co-injected with rol-6 (su1106)

(to 50 ng). The truncated gpa-4::nlp-14(1-95)::gfp construct contains 2.6 kb gpa-4 promoter, the first 285bp nlp-14 cDNA and gfp::unc-54 3’ UTR and was co-injected with unc-122::rfp (to 50 ng). The truncated gpa-4::nlp-14(1-170)::gfp construct contains 2.6 kb gpa-4 promoter, the first 510bp nlp-14 cDNA and gfp::unc-54 3’ UTR and was co- injected with unc-122::rfp (to 50 ng). The transcriptional Pgpa-4::gfp contains 2.6 kb gpa-4 promoter and gfp::unc-54 3’ UTR and was co-injected with the selectable marker unc-122::rfp (to 50 ng). All transcriptional or translational constructs were generated by

PCR fusion as described previously (Hobert, 2002), pooled from at least 3 separate reactions and confirmed by restriction digest.

All ASI-specific RNAi transgenes were generated by PCR fusion using either the gpa-4 or srg-47 promoter, as described previously (Esposito et al., 2007; Hapiak et al.,

2013) and co-injected with f25b3.3::gfp (to 100 ng). Briefly, the exon region of the gene of interest and the neuron selective promoter is amplified by PCR. The neuron-selective promoter is amplified with an overlap for either the 5’ or 3’ end of the exon region, generating two different products. The neuron-selective promoters are fused to their respective exons of interest to generate both sense and antisense products. These products are then mixed and injected into worms. All carrier DNA was injected into the gonads of

C. elegans wild-type or mutant backgrounds by standard techniques (Mello & Fire,

1995).

2.4. Behavioral Assays

28 Octanol avoidance assays were performed as described previously (Chao et al.,

2004; Hapiak et al., 2013). Briefly, L4 animals were picked 24 hrs prior to assay and kept at 16°C on standard NGM plates seeded with Escherichia coli strain OP50 to generate age-matched, well-fed young adults. NGM plates were prepared daily by adding 5-HT

(final concentration, 4 mM) and/or TA/OA (final concentration, 4 mM) to liquid media before pouring. A hair (Loew-Cornell 9000 Kolinsky 8 paintbrush) was fused to a P200 pipette tip and dipped in either dilute (30% v/v in 100% ethanol) or 100% 1-octanol and placed in front of an animal exhibiting forward locomotion. The time taken to initiate backward locomotion was scored for each animal and averaged. Assays were terminated after 20 sec as wild-type animals are known to spontaneously reverse ~20 sec (Chao et al., 2004). In all assays, animals were transferred to intermediate, non-seeded NGM plates and left for 30 secs, then transferred to assay plates and left for 30 min before being scored for 1-octanol avoidance. All strains were blinded and assays repeated in triplicate. Data are presented as a mean  SE and analyzed using two-tailed Student’s t test.

2.5. Confocal Microscopy

To localize the various full length, chimeric or truncated neuropeptides used in this study, each transgene was injected into the respective null animal, and well fed young adults were scanned using standard GFP filters. To co-localize NLP-9 and RAB-3, two different ASI promoters were used; gpa-4::nlp-9::gfp and srg-47::mCherry::rab-3 and co-injected into nlp-9 null animals. A similar approach was used to co-localize NLP-9 and NLP-14 using srg-47::nlp-9::mCherry and gpa-4::nlp-14::gfp, and then imaged for

GFP and mCHERRY using sequential scanning. All animals were immobilized in 3 μl

29 sodium azide (20 mM) and mounted on 3% agarose pads. All microscopy was performed on an Olympus IX81 inverted confocal microscope.

2.6. Neuropeptide Fluorescence Assay

For all neuropeptide fluorescence assays, transgenic L4 animals expressing either;

Pgpa-4::gfp, gpa-4::nlp-9::gfp, gpa-4::nlp-14::gfp, gpa-4::nlp-18::gfp, gpa-4::nlp-

14[leader sequence]::nlp-9::gfp, gpa-4::nlp-9[leader sequence]::nlp-14::gfp, gpa-

4::nlp-14(1-95)::gfp, gpa-4::nlp-14(1-170)::gfp or gpa-4::nlp-14(C-terminus)::gfp were picked

24 hrs prior to assaying and kept at 16°C. NGM plates containing either TA or OA (final concentration, 10 mM) were prepared daily. Animals were incubated on NGM plates seeded with E. coli strain OP50 or transferred to assay plates for 1 hr, prior to imaging.

Images (0.4µm Z-stacks) were captured using Olympus Fluoview FV1000 software and fluorescence was quantified using Volocity Software Version 4.0.0 Build 314

(Improvision Ltd). To quantify neuropeptide fluorescence, both the right and left ASIs were tightly cropped and divided into the soma and axon. All images were then blinded and fluorescence was filtered by intensity. The first 10 blinded images were used to generate an average threshold separating signal from noise, which was applied to all images within the experiment. Object intensities were summed for each image and objects < 3 voxels were regarded as non-specific background and excluded. The summed, arbitrary fluorescence of each treatment was normalized to the summed arbitrary fluorescence of untreated (control) animals. All assays were repeated at least two times, and statistical analysis was based on individual responses to treatment with n values given where appropriate. Data are presented as a mean  SE and analyzed using an one- way ANOVA test, followed up post-hoc with either Dunnett T3 or Dunnett t (2-sided)

30 tests. All imaging was performed on an Olympus IX81 inverted confocal microscope and animals were immobilized in 3 μl sodium azide (20 mM) and mounted on 3% agarose pads.

2.7. RNA Isolation and cDNA Synthesis

RNA was isolated as outlined previously (Portman, 2006) with minor revisions.

Briefly, wild-type animals were synchronized using alkaline hypochlorite and arrested at

L1 by starvation. Following arrest, L1s were moved to fresh NGM plates seeded with E. coli strain OP50 and maintained at 16°C until young adulthood (~2 days). Young adults were then washed in M9 and transferred to either fresh NGM plates seeded with E. coli strain OP50 (untreated population), fresh non-seeded NGM plates (control population), fresh OA (final concentration, 10 mM) or fresh TA (final concentration, 10 mM) NGM plates for 1 hr. Following incubation, animals were then washed in M9 and suspended in

Trizol. Animals were frozen overnight at -80°C and lysed via freeze-thaw cracking by cycling between a 37°C water bath and liquid nitrogen, with intermittent vortexing. RNA was isolated using standard Trizol techniques and assessed for quality/quantity on an agarose gel and using the Nanodrop ND-1000 Spectrophotometer. cDNA synthesis was performed using the SuperScript III First-Strand Synthesis system (ThermoFisher

Scientific) as per the manufactures instructions.

2.8 Quantitative PCR

cDNA pools from either untreated, control, OA (final concentration, 10 mM) or

TA (final concentration, 10 mM) populations were diluted to 50 ng/l and 2 l added for reaction setup. qPCR studies were conducted using SsoFast EvaGreen Supermix

(Bio-Rad) and measurements obtained using the Bio-Rad CFX96 Real-Time PCR

31 System. qPCR reactions were setup as per manufactures instructions. At least three separate pools of cDNA were analyzed, each from a different age-matched population.

Cycling conditions were used as per the manufactures instructions. All results were normalized to the reference gene act-1 and relative expression determined using the

Livak ∆Ct method (Livak & Schmittgen, 2001). Data are presented as a mean  SE and analyzed using an one-way ANOVA test, followed up post-hoc with either Dunnett T3 or

Dunnett t (2-sided) tests.

2.9 Bioinformatic Analysis

Predicted signal sequence cleavage sites were obtained using SignalP V4.1

(http://www.cbs.dtu.dk/services/SignalP/) and confirmed using Signal-BLAST

(sigpep.services.came.sbg.ac.at/signalblast.html). Discrepancies between algorithms were resolved by selecting the most likely cleavage site based on the Edman degradation/MS data detailing discovered neuropeptides in C. elegans (Husson et al., 2010). Neuropeptide preproproteins sequences were taken from (Husson et al., 2010) and analyzed using

Protean software (DNAStar suite, version 14.1.0).

2.10 Statistics

All data was initially assessed with either a Student’s t-test or a One-way ANOVA and when appropriate, post-hoc analysis was carried out using a Dunnett or Dunnett T3 test. All statistics were performed using SPSS software v24 (IBM).

32 Chapter 3

Results

3.1. Distinct Subsets of ASI Neuropeptides Are Essential for the TA- and OA-

Dependent Inhibition of Aversive Responses to 1-octanol

The two ASI sensory neurons express over 20 neuropeptide genes that encode over

60 predicted neuropeptides (Husson et al., 2010; Li & Kim, 2008; Nathoo et al., 2001).

Our previous work using primarily null mutants and ASI-specific rescue demonstrated that the serotonergic, tyraminergic and octopaminergic modulation of ASH-dependent nociception requires distinct subsets of these neuropeptide-encoding genes, and suggested that monoamines might differentially modulate neuropeptide release, as outlined in Fig

3.1.1 (Hapiak et al., 2013; Harris et al., 2010; Mills et al., 2016; Mills et al., 2012b). For example, the Gαq-coupled TA receptor, TYRA-3, and ASI neuropeptides encoded by nlp-

1, -14 and -18 are required for the TA inhibition of 5-HT stimulation (Hapiak et al.,

2013). In agreement, RNAi knockdown of nlp-14 and -18 using a different ASI-specific promoter abolished the TA inhibition of 5-HT-stimulated aversive responses (Student’s t- test: srg-47::nlp-14 p = 0.0001; srg-47::nlp-18 p = 0.0001; Fig 3.1.2A). It was important

33 to use a second ASI-specific promoter to confirm previous results in order to reduce the potential for any artifacts associated with RNAi before we proceeded further. For example, even though the two promoters exhibit ASI specificity when coupled to sequence coding for GFP, it is possible that the additional sequence coding for the RNAi could alter this specificity. Similarly, the α1-adrenergic-like Gαq-coupled OA receptor,

SER-6, and ASI neuropeptides encoded by nlp-6, -7 and -9 are required for the OA inhibition of aversive responses to higher 1-octanol concentrations. Again, in agreement,

RNAi knockdown of nlp-9 abolished the OA inhibition of the aversive response

(Student’s t-test: srg-47::nlp-9 p = 0.0001; Fig 3.1.2B; (Mills et al., 2012b)). Together, these data suggest that monoamine-dependent Gαq-coupled signaling in the ASIs differentially modulates ASI neuropeptide signaling and subsequently aversive behavior.

Therefore, the present study was designed to build upon our previous findings to understand how TA and OA might alter neuropeptide signaling from the ASIs, focusing specifically on ASI neuropeptides encoded nlp-9, -14 and -18.

34

Fig 3.1.1. The two ASI Sensory Neurons Integrate Both Monoaminergic and Peptidergic Signaling to Modulate Nociceptive Behavior

The two ASI sensory neurons express Gαq-coupled TA, OA and 5-HT receptors that differentially modulate neuropeptide release and, in turn, differentially modulate aversive responses mediated primarily by the two ASH sensory neurons. For example: 1) the ASI OA receptor, SER-6, and neuropeptides encoded by nlp-6, -7 and -9 are essential for the OA inhibition of aversive responses to 100% 1-octanol (Mills et al., 2012b), 2) the ASI TA receptor, TYRA-3, and neuropeptides encoded by nlp-1, -14 and -18 are essential to inhibit the 5-HT stimulation of aversive responses to 30% 1-octanol (Hapiak et al., 2013), and 3) the ASI 5-HT receptor, SER-1, appears to stimulate the release of neuropeptides encoded by nlp-24 that activate the Gαo-coupled ASI opiate receptor, NPR-17, to interfere with TYRA-3 signaling in response to 30% 1-octanol (Mills et al., 2016).

35

Fig 3.1.2. The TA- and OA-dependent Inhibition of Aversive Responses Requires Different Subsets of ASI Neuropeptides A. The initiation of aversive responses to 30% (dilute) 1-octanol were assayed in the presence and absence of 5-HT and/or 5-HT + TA, as described previously (Hapiak et al., 2013; Hapiak et al., 2009). B. The initiation of aversive responses to 100% 1-octanol were assayed in the presence and absence of OA, as described previously (Mills et al., 2012b; Wragg et al., 2007). The srg-47 promoter was used to drive specific ASI RNAi expression. Monoamine concentrations were 4 mM. * Denotes significantly different from wild-type (N2) animals under the same conditions (p = 0.0001). Data are presented as a mean ±SE (n) and were analyzed by two-tailed Student’s t test (2A n = 29 – 34. 2B n = 31 – 36).

36 3.2 TA- and OA-dependent Neuropeptides Appear to Co-localize at Individual ASI

Synapses

The ASIs contain 7-9 synapses in the distal portion of the axonal process based on serial electron microscopy reconstructions and confirmed by 7-9 fluorescent puncta found after ASI expression of synaptobrevin::GFP, a synaptic marker (Crump et al., 2001;

White et al., 1986). In addition, we previously demonstrated NLP-14::GFP fluorescence was observed in 7-9 discrete puncta along the distal portion of ASI axons that co- localized with the synaptic marker, RAB-3 (Hapiak et al., 2013). However, in this previous study GFP was fused to the C-terminus of the NLP-14 proprotein and not directly to an individual neuropeptide. To determine if the placement of GFP affected the correct packaging/localization/release, we quantified the number of axonal fluorescent puncta and mean intensity for an NLP-14::GFP with GFP fused directly to the last predicted peptide or at the C-terminus of the proprotein (Fig 3.2.1). We found no differences between the placement of fluorescent puncta along the ASI axonal process or their mean intensities before and after treatment with TA, suggesting that the placement of GFP did not affect packaging, trafficking or release (Fig 3.2.1A-B). All subsequent experiments were conducted with GFP fused to the last peptide to increase our confidence that the fluorescent signal emitted by the fluorophore represented the dynamics/action of the neuropeptide of interest.

37

Fig 3.2.1. Position of C-terminal GFP does not Affect DCV Packaging or Localization GFP was fused either in frame to the last predicted C-terminal peptide-encoding sequence or last amino acid. A. The number of fluorescent DCVs within the ASI axon were counted for both GFP locations. B. The fluorescence of each individual puncta along the ASI axon were quantified for both GFP locations. C. Straightened axons of transgenic animals expressing GFP fused in frame at the last C-terminal predicted peptide (top) or to the last amino acid (bottom). Data are presented as a mean ±SE (n) (3A n = 31 – 35. 3B n = 29 – 33).

38 In the present study, we have confirmed previous published observations with

NLP-14::GFP and demonstrated that fluorescence from NLP-18:: and NLP-9::GFP was also observed in 7-9 fluorescent puncta in the distal portions of ASI axons (Fig 3.2.2B-D circles). Importantly, all three GFP fusion constructs rescued the appropriate neuropeptide null phenotypes. As observed previously for NLP-14::GFP, NLP-9::GFP also co-localized with a synaptic marker, mCHERRY::RAB-3, within these predicted synaptic regions at least at the level of fluorescence microscopy (Fig 3.2.2E circles;

(Hapiak et al., 2013)). However, since neuropeptide-transporting DCVs often localize perisynaptically, the potential for a non-overlapping perisynaptic localization of the individual peptide-containing vesicles should not be ruled out (Hammarlund et al., 2008;

Salio et al., 2006). NLP-14::GFP and NLP-9::mCHERRY also co-localized at fluorescent puncta within predicted synaptic/perisynaptic regions of the ASIs, as anticipated, based on their co-localization with RAB-3 (Fig 3.2.2F circles; (Hapiak et al., 2013)). Together, these data suggest that both TA- and OA-dependent neuropeptides co-localize to each of

7-9 synaptic/perisynaptic puncta within the distal portion of right and left ASI axons.

Interestingly, although all of the animals expressing ASI::peptide::GFP exhibited 7-9 fluorescent synaptic/perisynaptic puncta, the spacing of these puncta in either the right or left ASIs often varied significantly from animal to animal, making the correlation of individual fluorescent puncta with specific downstream synaptic partners problematic.

Whether these differences result from preparation artifacts is unclear, but it is worth noting that most of what we know about synaptic connectivity in C. elegans is derived from very few animals and it would not be surprising if the physical relationship among

ASI synapses varied from animal to animal.

39 Additional peptide::GFP and mCHERRY::RAB-3 fluorescence was also observed in the soma and co-localized in the axon proximal to the cell body, where no synapses have been described by electron microscopy (Fig 3.2.2B-F, arrowheads). Whether these axonal fluorescent puncta represent trafficking vesicles, additional synapses not observed by electron microscopy or sites of extra-synaptic release remains to be determined, but the localization of at least some of these puncta appeared to be consistent in ASIs expressing different peptide::GFPs. For example, fluorescence from NLP-9::, NLP-14:: and NLP-18::GFP was consistently observed in 3-4 distinct puncta in the proximal portion of the axon near the soma (Fig 3.2.2B-D, arrowheads). Similar ASI fluorescent puncta have also been observed by others after ASI expression of synaptobrevin::GFP, another synaptic marker (Crump et al., 2001). Interestingly, the ratio of GFP fluorescence in synaptic and predicted extra-synaptic puncta differed significantly in ASIs from animals expressing either the TA-dependent NLP-14::GFP or the OA-dependent NLP-

9::mCHERRY, suggesting that these TA- and OA-dependent neuropeptides may be trafficked in different vesicles or preferentially localized to different sites (Student’s t- test: nlp-9 p = 0.0001; Fig 3.2.2G).

40

Fig 3.2.2. TA- and OA-dependent Neuropeptides Co-localize at ASI Synapses A. Schematic representation of an ASI neuron highlighting the predicted synaptic/perisynaptic and extra-synaptic sites. B-D. Translational neuropeptide fusions (ASI::nlp-14::gfp, ASI::nlp-18::gfp or ASI::nlp-9::gfp) expressed in their respective null backgrounds in the left ASI sensory neurons driven by the ASI-specific promoter gpa-4. Anterior is to the left and dorsal is up. E. Straightened axons of nlp-9 null animals co- expressing gpa-4::nlp-9::gfp and srg-47::mCherry::rab-3 transgenes. Circles indicate the sites of predicted ASI synapses based on electron microscopy. Arrowheads denote potential additional extra-synaptic sites of release. F. Straightened axons of an animal co- expressing gpa-4::nlp-14::gfp and srg-47::nlp-9::mCherry transgenes. G. Extra- synaptic/synaptic ratios of ASI::nlp-14::gfp or ASI::nlp-9::gfp expressed in their respective null backgrounds. * Denotes significantly different to nlp-14 (p = 0.0001). Data are presented as a mean ±SE (n) and were analyzed by two-tailed Student’s t test (n = 28 – 55).

41 3.3 TA and OA Stimulate the Release of Distinct Subsets of ASI Neuropeptides

No methods have been described to measure acute neuropeptide release in C. elegans, but two indirect methods have been used previously to approximate release; treatment-dependent decreases in neuronal-specific peptide::GFP fluorescence or increases in fluorophore uptake by coelomocytes, scavenger cells located in the pseudocoelmic fluid (Bhattacharya et al., 2014; Hu et al., 2011; Sieburth et al., 2007;

Speese et al., 2007). Unfortunately, the coelomocyte uptake assay is not effective for all neuropeptides because uptake is dependent on the amount of neuropeptide released and the location of the neuropeptide-releasing cells (Hu et al., 2011). Indeed, the coelomocyte uptake assay was not useful for measuring the release of ASI neuropeptides encoded by nlp-9, -14 or -18 in the present study, as coelomocyte uptake was low, inconsistent and different pairs of coelomocytes sometimes yielded different results. Therefore, we focused on monoamine-dependent decreases in fluorescence from GFP-tagged neuropeptides to approximate peptide release, using a monoamine-insensitive ASI- specific promoter, gpa-4. Worms were transferred to a monoamine-supplemented plate for 1 hr prior to imaging for all assays, and as noted in Fig 3.3.1A, TA and OA had no effect on ASI gpa-4::GFP expression. In addition, TA and OA also had no significant effect on gpa-4, nlp-14, -18 and -9 expression as assayed by qPCR, not surprisingly given the short time course of the experiment (Fig 3.3.1B).

Our behavioral data demonstrated that neuropeptides encoded by both nlp-14 and nlp-18 are required for the TA-inhibition of aversive responses (Fig 3.1.2A; (Hapiak et al., 2013)). As predicted, TA significantly decreased total NLP-14::GFP fluorescence of nlp-14 null animals expressing ASI::NLP-14::GFP (One-way ANOVA: Total p = 0.020;

42 Fig 3.3.1C). TA-dependent decreases in total ASI::NLP-14::GFP fluorescence required the TA receptor TYRA-3, as TA had no effect in tyra-3;nlp-14 null animals lacking the key TA receptor essential for the TA inhibition of aversive responses (Fig 3.3.1C;

(Hapiak et al., 2013)). In contrast, OA had no effect on ASI::NLP-14::GFP fluorescence

(Fig 3.3.1C). Together, these data demonstrate that decreases in ASI::NLP-14::GFP fluorescence were both TA-specific and TYRA-3-dependent. Importantly, TA, but not

OA, also specifically decreased axonal GFP fluorescence in predicted synaptic/perisynaptic, but not extra-synaptic regions (One-way ANOVA: Axonal p =

0.006; Synaptic p = 0.006; Fig 3.3.1C). These TA-dependent decreases in ASI::NLP-

14::GFP axonal fluorescence suggest either that TA reduces NLP-14::GFP expression and/or stimulates NLP-14::GFP release, but since TA had no effect on the ASI::GFP fluorescence driven by the gpa-4 promoter, we postulate that these decreases in peptide::GFP fluorescence were due to TA-dependent increases in synaptic/perisynaptic neuropeptide release. Indeed, neuropeptide release has been previously observed in vitro using C. elegans neurons. For example, Smith et al., observed stimulus-dependent decreases in FLP-17::VENUS fluorescence from cultured BAG neurons (Smith et al.,

2013). Similarly, Zhou et al., employed total internal reflection fluorescence microscopy combined with capacitance measurements to observe DCV exocytosis in cultured C. elegans neurons (Zhou et al., 2007).

TA decreased total and somatic ASI::NLP-18::GFP fluorescence, but had no effect on fluorescence in ASI axonal regions (One-way ANOVA: Total p = 0.045; Somatic p =

0.035; Fig 3.3.1D). In contrast, OA had no effect on ASI::NLP-18::GFP fluorescence

(Fig 3.3.1D). These observations suggest that the sites of TA-dependent ASI::NLP-

43 18::GFP release may be somatic and different from those of NLP-14::GFP. Indeed, stimulus-dependent somatic DCV exocytosis has been observed in other systems

(Fuenzalida et al., 2011; Xia et al., 2009).

44

Fig 3.3.1. TA Modulates ASI Neuropeptide Release A. Wild-type animals expressing Pgpa-4::gfp were incubated with either TA or OA and analyzed for GFP fluorescence. B. Quantitative PCR of several ASI neuropeptide- encoding genes and gpa-4 in wild-type animals. C-D. Animals expressing ASI::nlp- 14::gfp or ASI::nlp-18::gfp transgenes in their respective null backgrounds were incubated with either TA or OA and analyzed for GFP fluorescence. C. ASI::nlp-14::gfp in nlp-14 or nlp-14;tyra-3 null animals. D. ASI::nlp-18::gfp in nlp-18 null animals. The gpa-4 promoter was used to drive ASI expression. All monoamine concentrations were 10 mM. * Denotes significantly different from control (p ≤ 0.05). Data are presented as a mean ± SE (n) and were analyzed by One-way ANOVA (5A n = 31 – 33. 5B n = 9. 5C n = 22 – 28 One-way ANOVA: Total = p 0.020, Dunnett t (2-sided) = p 0.037, Axon = p 0.006, Dunnett T3 = p 0.011, Synaptic = p 0.006, Dunnett T3 = p 0.012. 5D n = 17 – 45 One-way ANOVA: Total = p 0.045, Dunnett t (2-sided) = p 0.025, Soma = p 0.035, Dunnett t (2-sided) = p 0.019). 45 Although OA had no effect on ASI::NLP-14::GFP or ASI::NLP-18::GFP fluorescence, OA significantly reduced total and axonal ASI::NLP-9::GFP fluorescence

(One-way ANOVA: Total p = 0.0001; Axon p = 0.0001; Fig 3.3.2A). Interestingly, this decrease in NLP-9::GFP axonal fluorescence was observed only in synaptic/perisynaptic puncta in the right, but not left ASI, suggesting that ASI signaling was asymmetric (One- way ANOVA: Synaptic p = 0.0001; ASIR p = 0.0001; Fig 3.3.2B). As predicted, TA had no effect on total, somatic or axonal NLP-9::GFP fluorescence. Together, these studies demonstrate TA and OA differentially modulate the release of distinct subsets of neuropeptides from different sites with the ASIs.

46

Fig 3.3.2. OA Modulates ASI Neuropeptide Release Animals expressing ASI::nlp-9::gfp transgenes in their respective null backgrounds were incubated with either TA or OA and analyzed for GFP fluorescence. A. ASI::nlp-9::gfp in nlp-9 null animals. B. Axonal analysis of ASI::nlp-9::gfp fluorescence in extra- synaptic/synaptic sites and right/left ASIs. The gpa-4 promoter was used to drive ASI expression. All monoamine concentrations were 10 mM. * Denotes significantly different from control (p ≤ 0.05). Data are presented as a mean ± SE (n) and were analyzed by One-way ANOVA (6A n = 38 – 55 One-way ANOVA: Total p = 0.000, Dunnett T3 = p 0.028, Axon p = 0.000, Dunnett t (2-sided) = 0.004. 6B n = 20 – 55 One-way ANOVA: Synaptic p = 0.000, Dunnett t (2-sided) = p 0.003, ASIR p = 0.000, Dunnett T3 p = 0.000).

47 3.4 ASI Neuropeptide Proproteins Required for Either TA- or OA-dependent

Inhibition Contain Different Leader Sequences

The N-terminal sequence of neuropeptide preproproteins can be critical for efficient packaging and sorting into DCVs (Blanco et al., 2013; Brakch et al., 2002; Zhang et al.,

2005). Since we hypothesized that the ASI neuropeptides released by TA and OA might be localized to different populations of DCVs, we analyzed each of the predicted neuropeptide proproteins for potential sorting signals/motifs using the protein structure and prediction software, Protean (DNA Star software). Interestingly, each of the proproteins contained a mixture of predicted sorting motifs, such as α-helices and dibasic cleavage sites (Fig 3.4.1). Importantly, the identity of the N-terminal mature peptides encoded by nlp-1, -14, -18 (involved in TA-dependent behavior), and nlp-6, -7 and -9

(involved in OA-dependent behavior) have each been confirmed by mass spectrometry

(Husson et al., 2010; Li & Kim, 2008). Interestingly, the NLP-9, -14 and -18 preproproteins all contained a predicted N-terminal α-helix, but their sizes differed. NLP-

9 had the largest relative α-helix that spanned the first 16 (12.4%) amino acids, in contrast to the smaller α-helices observed for NLP-14 (13 residues, 5.7%) and NLP-18 (4 residues, 4%) (Fig 3.4.1). In addition, we analyzed the charge with the N-termini as it is important in sorting pro-tachykinin into DCVs, and neutralization of charged elements dramatically reduced sorting efficiency (Ma et al., 2008). We found that NLP-9 contained a positive charge within the α-helix, in contrast to NLP-14 and NLP-18 that were uncharged (Fig 3.4.1). In contrast, all three preproproteins contained either a positive or negative charge within the neuropeptide-encoding regions, with no obvious association within or between groups. Lastly, we scanned for dibasic residues. Each of

48 the three TA-dependent preproproteins (encoded by nlp-1, -14 and -18) possessed dibasic cleavage sites upstream of the first N-terminal peptide, and after removal of the predicted , each of the three TA-dependent proproteins is predicted to contain a short, charged, N-terminal prosequence upstream of the first dibasic cleavage site (Fig 3.4.2A).

In contrast, each of the three OA-dependent preproproteins (encoded by nlp-6, -7 and -9) lack dibasic cleavage sites upstream of the first N-terminal peptide (Fig 3.4.2A).

Presumably, the signal peptidase truncates these three preproproteins immediately upstream of the first mature neuropeptide, leaving no charged prosequence, in contrast to proproteins encoded by nlp-1, -14 and -18. These results suggest that these different preprosequences might play a role in TA or OA dependent release.

To determine if these different preprosequences were involved in the differential action of TA and OA signaling, we constructed chimeric neuropeptide preproproteins by placing the predicted NLP-9 preprosequence upstream of the region coding for NLP-14 neuropeptides (ASI::NLP-9LS::NLP-14::GFP) and the predicted NLP-14 preprosequence upstream of the region coding for the NLP-9 neuropeptides (ASI::NLP-14LS::NLP-

9::GFP) (Fig 3.4.2B). Both chimeric neuropeptides localized to the 7-9 synaptic/perisynaptic axonal puncta observed for the native GFP-tagged neuropeptides, but TA or OA did not selectively decrease axonal fluorescence from either chimera (One- way ANOVA: Native NLP-14 p = 0.012 Fig 3.4.2C; Native NLP-9 p = 0.003 Fig

3.4.2D). Together, these data suggest that the differential effects of TA and OA on neuropeptide release do not lie exclusively within the different preprosequences. If all the information required for monoamine-specific neuropeptide release was contained within the preprosequence, we predict that the chimeric peptides would be differentially released

49 in response to the appropriate monoamine-stimulus i.e. ASI::NLP-9LS::NLP-14::GFP should be sensitive to OA, not TA, and vice versa. However, our data did not reflect this, suggesting additional sequence within the peptide-encoding region are required.

Alternatively, the chimeric peptides may not be properly processed and/or have altered folding, with either outcome inhibiting peptide release. Further investigation is required to understand how the N-terminal sequence functions in monoamine-specific neuropeptide release, as these properties may occur at the level of DCV packaging or downstream at the site of DCV localization/exocytosis

50

Fig 3.4.1. Both the TA- and OA-dependent Preproprotein Sequences Contain Several Predicted Sorting Sequences Involved in DCV Packaging Each preproprotein sequence was analyzed for α-helices, positive, negative and average charge using Protean (DNA Star software). Sequenced outline in green represent confirmed neuropeptides by either Edman degradation, Matrix Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry, or Quadrupole-Time of Flight Mass Spectrometry, and blue outline represent predicted neuropeptides (Husson et al., 2010). Red bars represent predicted α-helical regions and numbers along the x-axis represent amino acid location, from N- to C-terminus. 51

Fig 3.4.2. Effect of TA and OA on ASI GFP Fluorescence in Animals Expressing NLP-14/NLP-9 Chimeric Neuropeptides A. N-terminal amino acid sequences of the TA- and OA-specific neuropeptide preproproteins adapted from Husson et al., 2010. B. Schematic representation depicting chimeric NLP-14/NLP-9 preproproteins after swapping N-terminal preprosequences. C- D. Chimeric ASI::neuropeptide::gfp transgenes expressed in their respective null backgrounds. Anterior is to the left and dorsal is up. Circles indicate the sites of predicted ASI synapses based on electron microscopy. Arrowheads denote potential additional extra-synaptic sites of release. Chimeric fusions were incubated with either TA or OA and analyzed for GFP fluorescence. C. Chimeric ASI::nlp-9LS::nlp-14::gfp. D. Chimeric ASI::nlp-14LS::nlp-9::gfp. The gpa-4 promoter was used to drive ASI expression. All monoamine concentrations were 10 mM. * Denotes significantly different from control (p ≤ 0.05). Data are presented as a mean ± SE (n) and were analyzed by One-way ANOVA (8C n = 22 – 36, One-way ANOVA: NLP-14 p 0.012, Dunnett T3 = p 0.025. 8D n = 31 – 55, One-way ANOVA: NLP-9 p 0.003, Dunnett t (2-sided) = p 0.014).

52 To further assess the importance of sequence downstream of the preprosequences, we generated truncated ASI::NLP-14::GFP proteins. The nlp-14 gene encodes seven predicted and five confirmed neuropeptides (Fig 3.4.3A; (Husson et al., 2010)). The first truncation, NLP-14(1-95)::GFP, contains the complete N-terminal sequence, three predicted neuropeptides and two confirmed neuropeptides. The longer truncation, NLP-

14(1-170)::GFP, contains the complete N-terminal sequence but five predicted and five confirmed neuropeptides. Both truncations mimicked the full-length ASI::NLP-14::GFP and exhibited decreased axonal fluorescence following incubation in TA, but not OA

(One-way ANOVA: Native NLP-14: p = 0.012; NLP-14(1-95) p = 0.022; NLP-14(1-170) p =

0.013; Fig 3.4.3D). These data suggest that specific amino acid arrangements between the leader sequence and peptide-encoding regions are important for correct packaging, processing and/or monoamine sensitivity for release, i.e. in the case of NLP-14, the first

95 amino acids confer TA-dependent release and, in contrast, disruption of this sequence, as shown by the chimeric proproteins, abolishes neuropeptide release. The specific amino acids required for monoamine-sensitive release will no doubt vary between proproteins and will require further studies to confirm the key residues involved.

53

Fig 3.4.3. Truncation of the ASI::NLP-14 Preproprotein does not Alter TA and OA Sensitivity A. Amino acid sequence for the full length NLP-14 preproprotein. B-C. Amino acid sequence for the different truncated NLP-14 constructs. Bold denotes a dibasic cleavage site, blue denotes putative neuropeptides, red denotes neuropeptides confirmed by either Edman degradation, Matrix Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry, or Quadrupole-Time of Flight Mass Spectrometry and GFP sequence is underlined green. Amino acid sequences adapted from Husson et al., 2010. D. Animals expressing the truncated transgene ASI::nlp-14(1-95)::gfp or ASI::nlp-14(1-170)::gfp in an nlp-14 null background, were incubated with either TA or OA and analyzed for GFP fluorescence. The gpa-4 promoter was used to drive ASI expression. * Denotes significantly different from control (p ≤ 0.05). Data are presented as a mean ± SE (n) and were analyzed by One-way ANOVA (9D n = 22 – 42, One-way ANOVA: Native NLP- 14: p = 0.012, Dunnett T3 = p 0.025, NLP-14(1-95) p = 0.022, Dunnett T3 = p 0.012).

54 Chapter 4

Discussion

The present study highlights the crosstalk between monoaminergic and peptidergic signaling in the modulation of a key C. elegans behavior, and has begun to dissect the mechanism of differential monoamine-dependent neuropeptide release from individual pairs of sensory neurons. Our previous work has demonstrated that the monoaminergic modulation of the aversive responses to 1-octanol is dependent on differential peptidergic signaling from the two ASI sensory neurons (Hapiak et al., 2013;

Mills et al., 2016; Mills et al., 2012b). The present study has expanded these observations to suggest the individual monoamines selectively stimulate the release of distinct subsets of ASI neuropeptides from unique sites within the ASIs. Our data suggest TA-dependent neuropeptides encoded by nlp-14 and nlp-18 are released at synaptic/perisynaptic sites, or the ASI soma, respectively, and OA-dependent neuropeptides encoded by nlp-9 are released asymmetrically from synaptic/perisynaptic sites in the right axon (Fig 4.1).

55

Fig 4.1. Model of Monoamine-specific ASI Neuropeptide Release Neuropeptides encoded by nlp-9, -14 and -18 are expressed in the soma, predicted synaptic/perisynaptic and extra-synaptic sites in both ASI neurons. TA selectively stimulates the release of NLP-18::GFP from the ASI soma and NLP-14::GFP from synaptic/presynaptic regions of both the right and left ASI axons. In contrast, OA selectively stimulates the asymmetric release of NLP-9::GFP from only synaptic/perisynaptic regions in the right ASI axon.

56 4.1 Monoaminergic/peptidergic Crosstalk is Extensive and Modulates Most Key C. elegans Behaviors

Monoamines and neuropeptides extensively modulate ASH and ASI signaling throughout the ASH-mediated aversive circuit in response to 1-octanol. The ASHs express antagonistic pairs of 5-HT, DA, and OA receptors that differentially modulate

ASH signaling directly, and also stimulate the release of neuropeptides that differentially modulate signaling at multiple levels within the locomotory circuit. For example, the 5-

HT stimulation of aversive responses requires at least three distinct 5-HT receptors expressed at different levels within the ASH-mediated aversive circuit, including SER-5 on the ASHs, and neuropeptides encoded by nlp-3 in the ASHs (Harris et al., 2010;

Harris et al., 2009). Ultimately, 5-HT stimulates the initiation of an aversive response through a complex pathway resulting in decreased ASH Ca2+ and increased ASH depolarization (Harris et al., 2009; Zahratka et al., 2015). This 5-HT stimulation is inhibited by either OA or TA, and the OA inhibition is dependent on the levels of endogenous OA (Wragg et al., 2007). For example, low levels of OA inhibit the initiation of aversive responses to 1-octanol and require the ASH-expressed Gαo-coupled OA receptor, OCTR-1, while higher levels of OA have no effect, as the ASH-expressed Gαq- coupled OA receptor, SER-6, appears to antagonize OCTR-1 signaling (Mills et al.,

2012b; Wragg et al., 2007). A similar antagonism appears to operate between DOP-3 and

DOP-4 in the ASHs in the DA inhibition of food or 5-HT stimulation, although the involvement of neuropeptides in this antagonism remains to be explored (Ezak & Ferkey,

2010; Ezcurra et al., 2011).

57 Similarly, the ASIs also express several monoamine receptors, including Gαq- coupled 5-HT, TA and OA receptors that selectively stimulate the release of different subsets of ASI neuropeptides to modulate the initiation of aversive responses mediated by the two ASH sensory neurons (Hapiak et al., 2013; Harris et al., 2010; Mills et al., 2016;

Mills et al., 2012b; Wragg et al., 2007). These neuropeptides modulate signaling at multiple levels within the ASH-mediated locomotory circuit and are part of large modulatory network of monoamines and neuropeptides that define key behavioral states, such as nutritional status (Gray et al., 2005; Hapiak et al., 2013; Harris et al., 2010;

Harris et al., 2009; Luedtke et al., 2010; Mills et al., 2016; Mills et al., 2012b; Wragg et al., 2007). For example, in addition to the ASI neuropeptides described in the present study, ASH neuropeptides encoded by nlp-3 are also required for 5-HT stimulation and their overexpression from a number of different neurons dramatically decreases the time taken to initiate reversal (Harris et al., 2010). The level of crosstalk among these different signaling components is extensive and complex, with monoamines and neuropeptides modulating the activity of individual neurons, entire circuits and, as described in the present study, interacting directly, with monoamines differentially regulating the release of an array of modulatory neuropeptides from somal and synaptic sites within a single pairs of neurons. Lastly, additional ASH-ASI crosstalk in response to a noxious stimulus has been described elsewhere. For example, the ASHs detect Cu2+ and stimulate the release of OA from the octopaminergic RICs that, in turn, inhibits the peptidergic ASIs via the OA receptor, SER-3. Conversely, the ASIs also sense Cu2+ and stimulate 5-HT release from the serotonergic ADFs that, in turn, activates SER-5 on the glutamatergic

58 ASHs to stimulate aversive responses (Guo et al., 2015). Unfortunately, the exact role of neuropeptides in modulating this complex network have yet to be examined further.

Many other examples of monoaminergic/peptidergic interactions have been described in C. elegans. For example, feeding and egg-laying are also stimulated by 5-HT and inhibited by TA and OA, with a complex arrangement of multiple monoamine and neuropeptide receptors mediating the process (Alkema et al., 2005; Hapiak et al., 2009;

Hobson et al., 2006; Horvitz et al., 1982; Xiao et al., 2006). In fact, 5-HT can have both excitatory and inhibitory inputs into both processes, with the balance of signaling between different 5-HT receptor subtypes dictating behavior (Hapiak et al., 2009).

Indeed, a similar observation is paralleled in mammals where the involvement of 5-HT receptors in the antidepressant-like effects of selective 5-HT reuptake inhibitors is complex and involves the orchestrated stimulation and blockade of different 5-HT receptor subtypes (Carr & Lucki, 2011). Similarly, neuropeptides encoded by flp-7, -10, -

11 and -17 modulate both feeding and egg-laying, although the specifics of these complex monoamine/neuropeptide interactions have yet to be fully described (Ringstad

& Horvitz, 2008; Zang et al., 2017).

Monoamines and neuropeptides also modulate C. elegans mating, but again the specifics of the interactions are poorly understood. For example, vulva location and spicule insertion, key steps in mating behavior, require the male-specific serotonergic CP neurons and both 5-HT receptors, SER-1 and MOD-1, to modulate the turning behavior required for mating (Carnell et al., 2005; Loer & Kenyon, 1993). In addition, several neuropeptide-encoding genes are also required, as flp-8, -10, -12 and -20 mutants exhibit repetitive turns, diminishing an animal’s ability to efficiently find the vulva (Li & Kim,

59 2014; Liu et al., 2007). Similarly, defects of /vasopressin signaling, involved in mating behavior in many species, also interfere with reproductive behavior in C. elegans.

For example, C. elegans expresses an oxytocin/vasopressin-related neuropeptide- encoding gene, ntc-1, that activates receptors encoded by ntr-1 and -2, and ntc-1, ntr-1 or

-2 mutants exhibit a range of turning phenotypes that cause inefficient mating (Garrison et al., 2012).

Monoamines and neuropeptides also function at multiple levels within the locomotory circuit to modulate all aspects of locomotory behavior, from sensory integration at the level of sensory and interneurons, to motor neurons and body wall muscle (Choi et al., 2015; Churgin et al., 2017; Gjorgjieva et al., 2014; Komuniecki et al., 2014; Komuniecki et al., 2012; Laurent et al., 2015; Lim et al., 2016). Locomotion is dependent on coordinated muscle contraction and regulated by the excitatory cholinergic and the inhibitory GABAergic motor neurons. C. elegans exhibits a range of distinct monoamine and neuropeptide-dependent locomotory behaviors, such as roaming and dwelling, to explore their surroundings (Ben Arous et al., 2009). On food, 5-HT release from the serotonergic NSM neurons decreases locomotion and increases turning, while off food or during starvation, TA and OA release from the RIM and RIC neurons, respectively, increases locomotory rate and decreases turning (Alkema et al., 2005;

Churgin et al., 2017; Gray et al., 2005; Harris et al., 2011; Pirri et al., 2009). Upon encountering food, animals exhibit 5-HT and DA-dependent decreases in locomotion that require the Gαo-coupled SER-4 and DOP-3, respectively (Chase et al., 2004; Gurel et al.,

2012; Hapiak et al., 2009). In fact, SER-4 activation in the two AIB interneurons initiates a phenomenon known as “locomotory confusion”. The AIBs are unable to integrate

60 antagonistic sensory signals to move forward or backward, leaving animals confused and unable to move productively, but not actually paralyzed, as they can move forward for a few body bends when touched (Law et al., 2015). In contrast, starved worms exhibit a 5-

HT and OA-dependent enhanced locomotion with reduced turning, requiring SER-1 and as yet unidentified OA receptors (Dernovici et al., 2007; Komuniecki et al., 2004). For example, tbh-1 null animals that lack a key biosynthetic enzyme required for OA synthesis appear to move more rapidly than wild-type animals. In contrast, TA and DA inhibit locomotion and cause paralysis when added exogenously, through complex pathways involving multiple TA and DA receptors (Chase et al., 2004; Donnelly et al.,

2013; Felton & Johnson, 2011; Pirri et al., 2009). For example, TA inhibition requires the

- Gαo-coupled TA receptor, SER-2 and the TA-gated Cl channel, LGC-55, expressed in neck muscles and cholinergic motor neurons, while DA inhibition involves an antagonistic interaction between the DOP-1 and DOP-3 receptors expressed in cholinergic motor neurons, with dop-3 null animals resistant to DA-dependent paralysis

(Chase et al., 2004; Donnelly et al., 2013; Suo et al., 2009).

A wide range of neuropeptides also appear to interact with monoamines in modulating most aspects of locomotory behavior, as described by the identification of locomotory phenotypes in neuropeptide and neuropeptide receptor null mutants (Chen et al., 2016; Choi et al., 2015; Hu et al., 2011). For example, dwelling/roaming behavior is differently modulated by both monoamines and neuropeptides. 5-HT promotes dwelling by activating the 5-HT gated Cl- channel, MOD-1, to inhibit signaling from the AIY, RIF and ASI neurons (Flavell et al., 2013). In contrast, OA inhibits quiescence and promotes roaming by activating both SER-3 and SER-6 on the SIA neurons (Churgin et al., 2017).

61 Peptidergic signaling contributes significantly to this balance. For example, peptidergic signaling from the ASIs appears to be required for basal exploratory behavior, as ASI expression of the tetanus light chain reduces neuropeptide release and subsequently reduces exploration (Greene et al., 2016). Similarly, PDF-1 release from the PVP neurons also promotes roaming and arousal by activating PDFR-1 on several neurons (Choi et al.,

2013; Flavell et al., 2013; Ranganathan et al., 2000). Together, these studies demonstrate the extensive roles for both monoamines and neuropeptides in the modulation of most key C. elegans behaviors and highlight how much more remains to be learned about how these different signaling systems interact to modulate signaling, from individual neurons to complete circuits.

4.2 Monoaminergic/peptidergic Interactions Are Also Important in Other

Organisms

As in C. elegans, serotonergic/noradrenergic and peptidergic signaling can have complex effects on individual behaviors in mammals. For example, anxiety and stress- induced anxiety-like behaviors are poorly understood, but the balance of 5-HT signaling plays a key role in mammals. In rats, inescapable tail shock elicits exaggerated anxiety- related behaviors compared to escapable shock, and is postulated to be the result of an intense activation of 5-HT neurons in the dorsal raphe nucleus (DRN) (Maier & Watkins,

2005). A subset of the DRN neurons respond differently to inescapable shock and escapable shock, and this subset project to the basolateral amygdala, a key area involved in anxiety that is modulated by 5-HT. Interestingly, inescapable shock increased both 5-

HT efflux into the basolateral amygdala and anxiety-like behavior. In contrast, the anxiety-like behavior caused by inescapable tail shock could be reversed by either,

62 inhibiting the DRN neurons using the 5-HT1A agonist 8-OH-DPAT, or inhibiting the basolateral amygdala via 5-HT2C receptor antagonists (Christianson et al., 2010). In contrast, neuropeptide S (NPS) signaling in the lateral amygdala prevents stress-induced behavioral changes, with amygdala NPS receptors inhibiting depolarization-evoked 5-HT release in isolated amygdala synaptosomes, suggesting that peptidergic signaling modulates serotonergic terminals (Chauveau et al., 2012; Gardella et al., 2013). NPY signaling also plays a similar antagonistic role in modulating mood, as NPY Y2 and Y5 receptor agonists increase and decrease anxiety, respectively (Tasan et al., 2010; Walker et al., 2009).

Dysfunctional serotonergic and peptidergic signaling are also associated with depression. For example, depression has been linked with genetic variation in tryptophan hydroxylase 2, postsynaptic 5-HT receptors or 5-HT transporter genes (Belmaker &

Agam, 2008; Blier & El Mansari, 2013; Kohler et al., 2016). Additionally, antidepressants increase 5-HT concentrations by inhibiting monoamine oxidase, selective serotonin reuptake and/or modulating 5-HT auto-receptor responsiveness (Blier & El

Mansari, 2013). Similarly, tricyclic antidepressants increase net 5-HT transmission by blocking NA reuptake and sensitizing α2-adrenoceptor responsiveness on 5-HT terminals

(Blier & El Mansari, 2013). DA D2/D3 receptor agonists also increase 5-HT concentrations by desensitizing 5-HT1A auto-receptors (Chernoloz et al., 2009; Chernoloz et al., 2012). Dysfunctional neuropeptide systems also contribute to depression, but much remains to be learned about the specifics of how monoaminergic and peptidergic signaling interact, not surprisingly, given the complexity of the interaction and size of the mammalian nervous system. For example, elevated corticotropin-releasing factor (CRF)

63 and a hyperactive hypothalamus–pituitary–adrenal axis is common in patients suffering depression and, like 5-HT, the site of CRF signaling is also important (Holsboer, 2000).

For example, CRF overexpression in the central nucleus of the amygdala decreases stress-induced anxiety, while overexpression in the stria terminalis increases depressive- like behaviors (Regev et al., 2011). Similarly, NPY exerts antidepressant effects by mimicking the action of selective 5-HT reuptake inhibitors in a rat model for depression

(Stogner & Holmes, 2000). Collectively, these studies demonstrate that monoamines and neuropeptides interact to modulate many of the same behaviors and highlight the need for additional studies to define the specifics of these complex interactions.

4.3 Both TA- and OA-dependent ASI Neuropeptides Co-localize in the ASIs

The ASIs express over twenty neuropeptide-encoding genes and since monoamines appear to selectively stimulate the release of at least some, if not all, of these neuropeptides to modulate a wide range of behaviors, we predicted that neuropeptide- containing DCVs would be differentially localized in the ASIs (Cheong et al., 2015; Guo et al., 2015; Nathoo et al., 2001; Palamiuc et al., 2017). In fact, in the present study both

TA- and OA-dependent neuropeptides co-localized with synaptic markers, although whether this localization is synaptic or perisynaptic is unclear. Indeed, a perisynaptic localization of neuropeptide-containing DCVs has been reported in a number of systems

(Hammarlund et al., 2008; Nassel, 2009). For example, DCVs localize to distinct sites separate from small clear vesicles (SCVs) and the synaptic zone in mammalian dorsal root ganglion (Salio et al., 2006). Similarly, DCVs in Aplysia californica motor neurons are practically excluded from active zones, in contrast to SCVs (Karhunen et al., 2001).

In C. elegans cholinergic motor neurons, DCVs exhibit a perisynaptic localization, as

64 they do not cluster at active zones, but are in close proximity with, and in some instances contact, SCVs at synaptic sites (Hammarlund et al., 2008; Stigloher et al., 2011). More importantly, populations of different neuropeptide-containing DCVs have been observed.

Prothyrotropin-releasing hormone is differentially cleaved into different N- and C- terminal products, and DCV populations containing either N-, C- or a mixture of both terminal products were present in AtT20 cells. These in vitro observations parallel those observed in vivo in the median eminence of hypothalamic explants from rats, suggesting that cells can independently and specifically regulate the release of DCV containing different populations of bioactive peptides (Perello et al., 2008).

In the present study, RAB-3/neuropeptide-positive, fluorescent puncta were also observed along the proximal portion of the ASI axons near the soma, where synapses have not been identified anatomically. Interestingly, although ASI::NLP-14::GFP and

ASI::NLP-9::mCHERRY appear to co-localize in the same synaptic regions, the ratio of synaptic to extra-synaptic fluorescence for ASI::NLP-14::GFP and ASI::NLP-9::mCherry differed significantly. This difference in fluorescence suggests the DCVs containing the neuropeptides encoded by these two genes may be trafficked independently to different sites, accounting for their differential monoamine-specificity. Indeed, extra-synaptic neuropeptide release has been demonstrated in C. elegans and in other systems (Koelle,

2016; Tobin et al., 2012; Trueta & De-Miguel, 2012). In fact, many C. elegans monoamine receptors are expressed on neurons that are not connected synaptically to monoamine-producing cells, and a substantial portion of monoaminergic modulation may be extra-synaptic (Koelle, 2016). For example, tyraminergic and peptidergic signaling from the C. elegans UV1s inhibits egg-laying, even though the UV1s do not form direct

65 synaptic connections with the egg-laying circuit. In addition, neuropeptides encoded by nlp-40 that regulate the defecation program are expressed in intestinal cells and secreted humorally to activate AEX-2, the NLP-40 receptor, expressed on GABAergic motor neurons (Banerjee et al., 2017; Jose et al., 2007; Wang et al., 2013). Whether these additional ASI neuronal puncta are associated with DCV trafficking or novel sites of release requires further investigation; however, they do not appear to be sites of monoamine-dependent release.

As in C. elegans, neuronal co-localization of neurotransmitters has been in observed in several systems, including neuropeptides with classical, small molecule neurotransmitters. These systems include extensive neuropeptide release from peptidergic neurons, such as the anterior pituitary, where both pre- and post-synaptic GPCR- mediated neuropeptide signaling modulates the opening of ion channels. Initially, it was assumed that neurons co-stored and co-released the same set of neurotransmitters at all sites. However, it is now clear that neurotransmitters can be stored and released independently, and at distinct sites within single neurons, supporting the hypothesis that neurons differentially sort neurotransmitters to independent sites of release. For example, in C. elegans the ASHs release both glutamate and neuropeptides encoded by nlp-3 and nlp-15 to modulate nociceptive behavior (Harris et al., 2010). Interestingly, the ASH peptides encoded by nlp-3 and nlp-15 either enhance or inhibit, respectively, aspects of this response, suggesting that they may function antagonistically to fine tune ASH responses. As mentioned prior, 5-HT stimulates the initiation of aversive responses to dilute 1-octanol, and ASH peptides encoded by nlp-3 are required for this 5-HT stimulation, suggesting a complex interaction between monoaminergic, peptidergic and

66 glutamatergic signaling (Harris et al., 2010; Mills et al., 2016). Conversely, nlp-15 null animals initiate aversive responses much more rapidly than wild-type animals and animals overexpressing nlp-15 encoded peptides in the ASHs respond to 1-octanol much more slowly than wild-type animals (Hapiak, Oakes & Komuniecki, unpublished).

Similarly, AWC glutamatergic signaling stimulates food-search behavior that is simultaneously regulated by AWC peptidergic signaling evoking a negative feedback loop (Chalasani et al., 2010). In Drosophila, short neuropeptide F and acetylcholine co- released from mushroom body Kenyon cells stimulate mushroom body output neurons to modulate odor-driven behavior (Barnstedt et al., 2016). The crab modulatory commissural neuron 1 (MCN1) is also capable of co-release, with the neuropeptides proctolin and CabTRP Ia modulating the pyloric and gastric mill rhythms (Nusbaum et al., 2017). Co-transmission is also common in mammalian neurons. For example, glutamate and the neuropeptide orexin co-localize within the lateral hypothalamus (LH) and their interaction is involved in the modulation of numerous behaviors, including sleep-wake cycles (Ma et al., 2018). Glutamatergic signaling from the LH initiates the fast excitation of downstream histaminergic neurons involved in sleep/wakefulness, while orexin-mediated excitation also excites these same neurons but requires sustained LH activation and is slower and longer-lived than responses to glutamate, as predicted for

GPCR mediated signaling (Schone et al., 2014; Schone et al., 2012).

Finally, although neuropeptides appear to co-localize, specific neuropeptide signaling may also be modulated by selective proteolysis after release, by specific neuropeptidases differentially expressed at different release sites, adding an additional layer of modulatory complexity (Isaac et al., 2009; Turner et al., 2001). For example, in

67 Cancer borealis, the neuropeptide proctolin is expressed in two different neurons; MCN1 and the modulatory proctolin neuron (MPN), and these neurons regulate the stomatogastric ganglia to elicit distinct versions of the pyloric rhythm. Proctolin release from these two different sites helps modulate the motor pattern of the pyloric rhythm, and altering the levels of proctolin, by inhibiting local aminopeptidase activity, changed the pyloric circuit response, making the responses exerted by MCN1 and MPN indistinguishable (Wood & Nusbaum, 2002). In agreement, mammalian peptidases are also responsible for specific modulation/termination of neuropeptide signaling. For example, several novel NPY-cleaving enzymes were recently discovered that yield different truncated forms of NPY, and the co-localization of these peptidases with NPY differed between cell types, suggesting distinct NPY processing/degradation is used to localize NPY signaling (Wagner et al., 2015). The C. elegans genome encodes a predicted 37 genes involved in neuropeptide degradation, including several dipeptidyl peptidases, although little is known about their function and localization (Hobert, 2013).

Recent evidence suggests the C. elegans homolog of the extracellular peptidase neprilysin, NEP-2, is involved in regulating olfactory plasticity by degrading the proposed environmental signal, SNET-1 peptide (Yamada et al., 2010). Given the number of genes that are predicted to function in neuropeptide degradation, it will not be surprising if several of these are identified in future as playing key roles in modulating neuropeptide signaling within neuronal networks.

4.4 TA and OA Differentially Modulate Neuropeptide Release from the ASIs

The monoamine selectivity of ASI neuropeptide release is dependent on the expression of different Gαq-coupled monoamine receptors. Since Gαq signaling increases

68 2+ IP3-mediated Ca release, we hypothesized that TA and OA stimulate local ASI Gαq signaling and increase Ca2+, stimulating the release of DCVs from distinct pools (Huang

& Thathiah, 2015; Stojilkovic, 2005). Together these observations suggest that ASI

GPCRs are differentially localized and ASI Ca2+ signaling compartmentalized. However, compartmentalized ASI GPCR signaling or Ca2+ signaling have yet to be characterized directly. In contrast, localized Ca2+ release has been observed in a variety of other systems, with G-protein-coupled receptors often in proximity to local Ca2+ stores (Jin et al., 2013). In C. elegans, a muscarinic-like G-protein-coupled receptor modulates local

Ca2+ dynamics in the RIA interneurons to coordinate locomotion (Hendricks et al., 2012).

In Drosophila, OA stimulates neuropeptide release by activating both a cAMP-dependent protein kinase and increasing Ca2+ release from internal stores (Shakiryanova et al.,

2011). In mammalian retinal starburst amacrine cells, a localized Ca2+ signal is also compartmentalized by mGluR2 signaling that restricts propagation (Euler et al., 2002;

Koren et al., 2017). Amacrine cells are similar to many C. elegans neurons in that they share both synaptic inputs and outputs along their neuronal processes. Similarly, many

GPCRs are localized subcellularly. For example, the cannabinoid receptor, CB1, and the , SSTR2, differentially localize to axonal and somatodendritic regions in mammalian cells, respectively (Simon et al., 2013). Interestingly, our data suggest TA stimulates the release of somatic ASI::NLP-18::GFP, as TA decreases somal, but not axonal, fluorescence. Somatic DCV release has been observed in other systems, with the release of peptides from different DCVs mediated by localized Ca2+ signals

(Trueta et al., 2012; Xia et al., 2009; Zhang et al., 2012). In contrast, although not significant but suggestive, TA appears to increase somatic NLP-9::GFP fluorescence,

69 suggesting that TA inhibits neuropeptide-specific DCV trafficking and/or release. Indeed, monoamine-mediated inhibition of neuropeptide release has also been observed. For example, the activation of the D2 DA receptor in PC12 cells inhibits the release of NPY

(Cao et al., 2007). Selectively modulating local Ca2+ dynamics could expand the signaling capacity of individual neurons, an approach potentially significant in an organism like C. elegans with only 302 neurons.

Monoamines also modulate neuropeptide release in other C. elegans behaviors/processes. For example, 5-HT stimulates the release of flp-7 encoded peptides to decrease body fat and peptides encoded by daf-7 and -28 to facilitate exit from dauer

(Cunningham et al., 2014; Noble et al., 2013; Palamiuc et al., 2017). Similarly, DA stimulates the release of nlp-12 encoded peptides to modulate local search behavior and inhibits the release of flp-8 encoded neuropeptides to modify copper aversion

(Bhattacharya et al., 2014; Ezcurra et al., 2016). In Drosophila, OA stimulates the release of GFP-tagged atrial natriuretic factor (Shakiryanova et al., 2011). In mammals, multiple

5-HT receptors also modulate neuropeptide release associated with feeding. 5-HT stimulates the release of α-MSH to stimulate feeding and inhibits the release of the orexigenic peptides, NPY and AgRP to inhibit feeding (Heisler et al., 2002; Heisler et al.,

2006; Sohn et al., 2011; Voigt & Fink, 2015).

Conversely, neuropeptides directly modulate monoamine release. For example, nlp-

7, flp-10, -11 and -17 encoded neuropeptides inhibit 5-HT release to inhibit egg-laying and DAF-7 inhibits TA/OA release to promote satiety and quiescence (Banerjee et al.,

2017; Emtage et al., 2012; Gallagher et al., 2013; Ringstad & Horvitz, 2008). In mammals, NPS inhibits the release of 5-HT and NA from mouse amygdala and prefrontal

70 cortex synaptosomes, but stimulates the release of DA when administered intracerebroventricularly, highlighting the complex interaction between the two signaling systems (Gardella et al., 2013; Raiteri et al., 2009; Si et al., 2010). Similarly, peptide YY

(3-36) inhibits the release of DA and NA, but not 5-HT from isolated hypothalamic synaptosomes, suggesting that neuropeptides can confer specificity to monoamine release

(Brunetti et al., 2005). Collectively, these studies demonstrate that monoaminergic and peptidergic signaling often modulate the same behaviors, and also interact directly to modulate each other’s release.

4.5 OA Asymmetrically Stimulates Neuropeptide Release from the ASIs

OA asymmetrically modulates the release of nlp-9 encoded neuropeptides, with OA only stimulating release from the right ASIs. Indeed, the right and left ASIs exhibit different synaptic connectivities (White et al., 1986). However, some/most neuropeptides and, indeed, most monoamines act at receptors distantly localized from their sites of release, bringing into question the potential significance of our observed spatial patterns and asymmetric peptide release, if this peptidergic signaling is extra-synaptic. Little is known about the localization of neuropeptides with their cognate receptors in C. elegans.

Certainly, in C. elegans some/most neuropeptides and, indeed, most monoamines appear to signal extra-synaptically and act at receptors distantly localized from their sites of release, based on the localization of monoamine synthesizing neurons and monoamine receptors. Indeed, most monoaminergic neurons do not synapse directly onto neurons expressing most monoamine receptors (Donnelly et al., 2013; Gurel et al., 2012; Koelle,

2016). For example, none of the monoamine-synthesizing neurons synapse directly on the ASIs and only the serotonergic ADFs synapse on the ASHs, even though these two

71 pairs of sensory neurons express a number of 5-HT, TA, OA and DA receptors (available from: http://wormweb.org/neuralnet#c=ASI&m=1). However, 1) some peptide receptors may be synaptic and focus more local, extra-synaptic, monoaminergic signaling or, more likely, 2) given the tight packaging of neurons in the C. elegans nerve ring, the site of neuropeptide or monoamine release may be relevant, even for extra-synaptically localized peptide/monoamine receptors. Indeed, peptide receptors have been identified in close proximity to sites of peptide release in other systems. For example, the tachykinin- related peptide 1 and its receptor, LTKR, arborize in close proximity in the cockroach brain (Johard et al., 2001). In Drosophila, immunoreactivity-labeling of tachykinins was distinct and closely resembled that of their receptor, DTKR, suggesting a local site of activation (Birse et al., 2006). In addition, as noted above, the selective localization of neuropeptidases could further modify peptidergic signaling, especially if the gene encodes 1) a mix of receptor agonists that activate different receptors or 2) agonists/antagonists that have the potential to be differentially degraded, as we have suggested previously for C. elegans opiate signaling (Mills et al., 2016). For example, nematode neprilysins are differentially localized and modulate the degradation of number of regulatory peptides (Isaac et al., 2009; Turner et al., 2001). No studies to date have demonstrated compartmentalization in the ASIs, but data from the RIAs and the functional asymmetry of other sensory neurons increases the plausibility of a similar situation for the ASIs. Indeed, asymmetry in both connectivity and function has been observed in other C. elegans neuron pairs. For example, the differential expression of guanylyl cyclases contributes to functional asymmetry in the two ASE sensory neurons

(Hobert, 2014; Ortiz et al., 2006; Suzuki et al., 2008; Yu et al., 1997). Similarly, the two

72 functionally distinct AWC sensory neurons asymmetrically express str-2, a putative olfactory receptor, specifying str-2-expressing cells as AWCON and non-str-2-expressing cells as AWCOFF (Chalasani et al., 2010; Troemel et al., 1999; Wes & Bargmann, 2001).

Collectively, these studies highlight the ability of individual pairs of neurons to differentially perceive and process sensory information, which likely results in the stimulated, neuron-specific release of neurotransmitters, in agreement with our observations with the ASIs.

4.6 Monoamines and Neuropeptides Interact Directly to Modulate Release

In addition to the effects of the monoamine-stimulated ASI neuropeptide release reported in the present study, monoamines also directly modulate neuropeptide release to modify other C. elegans behaviors. For example, the control of body fat is a complex process involving 5-HT, OA and neuropeptides. OA upregulates tph-1 expression increasing 5-HT levels, and 5-HT signaling results in the release of neuropeptides encoded by flp-7 from the ASIs, ultimately driving fat loss via the adipocyte triglyceride lipase, ATGL-1 (Noble et al., 2013; Palamiuc et al., 2017). In addition, exogenous 5-HT increases the coelomocyte fluorescence signal in animals expressing ASI::DAF-

7::mCHERRY and ASI::DAF-28::mCHERRY, suggesting 5-HT can also stimulate the release of the transforming growth factor beta-β ligand, and insulin-like peptide, respectively (Cunningham et al., 2014). Interestingly, DAergic and peptidergic signaling are closely tied to nutritional status and can shape context-dependent behaviors. For example, ASH-mediated escape responses to repellents, such as CuCl2, adapt at a slower rate on food, compared to those off food (Ezcurra et al., 2011). Within this neuronal circuit, DA is released in response to bacteria and is required for the adaptive response on

73 food, in contrast to neuropeptide signaling, which is required for the adaptive behaviors off food (Ezcurra et al., 2011). Further analysis suggests DA inhibits neuropeptide release from the AUA neurons via DOP-1 to indirectly modulate the ASHs, in contrast to the neuropeptide receptors NPR-1 and NPR-2, that act on the ASHs to increase adaptation off food (Ezcurra et al., 2016). In addition, DA and peptides encoded by nlp-

12 are involved in modulating nutritionally-dependent locomotion. Animals that have recently been removed from food exhibit local search behavior, increasing the frequency of reorientations, and these responses are fine-tuned by NLP-12 peptides. Interestingly, exogenous DA decreases punctate fluorescence on NLP-12::NLP-12::VenusYFP in the

DVA interneurons, and this decrease is dependent on the DA receptor dop-1, suggesting

DA-dependent neuropeptide release, much like we have observed with the ASIs

(Bhattacharya et al., 2014).

The crosstalk between monoaminergic and peptidergic signaling has also been reported outside of C. elegans. For example, at the Drosophila muscle 6/7 neuromuscular junction, OA stimulates the release of GFP-tagged atrial natriuretic factor, an artificial protein that is processed, localized and released like an endogenous neuropeptide (Rao et al., 2001; Shakiryanova et al., 2011). In mammals, monoaminergic/peptidergic crosstalk is complex and depends on the type of receptor being activated. For example, feeding behavior involves multiple 5-HT receptors that modulate neuropeptide release. 5-HT2C receptor agonists reduce food intake, body weight and chronic exposure increases POMC mRNA (Voigt & Fink, 2015). POMC neurons release the anorectic peptide, α-MSH, and up to 80% of α-MSH containing neurons express 5-HT2C receptors in the ARC, highlighting the ability of 5-HT to modulate peptidergic signaling (Heisler et al., 2002).

74 Alpha-MSH is an endogenous MC agonist, and mice lacking MC3 and/or MC4 no longer exhibit 5-HT2C receptor agonist-induced hypophagia, suggesting α-MSH signaling operates downstream of 5-HT (Lam et al., 2008; Rowland et al., 2010). In agreement, 5-

HT causes the release of α-MSH from hypothalamic slices, and taken together, these data suggest 5-HT can inhibit feeding by stimulating the release of α-MSH (Heisler et al.,

2003; Tiligada & Wilson, 1989). In contrast, 5-HT can also modulate feeding by inhibiting the release of the endogenous orexigenic peptides, NPY/AgRP. Co-localization of the Gi-coupled 5-HT1B receptor, and NPY/AgRP was observed in neurons of the ARC, and selective 5-HT1B agonists hyperpolarized these cells (Heisler et al., 2006).

Collectively, these data suggest 5-HT can selectively stimulate or inhibit neuropeptide release within the ARC. Similarly, in vitro studies using DA D2 receptor agonists and antagonists show D2 activation decreases NPY release from PC12 cells, suggesting that like OA and 5-HT, DA is also able to modulate stimulus-dependent neuropeptide release

(Cao et al., 2007). Lastly, 5-HT may also stimulate peptide release through a receptor- independent mechanism that involves the modification of intracellular proteins, in a process known as serotonylation. In pancreatic β-cells, serotonylation of the small

GTPases RAB-3a and RAB-27a caused constitutive activation, increasing insulin secretion (Paulmann et al., 2009). However, whether this mechanism functions in neurons is still unknown and it should be noted that this process is likely a supplement to the function of exocytosis within the cell.

Conversely, neuropeptides can also modulate monoamine release. For example, several cells involved in the C. elegans egg-laying circuit, including the neuroendocrine

UV1 cells and HSN neurons, are in close proximity. 5-HT from the HSNs stimulates egg-

75 laying, in contrast to activation of the UV1 cells, which inhibit egg-laying and increase the number of eggs contained within the uterus, via TAergic and peptidergic signaling

(Banerjee et al., 2017; Hapiak et al., 2009; Schafer, 2006). However, the UV1 activation of egg-laying was reduced in nlp-7 and flp-11 single deletion mutants, suggesting these neuropeptides regulate 5-HT release. Interestingly, nlp-7 and flp-11 are expressed in the

UV1 cells, and are proposed to alter synaptic vesicle abundance, as SNB-1::GFP fluorescence in the HSNs is increased in nlp-7 mutants (Banerjee et al., 2017). This suggests an interesting mechanism of HSN modulation and 5-HT release, independent of the TA-inhibition of egg-laying, which is mediated by the TA-sensitive Cl- channel,

LGC-55 expressed on the HSNs (Banerjee et al., 2017; Collins et al., 2016). Similarly, neuropeptides encoded by flp-10 and flp-17 also inhibit egg-laying (Ringstad & Horvitz,

2008). These peptides are predicted to activate the Gαo-coupled EGL-6 receptor expressed on the HSNs that, in turn, opens inhibitory inwardly rectifying K+ channels and thus inhibit 5-HT release/egg-laying, although the possibility of these pathways operating in parallel should not be ruled out (Emtage et al., 2012; Ringstad & Horvitz, 2008).

As mentioned previously, in mammals, NPS and its receptor, NPSR, are involved in anxiety (Xu et al., 2004). Interestingly, NPS exhibits different monoaminergic modulation depending on the site of activity and the monoamine in question. For example, in isolated synaptosomes of the mouse amygdala and prefrontal cortex, NPS inhibits the release of 5-HT and 5-HT/NA, respectively, and the inhibitory effects of NPS are abolished via NPSR antagonists (Gardella et al., 2013; Raiteri et al., 2009). In contrast, the central administration of NPS causes dose-dependent increases of DA in the medial prefrontal cortex, and suggest a possible mechanism for the anxiolytic effects of

76 NPS (Si et al., 2010). Taken together, these studies demonstrate how one neuropeptide can differentially modulate monoamine release in a spatially dependent manner.

Similarly, peptide YY (3-36) inhibits the stimulated release of DA and NA, but not 5-HT from isolated hypothalamic synaptosomes, further suggesting that neuropeptides can confer specificity to monoamine release (Brunetti et al., 2005). Furthermore, DA neuron stimulating peptide-11, an 11-mer peptide that is derived from glial cell line-derived neurotrophic factor, increases DA levels and this increase persists after 28 days, suggesting peptidergic signaling can chronically elevate monoamine levels (Bradley et al., 2010). Lastly, peptidergic signaling in fish also regulates monoamines levels and behavior. For example, CRF, a neuroactive peptide found in diverse vertebrates, interacts with brain 5-HTergic systems to mediate stress, anxiety, and aggression (Lowry &

Moore, 2006). Indeed, CRF administration inhibits the elevated 5-HT in the amygdalar subpallium of rainbow trout during aggressive social interactions (Carpenter et al., 2009).

Collectively, these studies demonstrate the complex interactions between monoaminergic and peptidergic signaling. Not only do both monoaminergic and peptidergic signaling systems often modulate the same behaviors, but they also directly modulate each other’s release, highlighting the interrelatedness of the two systems.

4.7 The TA- and OA-dependent ASI Neuropeptides Contain Different Predicted

Preprosequences

Neuropeptides are cleaved from large preproproteins that encode multiple, often distinct peptides. Interestingly, many processing enzymes are co-packed with neuropeptides and activated within the DCV (Li & Kim, 2008; Seidah & Prat, 2002;

Zhou et al., 1999). However, the exact mechanism of differential packaging of specific

77 proteins into DCVs is unclear and has been debated for some time. As outlined above, two mechanisms for packaging of cargo destined for DCVs have been proposed; sorting- by-entry and sorting-by-retention (Courel et al., 2008). The sorting-by-entry model proposes a particular motif or sequence is responsible within an aggregate of secretory proteins that leads to subsequent association with a receptor and/or lipid component of the TGN. In contrast, sorting-by-retention suggests that sorting/aggregation occurs post-

TGN between ISVs, refining regulated secretory proteins destined for DCVs from other aggregated cargo. Despite this debate, one mechanism must be true; proteins destined for the regulated secretory pathway are separated from the constitutively regulated pathway and are packaged into different vesicles. Several studies suggest that the N-terminus of the preproprotein is required for sorting into the regulated secretory pathway. For example, earlier studies focused on truncating POMC to determine a sequence capable of sorting to the regulated secretory pathway. Specifically, N-terminal truncations of POMC

(POMC10, 26, 50, 101) were fused to a reporter construct and assessed for localization and release, with only POMC10 lacking DCV localization and stimulated release, highlighting the functional importance of the N-terminus (Cool & Loh, 1994). In addition, the regulated secretory protein CgA was also truncated and amino acids 1-115 are required for sufficient trafficking and release (Taupenot et al., 2005). Similarly, VP was still efficiently sorted and released despite being truncated to the first 27 N-terminal residues, and sequence within the N-terminus of the cocaine and amphetamine regulated transcript proprotein was required for sorting into the regulatory secretory pathway (Blanco et al.,

2013; de Bree et al., 2000). Collectively, these studies show that N-terminal sequence is required for the packaging and secretion of various DCV cargo, but the exact motif or

78 structure that drives this process is still to be elucidated. However, a variety of different mechanisms have been proposed that function in packaging cargo to the regulated secretory pathway, many of which are found at the N-terminus, such as α-helices, charged residues and dibasic cleavage sites (Dikeakos & Reudelhuber, 2007; Lacombe et al., 2005; Ma et al., 2008). To functionally explore the differences in TA- and OA- dependent neuropeptide preproproteins, we used protein structure and prediction software to analyze the predicted preproproteins encoded nlp-9, -14 and -18.

Interestingly, all three preproproteins contained an N-terminal α-helix, but remarkably, when considering their overall size, the α-helix within NLP-9 was more than twice the size of the α-helix in NLP-14 or NLP-18. Whether this size difference in α- helices is involved in differential DCV packaging remains to be determined, but α-helices are important. For example, secretogranin II (SgII) is a member of the granin family of prohormones that contains α-helices and is found packaged into DCVs. N- and C- terminal truncation of SgII fused to GFP at the C-terminus of SgII(1-301) or SgII(302-587) were efficiently packaged into DCVs, suggesting domains in either portion we present that efficiently sorted SgII into the regulated secretory pathway. Interestingly, further truncations demonstrated that amino acids 1-41 and 334-348 were required for efficient localization and stimulus-dependent release, and both these sequences are predicted to contain α-helices (Courel et al., 2008). In agreement, truncation of the cocaine and amphetamine regulated transcript proprotein narrowed down the N-terminal requirement for efficient DCV sorting to the first 41 amino acids. Further analysis of this sequence predicted an α-helical structure with an amphipathic configuration (Blanco et al., 2013).

Lastly, an interesting study using compounding sorting signals found that the most

79 efficient DCV sorting required a paired, basic cleavage site taken from human prorenin, and an α-helical domain from the PC1/3 C-terminus (Lacombe et al., 2005).

In addition to α-helices, we also examined the TA- and OA-dependent preproproteins for charge distribution. The OA-dependent NLP-9 contained a positive region within the α-helix, in contrast to the TA-dependent NLP-14 and NLP-18, which contained no charge in their α-helices. However, no charge differences were identified in the neuropeptide-encoding sections, as all three preproproteins contained varying positive and negative charges within these regions. Again, whether these regions are involved in the differential packaging of neuropeptides requires further investigation, but the importance of charge distribution has been demonstrated previously. For example, preprotachykinin (PPT) encodes a signal peptide, propeptide, substance P and neurokinin

A. GFP was fused in frame to various truncated forms of PPT and assessed for DCV localization. Interestingly, these truncation experiments suggest sequences within the

PPT propeptide contribute to DCV sorting, and further analysis showed a polarized distribution of charged amino acids. These charged residues are important for DCV packaging, as neutralizing mutations decreased DCV sorting efficiency (Ma et al., 2008).

Lastly, as predicted, all three preproproteins contained several dibasic residues that are known targets of the proprotein convertase enzymes (EGL-3) involved in cleaving proproteins to yield functional peptides (Li & Kim, 2008). Interestingly, both NLP-14 and NLP-18 preproproteins had dibasic cleavage sites flank all the predicted and confirmed neuropeptides. In contrast, the first peptide contained within the NLP-9 preproprotein did not have any flanking dibasic residues at the N-terminus, and the last peptide only had 1 basic residue on the C-terminus. The functional importance of these

80 dibasic sites was outside the scope of this study, but such residues are important for efficient DCV packaging and it has been postulated that they mediate interactions with the respective proprotein convertase during packaging to increase efficiency (Dikeakos &

Reudelhuber, 2007). For example, the addition of dibasic cleavage sites increases DCV sorting efficiency, and mutation of dibasic sites interferes with sorting and results in accumulation at the Golgi (Lacombe et al., 2005; McGirr et al., 2013).

We investigated the importance of the N-terminal sequence in the monoamine- specific release of NLP-14 and NLP-9 by generating chimeric proteins that contained the opposite predicted preprosequence upstream of the first peptide, i.e. the TA-dependent

NLP-14 now had the N-terminus of the OA-dependent NLP-9 and vice versa. We hypothesized that if all the necessary sorting signals are located within the N-terminus, monoamine-specific neuropeptide release could be switched, making NLP-14 OA- dependent and NLP-9 TA-dependent. Each chimeric protein exhibited 7-9 fluorescent puncta that localized along the ASI axon, much like the native GFP-tagged neuropeptides. However, incubation with either TA or OA did not induce release of the respective chimeric protein, as no changes in fluorescence were observed. These data suggest that the N-terminal preprosequences are not sufficient to induce TA- or OA- specificity to the respective peptide, and additional, downstream unidentified sequences are involved. Alternatively, the chimeric peptides might not have been fit for release, even though they were trafficked correctly, either due to incorrect processing or a conformational change in tertiary structure. Further studies are required to assess exactly which motifs/sequences within the preproprotein are required for efficient processing and/or release of NLP-9, -14 and -18. However, based on our predictive protein analysis,

81 both chimeras had an N-terminal α-helix, and localized to distinct axonal puncta, suggesting the α-helix is important in DCV sorting, but not necessarily DCV exocytosis for these particular proteins.

To identify other potential N-terminal sorting sequences within NLP-14, we truncated NLP-14::GFP. Surprisingly, both the short and long truncated NLP-14 preproproteins, ASI::NLP-14(1-95)::GFP and ASI::NLP-14(1-170)::GFP, respectively, mimicked both ASI::NLP-14::GFP localization and TA-sensitivity, suggesting sequence within the first 95 amino acids is sufficient for correct neuropeptide packaging into DCVs and release. Interestingly, both ASI::NLP-14(1-95)::GFP and ASI::NLP-14(1-170)::GFP are truncated at predicted α-helices. However, these proteins have multiple α-helices, suggesting redundancy between α-helices, or that the α-helices upstream of these sites are important for DCV packaging. Furthermore, these results suggest that the preprosequence and additional amino acid sequence in the N-terminus, but not the C-terminus, was required for monoamine-specific release. These localization and release data are in agreement with other truncation studies. For example, protachykinin and proneuropeptide

Y truncated at the C-termini efficiently localized to DCVs and in the case of proneuropeptide Y could be secreted (El Meskini et al., 2001; Ma et al., 2008).

Furthermore, the N-terminal sequence is important for correct bioactive function, at least

2+ in some neuropeptides. For example, truncated NPS(1-10) was sufficient to mobilize Ca in HEK293 cells expressing NPSR, but this activation required Phe2, Arg3, and Asn4.

However, these findings do not directly translate in vivo, as NPS(1-10) did not mimic the locomotor activity of full length NPS given intracerebroventricularly, suggesting the C- terminal region is important for in vivo function (Roth et al., 2006). In addition, , a

82 29 amino acid peptide, widely expressed in the central and peripheral systems is found in various truncated states in vivo. Interestingly, galanin(1-13), -(1-16), -(1-20) but not -(6-20)

125 displaced receptor bound I-labeled galanin(1-29), suggesting these truncated forms are also functional (Ihnatko & Theodorsson, 2017). Collectively, our data suggest the preproprotein N-terminus sequence is important for DCV packaging, but not sufficient to confer monoamine-selectivity to neuropeptide release, and that additional N-terminal sequence is required for monoamine-specific DCV sorting/release. We are not the first to report the possibility of multiple sorting signals for correct sorting of cargo destined for the regulated secretory pathway. For example, sorting could be disrupted by knockdown of CPE, or mutation of dibasic sites, suggesting receptor-mediated interactions and/or processing contribute to DCV sorting. Furthermore, protein analysis of proglucagon suggest several α-helices are present, whose importance in DCV sorting have been described above (McGirr et al., 2013).

4.8 Summary

Monoamines and neuropeptides modulate behavior and monoaminergic-peptidergic cross talk is extensive, if still poorly understand at the molecular level. In C. elegans, the adrenergic-like ligands, TA and OA require distinct subsets of neuropeptides in the two

ASI sensory neurons to inhibit nociception. The present study agrees with previous findings and confirms that the TA or OA inhibition of aversive responses to 1-octanol requires downstream peptidergic signaling, specifically ASI neuropeptides encoded by nlp-14/nlp-18, or nlp-9, respectively. Neuropeptides encoded by nlp-9, -14 and -18 were expressed in the two ASIs with GFP attached at the C-terminus to the to the last predicted neuropeptide. NLP-9::, -14:: or -18::GFPs each localized to 7-9 ASI axonal puncta and

83 co-localized with the synaptic marker, mCHERRY::RAB-3 in the distal portion of the

ASI axons. The 7-9 GFP-positive axonal puncta correlated well with the 7-9 ASI synapses previously identified by electron microscopy and localization with the synaptic marker, synaptobrevin. Neuropeptide and RAB-3 positive axonal puncta were also consistently observed extra-synaptically near ASI soma. ASI::NLP-14::GFP and

ASI::NLP-9::mCHERRY co-localized to both synaptic and extra-synaptic puncta, but the

GFP/mCHERRY ratios in extra-synaptic/synaptic puncta differed significantly, suggesting that TA- and OA-dependent neuropeptides may be differentially trafficked and/or localized. Taken together, our observations suggest these neuropeptide-containing

DCVs localize to synaptic/perisynaptic regions along the ASI axons, but may be trafficked in different vesicles or localized to distinct subcellular sites.

TA, but not OA, selectively increased the release of ASI neuropeptides encoded by nlp-

14 or nlp-18 from either synaptic/perisynaptic regions of ASI axons or the ASI soma, respectively. In contrast, OA, but not TA, selectively increased the release of ASI neuropeptides encoded by nlp-9 asymmetrically, only from synaptic/perisynaptic regions of the right ASI axon. Together, these data demonstrate that neuropeptides encoded by nlp-9, -14 and -18 are released from distinct subcellular sites within the ASIs, and suggest that monoaminergic GPCR and Ca2+ signaling may be compartmentalized in the ASIs, although no evidence directly supports these later two assumptions.

The size and position of α-helices, charge distribution and frequency/location of dibasic residues, all of which are believed to be important in DCV packaging, differed markedly in the NLP-9, -14 and -18 preproproteins. However, no correlation between these differences and monoamine-specificity of release was noted. In addition, the

84 predicted preprosequences of the three TA-dependent neuropeptide-encoding genes also differed markedly from the three OA-dependent genes. However, these distinct preprosequences were not sufficient to confer monoamine-specificity, as the release of chimeric neuropeptides containing swapped preprosequences did not respond to the predicted monoamine. In contrast, the release of a truncated NLP-14::GFP containing only the first 95 amino acids and only five of a predicted 12 neuropeptides responded to

TA, but not OA, demonstrating that additional N-terminal sequence was required for monoamine-specificity.

Collectively, our results demonstrate that TA and OA differently modulate the release of distinct subsets of neuropeptides from different subcellular sites within the

ASIs and highlight the complexity of neuropeptide packaging, monoamine-dependent release and monoaminergic/peptidergic modulation, even in an animal with a relatively simple nervous system containing only 302 neurons.

85 References

Alkema, M. J., Hunter-Ensor, M., Ringstad, N., & Horvitz, H. R. (2005). Tyramine

Functions independently of octopamine in the Caenorhabditis elegans nervous

system. Neuron, 46(2), 247-260. doi:10.1016/j.neuron.2005.02.024

Avery, L. (2010). Caenorhabditis elegans behavioral genetics: where are the knobs?

BMC Biol, 8, 69. doi:10.1186/1741-7007-8-69

Avery, L., & Horvitz, H. R. (1990). Effects of starvation and neuroactive drugs on

feeding in Caenorhabditis elegans. J Exp Zool, 253(3), 263-270.

doi:10.1002/jez.1402530305

Aviram, J., & Samuelly-Leichtag, G. (2017). Efficacy of cannabis-based medicines for

pain management: A systematic review and meta-analysis of randomized

controlled trials. Pain Physician, 20(6), E755-E796.

Banerjee, N., Bhattacharya, R., Gorczyca, M., Collins, K. M., & Francis, M. M. (2017).

Local neuropeptide signaling modulates serotonergic transmission to shape the

temporal organization of C. elegans egg-laying behavior. PLoS Genet, 13(4),

e1006697. doi:10.1371/journal.pgen.1006697

Banerjee, S., Hsieh, Y. J., Liu, C. R., Yeh, N. H., Hung, H. H., Lai, Y. S., . . . Pan, C. Y.

(2016). Differential releases of dopamine and neuropeptide y from histamine-

86 stimulated PC12 cells detected by an aptamer-modified nanowire transistor. Small,

12(40), 5524-5529. doi:10.1002/smll.201601370

Barnstedt, O., Owald, D., Felsenberg, J., Brain, R., Moszynski, J. P., Talbot, C. B., . . .

Waddell, S. (2016). Memory-relevant mushroom body output synapses are

cholinergic. Neuron, 89(6), 1237-1247. doi:10.1016/j.neuron.2016.02.015

Belmaker, R. H., & Agam, G. (2008). Major depressive disorder. N Engl J Med, 358(1),

55-68. doi:10.1056/NEJMra073096

Ben Arous, J., Laffont, S., & Chatenay, D. (2009). Molecular and sensory basis of a food

related two-state behavior in C. elegans. PLoS One, 4(10), e7584.

doi:10.1371/journal.pone.0007584

Berglund, E. D., Liu, T., Kong, X., Sohn, J. W., Vong, L., Deng, Z., . . . Elmquist, J. K.

(2014). Melanocortin 4 receptors in autonomic neurons regulate thermogenesis

and glycemia. Nat Neurosci, 17(7), 911-913. doi:10.1038/nn.3737

Bhattacharya, R., Touroutine, D., Barbagallo, B., Climer, J., Lambert, C. M., Clark, C.

M., . . . Francis, M. M. (2014). A conserved dopamine- signaling

pathway shapes context-dependent Caenorhabditis elegans behavior. PLoS

Genet, 10(8), e1004584. doi:10.1371/journal.pgen.1004584

Birse, R. T., Johnson, E. C., Taghert, P. H., & Nassel, D. R. (2006). Widely distributed

Drosophila G-protein-coupled receptor (CG7887) is activated by endogenous

tachykinin-related peptides. J Neurobiol, 66(1), 33-46. doi:10.1002/neu.20189

Blanco, E. H., Lagos, C. F., Andres, M. E., & Gysling, K. (2013). An amphipathic alpha-

helix in the prodomain of cocaine and amphetamine regulated transcript peptide

87 precursor serves as its sorting signal to the regulated secretory pathway. PLoS

One, 8(3), e59695. doi:10.1371/journal.pone.0059695

Blazquez, M., & Shennan, K. I. (2000). Basic mechanisms of secretion: sorting into the

regulated secretory pathway. Biochem Cell Biol, 78(3), 181-191.

Blier, P., & El Mansari, M. (2013). Serotonin and beyond: therapeutics for major

depression. Philos Trans R Soc Lond B Biol Sci, 368(1615), 20120536.

doi:10.1098/rstb.2012.0536

Borowsky, B., Adham, N., Jones, K. A., Raddatz, R., Artymyshyn, R., Ogozalek, K. L., .

. . Gerald, C. (2001). Trace amines: identification of a family of mammalian G

protein-coupled receptors. Proc Natl Acad Sci U S A, 98(16), 8966-8971.

doi:10.1073/pnas.151105198

Bradley, J., Reisert, J., & Frings, S. (2005). Regulation of cyclic nucleotide-gated

channels. Curr Opin Neurobiol, 15(3), 343-349. doi:10.1016/j.conb.2005.05.014

Bradley, L. H., Fuqua, J., Richardson, A., Turchan-Cholewo, J., Ai, Y., Kelps, K. A., . . .

Gerhardt, G. A. (2010). Dopamine neuron stimulating actions of a GDNF

propeptide. PLoS One, 5(3), e9752. doi:10.1371/journal.pone.0009752

Brakch, N., Allemandou, F., Cavadas, C., Grouzmann, E., & Brunner, H. R. (2002).

Dibasic cleavage site is required for sorting to the regulated secretory pathway for

both pro- and neuropeptide Y. J Neurochem, 81(6), 1166-1175.

Brechler, V., Chu, W. N., Baxter, J. D., Thibault, G., & Reudelhuber, T. L. (1996). A

protease processing site is essential for prorenin sorting to the regulated secretory

pathway. J Biol Chem, 271(34), 20636-20640.

Brenner, S. (1974). The genetics of Caenorhabditis elegans. Genetics, 77(1), 71-94.

88 Brunetti, L., Orlando, G., Ferrante, C., Chiavaroli, A., & Vacca, M. (2005). Peptide YY

(3 -36) inhibits dopamine and norepinephrine release in the hypothalamus. Eur J

Pharmacol, 519(1-2), 48-51. doi:10.1016/j.ejphar.2005.07.008

Burns, C. M., Chu, H., Rueter, S. M., Hutchinson, L. K., Canton, H., Sanders-Bush, E., &

Emeson, R. B. (1997). Regulation of serotonin-2C receptor G-protein coupling by

RNA editing. Nature, 387(6630), 303-308. doi:10.1038/387303a0

Campbell, J. C., Chin-Sang, I. D., & Bendena, W. G. (2015). Mechanosensation circuitry

in Caenorhabditis elegans: A focus on gentle touch. Peptides, 68, 164-174.

doi:10.1016/j.peptides.2014.12.004

Cao, G., Gardner, A., & Westfall, T. C. (2007). Mechanism of dopamine mediated

inhibition of neuropeptide Y release from pheochromocytoma cells (PC12 cells).

Biochem Pharmacol, 73(9), 1446-1454. doi:10.1016/j.bcp.2007.01.003

Carnell, L., Illi, J., Hong, S. W., & McIntire, S. L. (2005). The G-protein-coupled

serotonin receptor SER-1 regulates egg laying and male mating behaviors in

Caenorhabditis elegans. J Neurosci, 25(46), 10671-10681.

doi:10.1523/JNEUROSCI.3399-05.2005

Carpenter, R. E., Korzan, W. J., Bockholt, C., Watt, M. J., Forster, G. L., Renner, K. J., &

Summers, C. H. (2009). Corticotropin releasing factor influences aggression and

monoamines: modulation of attacks and retreats. Neuroscience, 158(2), 412-425.

doi:10.1016/j.neuroscience.2008.10.014

Carr, G. V., & Lucki, I. (2011). The role of serotonin receptor subtypes in treating

depression: a review of animal studies. Psychopharmacology (Berl), 213(2-3),

265-287. doi:10.1007/s00213-010-2097-z

89 Carre-Pierrat, M., Baillie, D., Johnsen, R., Hyde, R., Hart, A., Granger, L., & Segalat, L.

(2006). Characterization of the Caenorhabditis elegans G protein-coupled

serotonin receptors. Invert Neurosci, 6(4), 189-205. doi:10.1007/s10158-006-

0033-z

Cawley, N. X., Wetsel, W. C., Murthy, S. R., Park, J. J., Pacak, K., & Loh, Y. P. (2012).

New roles of carboxypeptidase E in endocrine and neural function and cancer.

Endocr Rev, 33(2), 216-253. doi:10.1210/er.2011-1039

Chalasani, S. H., Kato, S., Albrecht, D. R., Nakagawa, T., Abbott, L. F., & Bargmann, C.

I. (2010). Neuropeptide feedback modifies odor-evoked dynamics in

Caenorhabditis elegans olfactory neurons. Nat Neurosci, 13(5), 615-621.

doi:10.1038/nn.2526

Challis, B. G., Pinnock, S. B., Coll, A. P., Carter, R. N., Dickson, S. L., & O'Rahilly, S.

(2003). Acute effects of PYY3-36 on food intake and hypothalamic neuropeptide

expression in the mouse. Biochem Biophys Res Commun, 311(4), 915-919.

Chang, Y. J., Burton, T., Ha, L., Huang, Z., Olajubelo, A., & Li, C. (2015). Modulation

of locomotion and reproduction by FLP neuropeptides in the nematode

Caenorhabditis elegans. PLoS One, 10(9), e0135164.

doi:10.1371/journal.pone.0135164

Chao, M. Y., Komatsu, H., Fukuto, H. S., Dionne, H. M., & Hart, A. C. (2004). Feeding

status and serotonin rapidly and reversibly modulate a Caenorhabditis elegans

chemosensory circuit. Proc Natl Acad Sci U S A, 101(43), 15512-15517.

doi:10.1073/pnas.0403369101

90 Charbogne, P., Kieffer, B. L., & Befort, K. (2014). 15 years of genetic approaches in vivo

for addiction research: and peptide gene knockout in mouse

models of drug abuse. Neuropharmacology, 76 Pt B, 204-217.

doi:10.1016/j.neuropharm.2013.08.028

Chase, D. L., Pepper, J. S., & Koelle, M. R. (2004). Mechanism of extrasynaptic

dopamine signaling in Caenorhabditis elegans. Nat Neurosci, 7(10), 1096-1103.

doi:10.1038/nn1316

Chauveau, F., Lange, M. D., Jungling, K., Lesting, J., Seidenbecher, T., & Pape, H. C.

(2012). Prevention of stress-impaired fear extinction through neuropeptide s

action in the lateral amygdala. Neuropsychopharmacology, 37(7), 1588-1599.

doi:10.1038/npp.2012.3

Chen, D., Taylor, K. P., Hall, Q., & Kaplan, J. M. (2016). The neuropeptides FLP-2 and

PDF-1 act in concert to arouse Caenorhabditis elegans locomotion. Genetics,

204(3), 1151-1159. doi:10.1534/genetics.116.192898

Cheong, M. C., Artyukhin, A. B., You, Y. J., & Avery, L. (2015). An opioid-like system

regulating feeding behavior in C. elegans. Elife, 4. doi:10.7554/eLife.06683

Chernoloz, O., El Mansari, M., & Blier, P. (2009). Sustained administration of

pramipexole modifies the spontaneous firing of dopamine, norepinephrine, and

serotonin neurons in the rat brain. Neuropsychopharmacology, 34(3), 651-661.

doi:10.1038/npp.2008.114

Chernoloz, O., El Mansari, M., & Blier, P. (2012). Long-term administration of the

dopamine D3/2 receptor agonist pramipexole increases dopamine and serotonin

91 neurotransmission in the male rat forebrain. J Psychiatry Neurosci, 37(2), 113-

121. doi:10.1503/jpn.110038

Choi, S., Chatzigeorgiou, M., Taylor, K. P., Schafer, W. R., & Kaplan, J. M. (2013).

Analysis of NPR-1 reveals a circuit mechanism for behavioral quiescence in C.

elegans. Neuron, 78(5), 869-880. doi:10.1016/j.neuron.2013.04.002

Choi, S., Taylor, K. P., Chatzigeorgiou, M., Hu, Z., Schafer, W. R., & Kaplan, J. M.

(2015). Sensory neurons arouse C. elegans locomotion via both glutamate and

neuropeptide release. PLoS Genet, 11(7), e1005359.

doi:10.1371/journal.pgen.1005359

Christianson, J. P., Ragole, T., Amat, J., Greenwood, B. N., Strong, P. V., Paul, E. D., . . .

Maier, S. F. (2010). 5-hydroxytryptamine 2C receptors in the basolateral

amygdala are involved in the expression of anxiety after uncontrollable traumatic

stress. Biol Psychiatry, 67(4), 339-345. doi:10.1016/j.biopsych.2009.09.011

Churgin, M. A., McCloskey, R. J., Peters, E., & Fang-Yen, C. (2017). Antagonistic

serotonergic and octopaminergic neural circuits mediate food-dependent

locomotory behavior in Caenorhabditis elegans. J Neurosci, 37(33), 7811-7823.

doi:10.1523/JNEUROSCI.2636-16.2017

Clapper, J. R., Moreno-Sanz, G., Russo, R., Guijarro, A., Vacondio, F., Duranti, A., . . .

Piomelli, D. (2010). Anandamide suppresses pain initiation through a peripheral

endocannabinoid mechanism. Nat Neurosci, 13(10), 1265-1270.

doi:10.1038/nn.2632

92 Cohn, R., Morantte, I., & Ruta, V. (2015). Coordinated and Compartmentalized

Neuromodulation Shapes Sensory Processing in Drosophila. Cell, 163(7), 1742-

1755. doi:10.1016/j.cell.2015.11.019

Collins, K. M., Bode, A., Fernandez, R. W., Tanis, J. E., Brewer, J. C., Creamer, M. S., &

Koelle, M. R. (2016). Activity of the C. elegans egg-laying behavior circuit is

controlled by competing activation and feedback inhibition. Elife, 5.

doi:10.7554/eLife.21126

Cool, D. R., Fenger, M., Snell, C. R., & Loh, Y. P. (1995). Identification of the sorting

signal motif within pro-opiomelanocortin for the regulated secretory pathway. J

Biol Chem, 270(15), 8723-8729.

Cool, D. R., & Loh, Y. P. (1994). Identification of a sorting signal for the regulated

secretory pathway at the N-terminus of pro-opiomelanocortin. Biochimie, 76(3-4),

265-270.

Cool, D. R., Normant, E., Shen, F., Chen, H. C., Pannell, L., Zhang, Y., & Loh, Y. P.

(1997). Carboxypeptidase E is a regulated secretory pathway sorting receptor:

genetic obliteration leads to endocrine disorders in Cpe(fat) mice. Cell, 88(1), 73-

83.

Courel, M., Vasquez, M. S., Hook, V. Y., Mahata, S. K., & Taupenot, L. (2008). Sorting

of the neuroendocrine secretory protein Secretogranin II into the regulated

secretory pathway: role of N- and C-terminal alpha-helical domains. J Biol Chem,

283(17), 11807-11822. doi:10.1074/jbc.M709832200

Crump, J. G., Zhen, M., Jin, Y., & Bargmann, C. I. (2001). The SAD-1 kinase regulates

presynaptic vesicle clustering and axon termination. Neuron, 29(1), 115-129.

93 Cunningham, K. A., Bouagnon, A. D., Barros, A. G., Lin, L., Malard, L., Romano-Silva,

M. A., & Ashrafi, K. (2014). Loss of a neural AMP-activated kinase mimics the

effects of elevated serotonin on fat, movement, and hormonal secretions. PLoS

Genet, 10(6), e1004394. doi:10.1371/journal.pgen.1004394 de Bree, F. M., Knight, D., Howell, L., & Murphy, D. (2000). Sorting of the vasopressin

prohormone into the regulated secretory pathway. FEBS Lett, 475(3), 175-180. de Wit, J., Toonen, R. F., & Verhage, M. (2009). Matrix-dependent local retention of

secretory vesicle cargo in cortical neurons. J Neurosci, 29(1), 23-37.

doi:10.1523/JNEUROSCI.3931-08.2009

Demaurex, N., Furuya, W., D'Souza, S., Bonifacino, J. S., & Grinstein, S. (1998).

Mechanism of acidification of the trans-Golgi network (TGN). In situ

measurements of pH using retrieval of TGN38 and furin from the cell surface. J

Biol Chem, 273(4), 2044-2051.

Dernovici, S., Starc, T., Dent, J. A., & Ribeiro, P. (2007). The serotonin receptor SER-1

(5HT2ce) contributes to the regulation of locomotion in Caenorhabditis elegans.

Dev Neurobiol, 67(2), 189-204. doi:10.1002/dneu.20340

Dikeakos, J. D., Di Lello, P., Lacombe, M. J., Ghirlando, R., Legault, P., Reudelhuber, T.

L., & Omichinski, J. G. (2009). Functional and structural characterization of a

dense core secretory granule sorting domain from the PC1/3 protease. Proc Natl

Acad Sci U S A, 106(18), 7408-7413. doi:10.1073/pnas.0809576106

Dikeakos, J. D., Lacombe, M. J., Mercure, C., Mireuta, M., & Reudelhuber, T. L. (2007).

A hydrophobic patch in a charged alpha-helix is sufficient to target proteins to

94 dense core secretory granules. J Biol Chem, 282(2), 1136-1143.

doi:10.1074/jbc.M605718200

Dikeakos, J. D., & Reudelhuber, T. L. (2007). Sending proteins to dense core secretory

granules: still a lot to sort out. J Cell Biol, 177(2), 191-196.

doi:10.1083/jcb.200701024

Donnelly, J. L., Clark, C. M., Leifer, A. M., Pirri, J. K., Haburcak, M., Francis, M. M., . .

. Alkema, M. J. (2013). Monoaminergic orchestration of motor programs in a

complex C. elegans behavior. PLoS Biol, 11(4), e1001529.

doi:10.1371/journal.pbio.1001529

El Meskini, R., Jin, L., Marx, R., Bruzzaniti, A., Lee, J., Emeson, R., & Mains, R. (2001).

A signal sequence is sufficient for green fluorescent protein to be routed to

regulated secretory granules. Endocrinology, 142(2), 864-873.

doi:10.1210/endo.142.2.7929

Emtage, L., Aziz-Zaman, S., Padovan-Merhar, O., Horvitz, H. R., Fang-Yen, C., &

Ringstad, N. (2012). IRK-1 potassium channels mediate peptidergic inhibition of

Caenorhabditis elegans serotonin neurons via a G(o) signaling pathway. J

Neurosci, 32(46), 16285-16295. doi:10.1523/JNEUROSCI.2667-12.2012

Esposito, G., Di Schiavi, E., Bergamasco, C., & Bazzicalupo, P. (2007). Efficient and cell

specific knock-down of gene function in targeted C. elegans neurons. Gene,

395(1-2), 170-176. doi:10.1016/j.gene.2007.03.002

Euler, T., Detwiler, P. B., & Denk, W. (2002). Directionally selective calcium signals in

dendrites of starburst amacrine cells. Nature, 418(6900), 845-852.

doi:10.1038/nature00931

95 Eyun, S. I., Moriyama, H., Hoffmann, F. G., & Moriyama, E. N. (2016). Molecular

evolution and functional divergence of trace amine-associated receptors. PLoS

One, 11(3), e0151023. doi:10.1371/journal.pone.0151023

Ezak, M. J., & Ferkey, D. M. (2010). The C. elegans D2-like dopamine receptor DOP-3

decreases behavioral sensitivity to the olfactory stimulus 1-octanol. PLoS One,

5(3), e9487. doi:10.1371/journal.pone.0009487

Ezcurra, M., Tanizawa, Y., Swoboda, P., & Schafer, W. R. (2011). Food sensitizes C.

elegans avoidance behaviours through acute dopamine signalling. EMBO J, 30(6),

1110-1122. doi:10.1038/emboj.2011.22

Ezcurra, M., Walker, D. S., Beets, I., Swoboda, P., & Schafer, W. R. (2016).

Neuropeptidergic signaling and active feeding state inhibit nociception in

Caenorhabditis elegans. J Neurosci, 36(11), 3157-3169.

doi:10.1523/JNEUROSCI.1128-15.2016

Farina, M., van de Bospoort, R., He, E., Persoon, C. M., van Weering, J. R., Broeke, J.

H., . . . Toonen, R. F. (2015). CAPS-1 promotes fusion competence of stationary

dense-core vesicles in presynaptic terminals of mammalian neurons. Elife, 4.

doi:10.7554/eLife.05438

Felton, C. M., & Johnson, C. M. (2011). Modulation of dopamine-dependent behaviors

by the Caenorhabditis elegans Olig homolog HLH-17. J Neurosci Res, 89(10),

1627-1636. doi:10.1002/jnr.22694

Flavell, S. W., Pokala, N., Macosko, E. Z., Albrecht, D. R., Larsch, J., & Bargmann, C. I.

(2013). Serotonin and the neuropeptide PDF initiate and extend opposing

96 behavioral states in C. elegans. Cell, 154(5), 1023-1035.

doi:10.1016/j.cell.2013.08.001

Fuenzalida, L. C., Keen, K. L., & Terasawa, E. (2011). Colocalization of FM1-43,

Bassoon, and GnRH-1: GnRH-1 release from cell bodies and their

neuroprocesses. Endocrinology, 152(11), 4310-4321. doi:10.1210/en.2011-1416

Gallagher, T., Kim, J., Oldenbroek, M., Kerr, R., & You, Y. J. (2013). ASI regulates

satiety quiescence in C. elegans. J Neurosci, 33(23), 9716-9724.

doi:10.1523/JNEUROSCI.4493-12.2013

Gardella, E., Romei, C., Cavallero, A., Trapella, C., Fedele, E., & Raiteri, L. (2013).

Neuropeptide S inhibits release of 5-HT and glycine in mouse amygdala and

frontal/prefrontal cortex through activation of the neuropeptide S receptor.

Neurochem Int, 62(4), 360-366. doi:10.1016/j.neuint.2013.02.003

Garfield, A. S., Li, C., Madara, J. C., Shah, B. P., Webber, E., Steger, J. S., . . . Lowell,

B. B. (2015). A neural basis for melanocortin-4 receptor-regulated appetite. Nat

Neurosci, 18(6), 863-871. doi:10.1038/nn.4011

Garrison, J. L., Macosko, E. Z., Bernstein, S., Pokala, N., Albrecht, D. R., & Bargmann,

C. I. (2012). Oxytocin/vasopressin-related peptides have an ancient role in

reproductive behavior. Science, 338(6106), 540-543.

doi:10.1126/science.1226201

Ghosh, D. D., Sanders, T., Hong, S., McCurdy, L. Y., Chase, D. L., Cohen, N., . . .

Nitabach, M. N. (2016). Neural architecture of hunger-dependent multisensory

decision making in C. elegans. Neuron, 92(5), 1049-1062.

doi:10.1016/j.neuron.2016.10.030

97 Gjorgjieva, J., Biron, D., & Haspel, G. (2014). Neurobiology of Caenorhabditis elegans

locomotion: Where do we stand? Bioscience, 64(6), 476-486.

doi:10.1093/biosci/biu058

Glombik, M. M., Kromer, A., Salm, T., Huttner, W. B., & Gerdes, H. H. (1999). The

disulfide-bonded loop of chromogranin B mediates membrane binding and directs

sorting from the trans-Golgi network to secretory granules. EMBO J, 18(4), 1059-

1070. doi:10.1093/emboj/18.4.1059

Godinho, R. O., Duarte, T., & Pacini, E. S. (2015). New perspectives in signaling

mediated by receptors coupled to stimulatory G protein: the emerging significance

of cAMP efflux and extracellular cAMP-adenosine pathway. Front Pharmacol, 6,

58. doi:10.3389/fphar.2015.00058

Gray, J. M., Hill, J. J., & Bargmann, C. I. (2005). A circuit for navigation in

Caenorhabditis elegans. Proc Natl Acad Sci U S A, 102(9), 3184-3191.

doi:10.1073/pnas.0409009101

Greene, J. S., Dobosiewicz, M., Butcher, R. A., McGrath, P. T., & Bargmann, C. I.

(2016). Regulatory changes in two chemoreceptor genes contribute to a

Caenorhabditis elegans QTL for foraging behavior. Elife, 5.

doi:10.7554/eLife.21454

Greer, E. R., Perez, C. L., Van Gilst, M. R., Lee, B. H., & Ashrafi, K. (2008). Neural and

molecular dissection of a C. elegans sensory circuit that regulates fat and feeding.

Cell Metab, 8(2), 118-131. doi:10.1016/j.cmet.2008.06.005

Guo, M., Wu, T. H., Song, Y. X., Ge, M. H., Su, C. M., Niu, W. P., . . . Wu, Z. X. (2015).

Reciprocal inhibition between sensory ASH and ASI neurons modulates

98 nociception and avoidance in Caenorhabditis elegans. Nat Commun, 6, 5655.

doi:10.1038/ncomms6655

Gurel, G., Gustafson, M. A., Pepper, J. S., Horvitz, H. R., & Koelle, M. R. (2012).

Receptors and other signaling proteins required for serotonin control of

locomotion in Caenorhabditis elegans. Genetics, 192(4), 1359-1371.

doi:10.1534/genetics.112.142125

Hammarlund, M., Watanabe, S., Schuske, K., & Jorgensen, E. M. (2008). CAPS and

syntaxin dock dense core vesicles to the plasma membrane in neurons. J Cell

Biol, 180(3), 483-491. doi:10.1083/jcb.200708018

Hannon, J., & Hoyer, D. (2008). Molecular biology of 5-HT receptors. Behav Brain Res,

195(1), 198-213. doi:10.1016/j.bbr.2008.03.020

Hapiak, V., Summers, P., Ortega, A., Law, W. J., Stein, A., & Komuniecki, R. (2013).

Neuropeptides amplify and focus the monoaminergic inhibition of nociception in

Caenorhabditis elegans. J Neurosci, 33(35), 14107-14116.

doi:10.1523/JNEUROSCI.1324-13.2013

Hapiak, V. M., Hobson, R. J., Hughes, L., Smith, K., Harris, G., Condon, C., . . .

Komuniecki, R. W. (2009). Dual excitatory and inhibitory serotonergic inputs

modulate egg laying in Caenorhabditis elegans. Genetics, 181(1), 153-163.

doi:10.1534/genetics.108.096891

Harris, G., Korchnak, A., Summers, P., Hapiak, V., Law, W. J., Stein, A. M., . . .

Komuniecki, R. (2011). Dissecting the serotonergic food signal stimulating

sensory-mediated aversive behavior in C. elegans. PLoS One, 6(7), e21897.

doi:10.1371/journal.pone.0021897

99 Harris, G., Mills, H., Wragg, R., Hapiak, V., Castelletto, M., Korchnak, A., &

Komuniecki, R. W. (2010). The monoaminergic modulation of sensory-mediated

aversive responses in Caenorhabditis elegans requires glutamatergic/peptidergic

cotransmission. J Neurosci, 30(23), 7889-7899. doi:10.1523/JNEUROSCI.0497-

10.2010

Harris, G. P., Hapiak, V. M., Wragg, R. T., Miller, S. B., Hughes, L. J., Hobson, R. J., . . .

Komuniecki, R. W. (2009). Three distinct amine receptors operating at different

levels within the locomotory circuit are each essential for the serotonergic

modulation of chemosensation in Caenorhabditis elegans. J Neurosci, 29(5),

1446-1456. doi:10.1523/JNEUROSCI.4585-08.2009

Hayashi, S., Ueda, M., Amaya, F., Matusda, T., Tamada, Y., Ibata, Y., & Tanaka, M.

(2001). Serotonin modulates expression of VIP and GRP mRNA via the 5-

HT(1B) receptor in the suprachiasmatic nucleus of the rat. Exp Neurol, 171(2),

285-292. doi:10.1006/exnr.2001.7759

Heisler, L. K., Cowley, M. A., Kishi, T., Tecott, L. H., Fan, W., Low, M. J., . . .

Elmquist, J. K. (2003). Central serotonin and melanocortin pathways regulating

energy homeostasis. Ann N Y Acad Sci, 994, 169-174.

Heisler, L. K., Cowley, M. A., Tecott, L. H., Fan, W., Low, M. J., Smart, J. L., . . .

Elmquist, J. K. (2002). Activation of central melanocortin pathways by

fenfluramine. Science, 297(5581), 609-611. doi:10.1126/science.1072327

Heisler, L. K., Jobst, E. E., Sutton, G. M., Zhou, L., Borok, E., Thornton-Jones, Z., . . .

Cowley, M. A. (2006). Serotonin reciprocally regulates melanocortin neurons to

modulate food intake. Neuron, 51(2), 239-249. doi:10.1016/j.neuron.2006.06.004

100 Hendricks, M., Ha, H., Maffey, N., & Zhang, Y. (2012). Compartmentalized calcium

dynamics in a C. elegans interneuron encode head movement. Nature, 487(7405),

99-103. doi:10.1038/nature11081

Heng, B. C., Aubel, D., & Fussenegger, M. (2013). An overview of the diverse roles of

G-protein coupled receptors (GPCRs) in the pathophysiology of various human

diseases. Biotechnol Adv, 31(8), 1676-1694.

doi:10.1016/j.biotechadv.2013.08.017

Hobert, O. (2002). PCR fusion-based approach to create reporter gene constructs for

expression analysis in transgenic C. elegans. Biotechniques, 32(4), 728-730.

Hobert, O. (2013). The neuronal genome of Caenorhabditis elegans. WormBook, 1-106.

doi:10.1895/wormbook.1.161.1

Hobert, O. (2014). Development of left/right asymmetry in the Caenorhabditis elegans

nervous system: from zygote to postmitotic neuron. Genesis, 52(6), 528-543.

doi:10.1002/dvg.22747

Hobson, R. J., Hapiak, V. M., Xiao, H., Buehrer, K. L., Komuniecki, P. R., &

Komuniecki, R. W. (2006). SER-7, a Caenorhabditis elegans 5-HT7-like

receptor, is essential for the 5-HT stimulation of pharyngeal pumping and egg

laying. Genetics, 172(1), 159-169. doi:10.1534/genetics.105.044495

Holsboer, F. (2000). The corticosteroid receptor hypothesis of depression.

Neuropsychopharmacology, 23(5), 477-501. doi:10.1016/S0893-133X(00)00159-

7

Hook, V., Funkelstein, L., Lu, D., Bark, S., Wegrzyn, J., & Hwang, S. R. (2008).

Proteases for processing proneuropeptides into peptide neurotransmitters and

101 hormones. Annu Rev Pharmacol Toxicol, 48, 393-423.

doi:10.1146/annurev.pharmtox.48.113006.094812

Horvitz, H. R., Chalfie, M., Trent, C., Sulston, J. E., & Evans, P. D. (1982). Serotonin

and octopamine in the nematode Caenorhabditis elegans. Science, 216(4549),

1012-1014.

Hu, Z., Pym, E. C., Babu, K., Vashlishan Murray, A. B., & Kaplan, J. M. (2011). A

neuropeptide-mediated stretch response links muscle contraction to changes in

neurotransmitter release. Neuron, 71(1), 92-102.

doi:10.1016/j.neuron.2011.04.021

Huang, Y., & Thathiah, A. (2015). Regulation of neuronal communication by G protein-

coupled receptors. FEBS Lett, 589(14), 1607-1619.

doi:10.1016/j.febslet.2015.05.007

Husson, S. J., Clynen, E., Boonen, K., Janssen, T., Lindemans, M., Baggerman, G., &

Schoofs, L. (2010). Approaches to identify endogenous peptides in the soil

nematode Caenorhabditis elegans. Methods Mol Biol, 615, 29-47.

doi:10.1007/978-1-60761-535-4_3

Ihnatko, R., & Theodorsson, E. (2017). Short N-terminal galanin fragments are occurring

naturally in vivo. Neuropeptides, 63, 1-13. doi:10.1016/j.npep.2017.03.005

Irminger, J. C., Verchere, C. B., Meyer, K., & Halban, P. A. (1997). Proinsulin targeting

to the regulated pathway is not impaired in carboxypeptidase E-deficient

Cpefat/Cpefat mice. J Biol Chem, 272(44), 27532-27534.

102 Irminger, J. C., Vollenweider, F. M., Neerman-Arbez, M., & Halban, P. A. (1994).

Human proinsulin conversion in the regulated and the constitutive pathways of

transfected AtT20 cells. J Biol Chem, 269(3), 1756-1762.

Isaac, R. E., Bland, N. D., & Shirras, A. D. (2009). Neuropeptidases and the metabolic

inactivation of insect neuropeptides. Gen Comp Endocrinol, 162(1), 8-17.

doi:10.1016/j.ygcen.2008.12.011

Jin, X., Shah, S., Liu, Y., Zhang, H., Lees, M., Fu, Z., . . . Gamper, N. (2013). Activation

of the Cl- channel ANO1 by localized calcium signals in nociceptive sensory

neurons requires coupling with the IP3 receptor. Sci Signal, 6(290), ra73.

doi:10.1126/scisignal.2004184

Johard, H. A., Muren, J. E., Nichols, R., Larhammar, D. S., & Nassel, D. R. (2001). A

putative in the cockroach brain: molecular cloning and

analysis of expression by means of antisera to portions of the receptor protein.

Brain Res, 919(1), 94-105.

Jose, A. M., Bany, I. A., Chase, D. L., & Koelle, M. R. (2007). A specific subset of

transient receptor potential vanilloid-type channel subunits in Caenorhabditis

elegans endocrine cells function as mixed heteromers to promote neurotransmitter

release. Genetics, 175(1), 93-105. doi:10.1534/genetics.106.065516

Karhunen, T., Vilim, F. S., Alexeeva, V., Weiss, K. R., & Church, P. J. (2001). Targeting

of peptidergic vesicles in cotransmitting terminals. J Neurosci, 21(3), RC127.

Kienzle, C., & von Blume, J. (2014). Secretory cargo sorting at the trans-Golgi network.

Trends Cell Biol, 24(10), 584-593. doi:10.1016/j.tcb.2014.04.007

103 Kim, T., Gondre-Lewis, M. C., Arnaoutova, I., & Loh, Y. P. (2006). Dense-core

secretory granule biogenesis. Physiology (Bethesda), 21, 124-133.

doi:10.1152/physiol.00043.2005

Kirkpatrick, D. R., McEntire, D. M., Hambsch, Z. J., Kerfeld, M. J., Smith, T. A.,

Reisbig, M. D., . . . Agrawal, D. K. (2015). Therapeutic basis of clinical pain

modulation. Clin Transl Sci, 8(6), 848-856. doi:10.1111/cts.12282

Kis, G. K., Ocsko, T., Galfi, M., Radacs, M., Molnar, Z., Rakosi, K., . . . Laszlo, F. A.

(2011). The effects of on monoaminerg-induced changes in vasopressin

level in rat neurohypophyseal cell cultures. Neuropeptides, 45(6), 385-389.

doi:10.1016/j.npep.2011.08.001

Knezevic, N. N., Yekkirala, A., & Yaksh, T. L. (2017). Basic/translational development

of forthcoming opioid- and nonopioid-targeted pain therapeutics. Anesth Analg,

125(5), 1714-1732. doi:10.1213/ANE.0000000000002442

Koelle, M. R. (2016). Neurotransmitter signaling through heterotrimeric G proteins:

insights from studies in C. elegans. WormBook, 1-78.

doi:10.1895/wormbook.1.75.2

Kohler, S., Cierpinsky, K., Kronenberg, G., & Adli, M. (2016). The serotonergic system

in the neurobiology of depression: Relevance for novel antidepressants. J

Psychopharmacol, 30(1), 13-22. doi:10.1177/0269881115609072

Komuniecki, R., Hapiak, V., Harris, G., & Bamber, B. (2014). Context-dependent

modulation reconfigures interactive sensory-mediated microcircuits in

Caenorhabditis elegans. Curr Opin Neurobiol, 29, 17-24.

doi:10.1016/j.conb.2014.04.006

104 Komuniecki, R., Law, W. J., Jex, A., Geldhof, P., Gray, J., Bamber, B., & Gasser, R. B.

(2012). Monoaminergic signaling as a target for anthelmintic drug discovery:

receptor conservation among the free-living and parasitic nematodes. Mol

Biochem Parasitol, 183(1), 1-7. doi:10.1016/j.molbiopara.2012.02.001

Komuniecki, R. W., Hobson, R. J., Rex, E. B., Hapiak, V. M., & Komuniecki, P. R.

(2004). Biogenic amine receptors in parasitic nematodes: what can be learned

from Caenorhabditis elegans? Mol Biochem Parasitol, 137(1), 1-11.

doi:10.1016/j.molbiopara.2004.05.010

Koren, D., Grove, J. C. R., & Wei, W. (2017). Cross-compartmental modulation of

dendritic signals for retinal direction selectivity. Neuron, 95(4), 914-927 e914.

doi:10.1016/j.neuron.2017.07.020

Kourtesis, I., Kasparov, S., Verkade, P., & Teschemacher, A. G. (2015). Ultrastructural

correlates of enhanced norepinephrine and neuropeptide y cotransmission in the

spontaneously hypertensive rat brain. ASN Neuro, 7(5).

doi:10.1177/1759091415610115

Krashes, M. J., Lowell, B. B., & Garfield, A. S. (2016). Melanocortin-4 receptor-

regulated energy homeostasis. Nat Neurosci, 19(2), 206-219. doi:10.1038/nn.4202

Kromer, A., Glombik, M. M., Huttner, W. B., & Gerdes, H. H. (1998). Essential role of

the disulfide-bonded loop of chromogranin B for sorting to secretory granules is

revealed by expression of a deletion mutant in the absence of endogenous granin

synthesis. J Cell Biol, 140(6), 1331-1346.

Kullyev, A., Dempsey, C. M., Miller, S., Kuan, C. J., Hapiak, V. M., Komuniecki, R. W.,

. . . Sze, J. Y. (2010). A genetic survey of fluoxetine action on synaptic

105 transmission in Caenorhabditis elegans. Genetics, 186(3), 929-941.

doi:10.1534/genetics.110.118877

Kushner, R. F., & Kahan, S. (2018). Introduction: The state of obesity in 2017. Med Clin

North Am, 102(1), 1-11. doi:10.1016/j.mcna.2017.08.003

Lacombe, M. J., Mercure, C., Dikeakos, J. D., & Reudelhuber, T. L. (2005). Modulation

of secretory granule-targeting efficiency by cis and trans compounding of sorting

signals. J Biol Chem, 280(6), 4803-4807. doi:10.1074/jbc.M408658200

Lam, D. D., Przydzial, M. J., Ridley, S. H., Yeo, G. S., Rochford, J. J., O'Rahilly, S., &

Heisler, L. K. (2008). Serotonin 5-HT2C receptor agonist promotes hypophagia

via downstream activation of melanocortin 4 receptors. Endocrinology, 149(3),

1323-1328. doi:10.1210/en.2007-1321

Laurent, P., Soltesz, Z., Nelson, G. M., Chen, C., Arellano-Carbajal, F., Levy, E., & de

Bono, M. (2015). Decoding a neural circuit controlling global animal state in C.

elegans. Elife, 4. doi:10.7554/eLife.04241

Law, W., Wuescher, L. M., Ortega, A., Hapiak, V. M., Komuniecki, P. R., &

Komuniecki, R. (2015). Heterologous expression in remodeled C. elegans: A

platform for monoaminergic agonist identification and anthelmintic screening.

PLoS Pathog, 11(4), e1004794. doi:10.1371/journal.ppat.1004794

Lehtonen, M., Reisner, K., Auriola, S., Wong, G., & Callaway, J. C. (2008). Mass-

spectrometric identification of anandamide and 2-arachidonoylglycerol in

nematodes. Chem Biodivers, 5(11), 2431-2441. doi:10.1002/cbdv.200890208

Lehtonen, M., Storvik, M., Malinen, H., Hyytia, P., Lakso, M., Auriola, S., . . . Callaway,

J. C. (2011). Determination of endocannabinoids in nematodes and human brain

106 tissue by liquid chromatography electrospray ionization tandem mass

spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci, 879(11-12), 677-

694. doi:10.1016/j.jchromb.2011.02.004

Leinwand, S. G., & Chalasani, S. H. (2013). Neuropeptide signaling remodels

chemosensory circuit composition in Caenorhabditis elegans. Nat Neurosci,

16(10), 1461-1467. doi:10.1038/nn.3511

Li, C., & Kim, K. (2008). Neuropeptides. WormBook, 1-36.

doi:10.1895/wormbook.1.142.1

Li, C., & Kim, K. (2010). Neuropeptide gene families in Caenorhabditis elegans. Adv

Exp Med Biol, 692, 98-137.

Li, C., & Kim, K. (2014). Family of FLP peptides in Caenorhabditis elegans and related

nematodes. Front Endocrinol (Lausanne), 5, 150. doi:10.3389/fendo.2014.00150

Li, C., Timbers, T. A., Rose, J. K., Bozorgmehr, T., McEwan, A., & Rankin, C. H.

(2013a). The FMRFamide-related neuropeptide FLP-20 is required in the

mechanosensory neurons during memory for massed training in C. elegans. Learn

Mem, 20(2), 103-108. doi:10.1101/lm.028993.112

Li, Y., Zhao, Y., Huang, X., Lin, X., Guo, Y., Wang, D., . . . Wang, D. (2013b).

Serotonin control of thermotaxis memory behavior in nematode Caenorhabditis

elegans. PLoS One, 8(11), e77779. doi:10.1371/journal.pone.0077779

Lim, M. A., Chitturi, J., Laskova, V., Meng, J., Findeis, D., Wiekenberg, A., . . . Zhen,

M. (2016). Neuroendocrine modulation sustains the C. elegans forward motor

state. Elife, 5. doi:10.7554/eLife.19887

107 Liu, T., Kim, K., Li, C., & Barr, M. M. (2007). FMRFamide-like neuropeptides and

mechanosensory touch receptor neurons regulate male sexual turning behavior in

Caenorhabditis elegans. J Neurosci, 27(27), 7174-7182.

doi:10.1523/JNEUROSCI.1405-07.2007

Livak, K. J., & Schmittgen, T. D. (2001). Analysis of relative gene expression data using

real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods, 25(4),

402-408. doi:10.1006/meth.2001.1262

Loer, C. M., & Kenyon, C. J. (1993). Serotonin-deficient mutants and male mating

behavior in the nematode Caenorhabditis elegans. J Neurosci, 13(12), 5407-5417.

Loh, Y. P., Maldonado, A., Zhang, C., Tam, W. H., & Cawley, N. (2002). Mechanism of

sorting proopiomelanocortin and proenkephalin to the regulated secretory

pathway of neuroendocrine cells. Ann N Y Acad Sci, 971, 416-425.

Lou, H., Park, J. J., Cawley, N. X., Sarcon, A., Sun, L., Adams, T., & Loh, Y. P. (2010).

Carboxypeptidase E cytoplasmic tail mediates localization of synaptic vesicles to

the pre-active zone in hypothalamic pre-synaptic terminals. J Neurochem, 114(3),

886-896. doi:10.1111/j.1471-4159.2010.06820.x

Lowry, C. A., & Moore, F. L. (2006). Regulation of behavioral responses by

corticotropin-releasing factor. Gen Comp Endocrinol, 146(1), 19-27.

doi:10.1016/j.ygcen.2005.12.006

Luedtke, S., O'Connor, V., Holden-Dye, L., & Walker, R. J. (2010). The regulation of

feeding and metabolism in response to food deprivation in Caenorhabditis

elegans. Invert Neurosci, 10(2), 63-76. doi:10.1007/s10158-010-0112-z

108 Ma, G. Q., Wang, B., Wang, H. B., Wang, Q., & Bao, L. (2008). Short elements with

charged amino acids form clusters to sort protachykinin into large dense-core

vesicles. Traffic, 9(12), 2165-2179. doi:10.1111/j.1600-0854.2008.00836.x

Ma, S., Hangya, B., Leonard, C. S., Wisden, W., & Gundlach, A. L. (2018). Dual-

transmitter systems regulating arousal, attention, learning and memory. Neurosci

Biobehav Rev, 85, 21-33. doi:10.1016/j.neubiorev.2017.07.009

Ma, Z., Guo, X., Lei, H., Li, T., Hao, S., & Kang, L. (2015). Octopamine and tyramine

respectively regulate attractive and repulsive behavior in locust phase changes.

Sci Rep, 5, 8036. doi:10.1038/srep08036

Magalhaes, C. P., de Freitas, M. F., Nogueira, M. I., Campina, R. C., Takase, L. F., de

Souza, S. L., & de Castro, R. M. (2010). Modulatory role of serotonin on feeding

behavior. Nutr Neurosci, 13(6), 246-255.

doi:10.1179/147683010X12611460764723

Mahapatra, N. R., Taupenot, L., Courel, M., Mahata, S. K., & O'Connor, D. T. (2008).

The trans-Golgi proteins SCLIP and SCG10 interact with chromogranin A to

regulate neuroendocrine secretion. Biochemistry, 47(27), 7167-7178.

doi:10.1021/bi7019996

Maier, S. F., & Watkins, L. R. (2005). Stressor controllability and learned helplessness:

the roles of the dorsal raphe nucleus, serotonin, and corticotropin-releasing factor.

Neurosci Biobehav Rev, 29(4-5), 829-841. doi:10.1016/j.neubiorev.2005.03.021

Marion, S., Oakley, R. H., Kim, K. M., Caron, M. G., & Barak, L. S. (2006). A beta-

arrestin binding determinant common to the second intracellular loops of

109 family G protein-coupled receptors. J Biol Chem, 281(5), 2932-2938.

doi:10.1074/jbc.M508074200

Martinez-Rubio, C., Serrano, G. E., & Miller, M. W. (2010). Octopamine promotes

rhythmicity but not synchrony in a bilateral pair of bursting motor neurons in the

feeding circuit of Aplysia. J Exp Biol, 213(Pt 7), 1182-1194.

doi:10.1242/jeb.040378

McCloskey, R. J., Fouad, A. D., Churgin, M. A., & Fang-Yen, C. (2017). Food

responsiveness regulates episodic behavioral states in Caenorhabditis elegans. J

Neurophysiol, 117(5), 1911-1934. doi:10.1152/jn.00555.2016

McGirr, R., Guizzetti, L., & Dhanvantari, S. (2013). The sorting of proglucagon to

secretory granules is mediated by carboxypeptidase E and intrinsic sorting

signals. J Endocrinol, 217(2), 229-240. doi:10.1530/JOE-12-0468

Mello, C., & Fire, A. (1995). DNA transformation. Methods Cell Biol, 48, 451-482.

Mills, H., Hapiak, V., Harris, G., Summers, P., & Komuniecki, R. (2012a). The

interaction of octopamine and neuropeptides to slow aversive responses in C.

elegans mimics the modulation of chronic pain in mammals. Worm, 1(4), 202-

206. doi:10.4161/worm.20467

Mills, H., Ortega, A., Law, W., Hapiak, V., Summers, P., Clark, T., & Komuniecki, R.

(2016). Opiates modulate noxious chemical nociception through a complex

monoaminergic/peptidergic cascade. J Neurosci, 36(20), 5498-5508.

doi:10.1523/JNEUROSCI.4520-15.2016

Mills, H., Wragg, R., Hapiak, V., Castelletto, M., Zahratka, J., Harris, G., . . .

Komuniecki, R. (2012b). Monoamines and neuropeptides interact to inhibit

110 aversive behaviour in Caenorhabditis elegans. EMBO J, 31(3), 667-678.

doi:10.1038/emboj.2011.422

Morvan, J., & Tooze, S. A. (2008). Discovery and progress in our understanding of the

regulated secretory pathway in neuroendocrine cells. Histochem Cell Biol, 129(3),

243-252. doi:10.1007/s00418-008-0377-z

Mulcahy, L. R., Vaslet, C. A., & Nillni, E. A. (2005). Prohormone-convertase 1

processing enhances post-Golgi sorting of prothyrotropin-releasing hormone-

derived peptides. J Biol Chem, 280(48), 39818-39826.

doi:10.1074/jbc.M507193200

Munoz-Llancao, P., Henriquez, D. R., Wilson, C., Bodaleo, F., Boddeke, E. W.,

Lezoualc'h, F., . . . Gonzalez-Billault, C. (2015). Exchange protein directly

activated by cAMP (EPAC) regulates neuronal polarization through Rap1B. J

Neurosci, 35(32), 11315-11329. doi:10.1523/JNEUROSCI.3645-14.2015

Murrough, J. W., Yaqubi, S., Sayed, S., & Charney, D. S. (2015). Emerging drugs for the

treatment of anxiety. Expert Opin Emerg Drugs, 20(3), 393-406.

doi:10.1517/14728214.2015.1049996

Nassel, D. R. (2009). Neuropeptide signaling near and far: how localized and timed is the

action of neuropeptides in brain circuits? Invert Neurosci, 9(2), 57-75.

doi:10.1007/s10158-009-0090-1

Nathoo, A. N., Moeller, R. A., Westlund, B. A., & Hart, A. C. (2001). Identification of

neuropeptide-like protein gene families in Caenorhabditis elegans and other

species. Proc Natl Acad Sci U S A, 98(24), 14000-14005.

doi:10.1073/pnas.241231298

111 Niacaris, T., & Avery, L. (2003). Serotonin regulates repolarization of the C. elegans

pharyngeal muscle. J Exp Biol, 206(Pt 2), 223-231.

Noble, T., Stieglitz, J., & Srinivasan, S. (2013). An integrated serotonin and octopamine

neuronal circuit directs the release of an endocrine signal to control C. elegans

body fat. Cell Metab, 18(5), 672-684. doi:10.1016/j.cmet.2013.09.007

Normant, E., & Loh, Y. P. (1998). Depletion of carboxypeptidase E, a regulated secretory

pathway sorting receptor, causes misrouting and constitutive secretion of

proinsulin and proenkephalin, but not chromogranin A. Endocrinology, 139(4),

2137-2145. doi:10.1210/endo.139.4.5951

Nusbaum, M. P., Blitz, D. M., & Marder, E. (2017). Functional consequences of

neuropeptide and small-molecule co-transmission. Nat Rev Neurosci, 18(7), 389-

403. doi:10.1038/nrn.2017.56

Oakes, M. D., Law, W. J., Clark, T., Bamber, B. A., & Komuniecki, R. (2017).

Cannabinoids activate monoaminergic signaling to modulate key C. elegans

behaviors. J Neurosci, 37(11), 2859-2869. doi:10.1523/JNEUROSCI.3151-

16.2017

Ortiz, C. O., Etchberger, J. F., Posy, S. L., Frokjaer-Jensen, C., Lockery, S., Honig, B., &

Hobert, O. (2006). Searching for neuronal left/right asymmetry: genomewide

analysis of nematode receptor-type guanylyl cyclases. Genetics, 173(1), 131-149.

doi:10.1534/genetics.106.055749

Paeger, L., Karakasilioti, I., Altmuller, J., Frommolt, P., Bruning, J., & Kloppenburg, P.

(2017). Antagonistic modulation of NPY/AgRP and POMC neurons in the arcuate

nucleus by noradrenalin. Elife, 6. doi:10.7554/eLife.25770

112 Palamiuc, L., Noble, T., Witham, E., Ratanpal, H., Vaughan, M., & Srinivasan, S. (2017).

A tachykinin-like neuroendocrine signalling axis couples central serotonin action

and nutrient sensing with peripheral lipid metabolism. Nat Commun, 8, 14237.

doi:10.1038/ncomms14237

Palouzier-Paulignan, B., Lacroix, M. C., Aime, P., Baly, C., Caillol, M., Congar, P., . . .

Fadool, D. A. (2012). Olfaction under metabolic influences. Chem Senses, 37(9),

769-797. doi:10.1093/chemse/bjs059

Park, H. Y., So, S. H., Lee, W. B., You, S. H., & Yoo, S. H. (2002). Purification, pH-

dependent conformational change, aggregation, and secretory granule membrane

binding property of secretogranin II (chromogranin C). Biochemistry, 41(4),

1259-1266.

Park, S. K., Link, C. D., & Johnson, T. E. (2010). Life-span extension by dietary

restriction is mediated by NLP-7 signaling and coelomocyte endocytosis in C.

elegans. FASEB J, 24(2), 383-392. doi:10.1096/fj.09-142984

Park, S. K., Tedesco, P. M., & Johnson, T. E. (2009). Oxidative stress and longevity in

Caenorhabditis elegans as mediated by SKN-1. Aging Cell, 8(3), 258-269.

doi:10.1111/j.1474-9726.2009.00473.x

Paulmann, N., Grohmann, M., Voigt, J. P., Bert, B., Vowinckel, J., Bader, M., . . .

Walther, D. J. (2009). Intracellular serotonin modulates insulin secretion from

pancreatic beta-cells by protein serotonylation. PLoS Biol, 7(10), e1000229.

doi:10.1371/journal.pbio.1000229

113 Perello, M., Stuart, R., & Nillni, E. A. (2008). Prothyrotropin-releasing hormone targets

its processing products to different vesicles of the secretory pathway. J Biol

Chem, 283(29), 19936-19947. doi:10.1074/jbc.M800732200

Perez-Caballero, L., Torres-Sanchez, S., Bravo, L., Mico, J. A., & Berrocoso, E. (2014).

Fluoxetine: a case history of its discovery and preclinical development. Expert

Opin Drug Discov, 9(5), 567-578. doi:10.1517/17460441.2014.907790

Pirri, J. K., McPherson, A. D., Donnelly, J. L., Francis, M. M., & Alkema, M. J. (2009).

A tyramine-gated chloride channel coordinates distinct motor programs of a

Caenorhabditis elegans escape response. Neuron, 62(4), 526-538.

doi:10.1016/j.neuron.2009.04.013

Portman, D. S. (2006). Profiling C. elegans gene expression with DNA microarrays.

WormBook, 1-11. doi:10.1895/wormbook.1.104.1

Ptak, K., Yamanishi, T., Aungst, J., Milescu, L. S., Zhang, R., Richerson, G. B., & Smith,

J. C. (2009). Raphe neurons stimulate respiratory circuit activity by multiple

mechanisms via endogenously released serotonin and substance P. J Neurosci,

29(12), 3720-3737. doi:10.1523/JNEUROSCI.5271-08.2009

Radacs, M., Galfi, M., Nagyeri, G., Molnar, A. H., Varga, C., Laszlo, F., & Laszlo, F. A.

(2008). Significance of the adrenergic system in the regulation of vasopressin

secretion in rat neurohypophyseal tissue cultures. Regul Pept, 148(1-3), 1-5.

doi:10.1016/j.regpep.2008.03.004

Raiteri, L., Luccini, E., Romei, C., Salvadori, S., & Calo, G. (2009). Neuropeptide S

selectively inhibits the release of 5-HT and noradrenaline from mouse frontal

114 cortex nerve endings. Br J Pharmacol, 157(3), 474-481. doi:10.1111/j.1476-

5381.2009.00163.x

Ranganathan, R., Cannon, S. C., & Horvitz, H. R. (2000). MOD-1 is a serotonin-gated

chloride channel that modulates locomotory behaviour in C. elegans. Nature,

408(6811), 470-475. doi:10.1038/35044083

Rao, S., Lang, C., Levitan, E. S., & Deitcher, D. L. (2001). Visualization of neuropeptide

expression, transport, and exocytosis in Drosophila melanogaster. J Neurobiol,

49(3), 159-172.

Regev, L., Neufeld-Cohen, A., Tsoory, M., Kuperman, Y., Getselter, D., Gil, S., & Chen,

A. (2011). Prolonged and site-specific over-expression of corticotropin-releasing

factor reveals differential roles for extended amygdala nuclei in emotional

regulation. Mol Psychiatry, 16(7), 714-728. doi:10.1038/mp.2010.64

Rex, E., Molitor, S. C., Hapiak, V., Xiao, H., Henderson, M., & Komuniecki, R. (2004).

Tyramine receptor (SER-2) isoforms are involved in the regulation of pharyngeal

pumping and foraging behavior in Caenorhabditis elegans. J Neurochem, 91(5),

1104-1115. doi:10.1111/j.1471-4159.2004.02787.x

Rindler, M. J. (1998). Carboxypeptidase E, a peripheral membrane protein implicated in

the targeting of hormones to secretory granules, co-aggregates with granule

content proteins at acidic pH. J Biol Chem, 273(47), 31180-31185.

Ringstad, N., & Horvitz, H. R. (2008). FMRFamide neuropeptides and acetylcholine

synergistically inhibit egg-laying by C. elegans. Nat Neurosci, 11(10), 1168-1176.

doi:10.1038/nn.2186

115 Rogers, C., Reale, V., Kim, K., Chatwin, H., Li, C., Evans, P., & de Bono, M. (2003).

Inhibition of Caenorhabditis elegans social feeding by FMRFamide-related

peptide activation of NPR-1. Nat Neurosci, 6(11), 1178-1185.

doi:10.1038/nn1140

Roth, A. L., Marzola, E., Rizzi, A., Arduin, M., Trapella, C., Corti, C., . . . Guerrini, R.

(2006). Structure-activity studies on neuropeptide S: identification of the amino

acid residues crucial for receptor activation. J Biol Chem, 281(30), 20809-20816.

doi:10.1074/jbc.M601846200

Rowland, N. E., Fakhar, K. J., Robertson, K. L., & Haskell-Luevano, C. (2010). Effect of

serotonergic anorectics on food intake and induction of Fos in brain of mice with

disruption of melanocortin 3 and/or 4 receptors. Pharmacol Biochem Behav,

97(1), 107-111. doi:10.1016/j.pbb.2010.03.008

Salio, C., Averill, S., Priestley, J. V., & Merighi, A. (2007). Costorage of BDNF and

neuropeptides within individual dense-core vesicles in central and peripheral

neurons. Dev Neurobiol, 67(3), 326-338. doi:10.1002/dneu.20358

Salio, C., Lossi, L., Ferrini, F., & Merighi, A. (2006). Neuropeptides as synaptic

transmitters. Cell Tissue Res, 326(2), 583-598. doi:10.1007/s00441-006-0268-3

Schafer, W. F. (2006). Genetics of egg-laying in worms. Annu Rev Genet, 40, 487-509.

doi:10.1146/annurev.genet.40.110405.090527

Scheiner, R., Reim, T., Sovik, E., Entler, B. V., Barron, A. B., & Thamm, M. (2017).

Learning, gustatory responsiveness and tyramine differences across nurse and

forager honeybees. J Exp Biol, 220(Pt 8), 1443-1450. doi:10.1242/jeb.152496

116 Schellekens, H., Clarke, G., Jeffery, I. B., Dinan, T. G., & Cryan, J. F. (2012). Dynamic

5-HT2C receptor editing in a mouse model of obesity. PLoS One, 7(3), e32266.

doi:10.1371/journal.pone.0032266

Schone, C., Apergis-Schoute, J., Sakurai, T., Adamantidis, A., & Burdakov, D. (2014).

Coreleased orexin and glutamate evoke nonredundant spike outputs and

computations in histamine neurons. Cell Rep, 7(3), 697-704.

doi:10.1016/j.celrep.2014.03.055

Schone, C., Cao, Z. F., Apergis-Schoute, J., Adamantidis, A., Sakurai, T., & Burdakov,

D. (2012). Optogenetic probing of fast glutamatergic transmission from

hypocretin/orexin to histamine neurons in situ. J Neurosci, 32(36), 12437-12443.

doi:10.1523/JNEUROSCI.0706-12.2012

Seidah, N. G., & Prat, A. (2002). Precursor convertases in the secretory pathway, cytosol

and extracellular milieu. Essays Biochem, 38, 79-94.

Shakiryanova, D., Zettel, G. M., Gu, T., Hewes, R. S., & Levitan, E. S. (2011). Synaptic

neuropeptide release induced by octopamine without Ca2+ entry into the nerve

terminal. Proc Natl Acad Sci U S A, 108(11), 4477-4481.

doi:10.1073/pnas.1017837108

Shen, F. S., & Loh, Y. P. (1997). Intracellular misrouting and abnormal secretion of

adrenocorticotropin and growth hormone in cpefat mice associated with a

carboxypeptidase E mutation. Proc Natl Acad Sci U S A, 94(10), 5314-5319.

Si, W., Aluisio, L., Okamura, N., Clark, S. D., Fraser, I., Sutton, S. W., . . . Reinscheid,

R. K. (2010). Neuropeptide S stimulates dopaminergic neurotransmission in the

117 medial prefrontal cortex. J Neurochem, 115(2), 475-482. doi:10.1111/j.1471-

4159.2010.06947.x

Sieburth, D., Madison, J. M., & Kaplan, J. M. (2007). PKC-1 regulates secretion of

neuropeptides. Nat Neurosci, 10(1), 49-57. doi:10.1038/nn1810

Simon, A. C., Loverdo, C., Gaffuri, A. L., Urbanski, M., Ladarre, D., Carrel, D., . . .

Lenkei, Z. (2013). Activation-dependent plasticity of polarized GPCR distribution

on the neuronal surface. J Mol Cell Biol, 5(4), 250-265. doi:10.1093/jmcb/mjt014

Smith, E. S., Martinez-Velazquez, L., & Ringstad, N. (2013). A chemoreceptor that

detects molecular carbon dioxide. J Biol Chem, 288(52), 37071-37081.

doi:10.1074/jbc.M113.517367

Smith, J. S., & Rajagopal, S. (2016). The beta-arrestins: Multifunctional regulators of G

protein-coupled receptors. J Biol Chem, 291(17), 8969-8977.

doi:10.1074/jbc.R115.713313

Smith, S. M., & Vale, W. W. (2006). The role of the hypothalamic-pituitary-adrenal axis

in neuroendocrine responses to stress. Dialogues Clin Neurosci, 8(4), 383-395.

Sobota, J. A., Ferraro, F., Back, N., Eipper, B. A., & Mains, R. E. (2006). Not all

secretory granules are created equal: Partitioning of soluble content proteins. Mol

Biol Cell, 17(12), 5038-5052. doi:10.1091/mbc.E06-07-0626

Sohn, J. W. (2015). Network of hypothalamic neurons that control appetite. BMB Rep,

48(4), 229-233.

Sohn, J. W., Elmquist, J. K., & Williams, K. W. (2013). Neuronal circuits that regulate

feeding behavior and metabolism. Trends Neurosci, 36(9), 504-512.

doi:10.1016/j.tins.2013.05.003

118 Sohn, J. W., Xu, Y., Jones, J. E., Wickman, K., Williams, K. W., & Elmquist, J. K.

(2011). Serotonin 2C receptor activates a distinct population of arcuate pro-

opiomelanocortin neurons via TRPC channels. Neuron, 71(3), 488-497.

doi:10.1016/j.neuron.2011.06.012

Sossin, W. S., Fisher, J. M., & Scheller, R. H. (1990). Sorting within the regulated

secretory pathway occurs in the trans-Golgi network. J Cell Biol, 110(1), 1-12.

Speese, S., Petrie, M., Schuske, K., Ailion, M., Ann, K., Iwasaki, K., . . . Martin, T. F.

(2007). UNC-31 (CAPS) is required for dense-core vesicle but not synaptic

vesicle exocytosis in Caenorhabditis elegans. J Neurosci, 27(23), 6150-6162.

doi:10.1523/JNEUROSCI.1466-07.2007

Srinivasan, S., Sadegh, L., Elle, I. C., Christensen, A. G., Faergeman, N. J., & Ashrafi, K.

(2008). Serotonin regulates C. elegans fat and feeding through independent

molecular mechanisms. Cell Metab, 7(6), 533-544.

doi:10.1016/j.cmet.2008.04.012

Stein, C. (2013). Opioids, sensory systems and chronic pain. Eur J Pharmacol, 716(1-3),

179-187. doi:10.1016/j.ejphar.2013.01.076

Stigloher, C., Zhan, H., Zhen, M., Richmond, J., & Bessereau, J. L. (2011). The

presynaptic dense projection of the Caenorhabditis elegans cholinergic

neuromuscular junction localizes synaptic vesicles at the active zone through

SYD-2/liprin and UNC-10/RIM-dependent interactions. J Neurosci, 31(12), 4388-

4396. doi:10.1523/JNEUROSCI.6164-10.2011

Stogner, K. A., & Holmes, P. V. (2000). Neuropeptide-Y exerts antidepressant-like

effects in the forced swim test in rats. Eur J Pharmacol, 387(2), R9-10.

119 Stojilkovic, S. S. (2005). Ca2+-regulated exocytosis and SNARE function. Trends

Endocrinol Metab, 16(3), 81-83. doi:10.1016/j.tem.2005.02.002

Suo, S., Culotti, J. G., & Van Tol, H. H. (2009). Dopamine counteracts octopamine

signalling in a neural circuit mediating food response in C. elegans. EMBO J,

28(16), 2437-2448. doi:10.1038/emboj.2009.194

Suo, S., Kimura, Y., & Van Tol, H. H. (2006). Starvation induces cAMP response

element-binding protein-dependent gene expression through octopamine-Gq

signaling in Caenorhabditis elegans. J Neurosci, 26(40), 10082-10090.

doi:10.1523/JNEUROSCI.0819-06.2006

Suzuki, H., Thiele, T. R., Faumont, S., Ezcurra, M., Lockery, S. R., & Schafer, W. R.

(2008). Functional asymmetry in Caenorhabditis elegans taste neurons and its

computational role in chemotaxis. Nature, 454(7200), 114-117.

doi:10.1038/nature06927

Sze, J. Y., Victor, M., Loer, C., Shi, Y., & Ruvkun, G. (2000). Food and metabolic

signalling defects in a Caenorhabditis elegans serotonin-synthesis mutant.

Nature, 403(6769), 560-564. doi:10.1038/35000609

Tasan, R. O., Nguyen, N. K., Weger, S., Sartori, S. B., Singewald, N., Heilbronn, R., . . .

Sperk, G. (2010). The central and basolateral amygdala are critical sites of

neuropeptide Y/Y2 receptor-mediated regulation of anxiety and depression. J

Neurosci, 30(18), 6282-6290. doi:10.1523/JNEUROSCI.0430-10.2010

Taupenot, L., Harper, K. L., & O'Connor, D. T. (2005). Role of H+-ATPase-mediated

acidification in sorting and release of the regulated secretory protein

120 chromogranin A: evidence for a vesiculogenic function. J Biol Chem, 280(5),

3885-3897. doi:10.1074/jbc.M408197200

Tiligada, E., & Wilson, J. F. (1989). Regulation of alpha-melanocyte-stimulating

hormone release from superfused slices of rat hypothalamus by serotonin and the

interaction of serotonin with the dopaminergic system inhibiting peptide release.

Brain Res, 503(2), 225-228.

Tobin, V., Leng, G., & Ludwig, M. (2012). The involvement of actin, calcium channels

and exocytosis proteins in somato-dendritic oxytocin and vasopressin release.

Front Physiol, 3, 261. doi:10.3389/fphys.2012.00261

Tong, J., Mannea, E., Aime, P., Pfluger, P. T., Yi, C. X., Castaneda, T. R., . . . Tschop,

M. H. (2011). enhances olfactory sensitivity and exploratory sniffing in

rodents and humans. J Neurosci, 31(15), 5841-5846.

doi:10.1523/JNEUROSCI.5680-10.2011

Tooze, S. A., Martens, G. J., & Huttner, W. B. (2001). Secretory granule biogenesis:

rafting to the SNARE. Trends Cell Biol, 11(3), 116-122.

Troemel, E. R., Sagasti, A., & Bargmann, C. I. (1999). Lateral signaling mediated by

axon contact and calcium entry regulates asymmetric odorant receptor expression

in C. elegans. Cell, 99(4), 387-398.

Trueta, C., & De-Miguel, F. F. (2012). Extrasynaptic exocytosis and its mechanisms: a

source of molecules mediating volume transmission in the nervous system. Front

Physiol, 3, 319. doi:10.3389/fphys.2012.00319

121 Trueta, C., Kuffler, D. P., & De-Miguel, F. F. (2012). Cycling of dense core vesicles

involved in somatic exocytosis of serotonin by leech neurons. Front Physiol, 3,

175. doi:10.3389/fphys.2012.00175

Turner, A. J., Isaac, R. E., & Coates, D. (2001). The neprilysin (NEP) family of zinc

metalloendopeptidases: genomics and function. Bioessays, 23(3), 261-269.

doi:10.1002/1521-1878(200103)23:3<261::AID-BIES1036>3.0.CO;2-K

Voigt, J. P., & Fink, H. (2015). Serotonin controlling feeding and satiety. Behav Brain

Res, 277, 14-31. doi:10.1016/j.bbr.2014.08.065

Wagner, L., Wolf, R., Zeitschel, U., Rossner, S., Petersen, A., Leavitt, B. R., . . . von

Horsten, S. (2015). Proteolytic degradation of neuropeptide Y (NPY) from head

to toe: Identification of novel NPY-cleaving peptidases and potential drug

interactions in CNS and Periphery. J Neurochem, 135(5), 1019-1037.

doi:10.1111/jnc.13378

Walker, M. W., Wolinsky, T. D., Jubian, V., Chandrasena, G., Zhong, H., Huang, X., . . .

Craig, D. A. (2009). The novel neuropeptide Y Y5 Lu

AA33810 [N-[[trans-4-[(4,5-dihydro[1]benzothiepino[5,4-d]thiazol-2-

yl)amino]cyclohexyl]me thyl]-methanesulfonamide] exerts anxiolytic- and

antidepressant-like effects in rat models of stress sensitivity. J Pharmacol Exp

Ther, 328(3), 900-911. doi:10.1124/jpet.108.144634

Wang, H., Girskis, K., Janssen, T., Chan, J. P., Dasgupta, K., Knowles, J. A., . . .

Sieburth, D. (2013). Neuropeptide secreted from a pacemaker activates neurons to

control a rhythmic behavior. Curr Biol, 23(9), 746-754.

doi:10.1016/j.cub.2013.03.049

122 Wes, P. D., & Bargmann, C. I. (2001). C. elegans odour discrimination requires

asymmetric diversity in olfactory neurons. Nature, 410(6829), 698-701.

doi:10.1038/35070581

White, J. G., Southgate, E., Thomson, J. N., & Brenner, S. (1986). The structure of the

nervous system of the nematode Caenorhabditis elegans. Philos Trans R Soc

Lond B Biol Sci, 314(1165), 1-340.

White, J. Q., & Jorgensen, E. M. (2012). Sensation in a single neuron pair represses male

behavior in hermaphrodites. Neuron, 75(4), 593-600.

doi:10.1016/j.neuron.2012.03.044

Wood, D. E., & Nusbaum, M. P. (2002). Extracellular peptidase activity tunes motor

pattern modulation. J Neurosci, 22(10), 4185-4195. doi:20026341

Wragg, R. T., Hapiak, V., Miller, S. B., Harris, G. P., Gray, J., Komuniecki, P. R., &

Komuniecki, R. W. (2007). Tyramine and octopamine independently inhibit

serotonin-stimulated aversive behaviors in Caenorhabditis elegans through two

novel amine receptors. J Neurosci, 27(49), 13402-13412.

doi:10.1523/JNEUROSCI.3495-07.2007

Wyler, S. C., Lord, C. C., Lee, S., Elmquist, J. K., & Liu, C. (2017). Serotonergic control

of metabolic homeostasis. Front Cell Neurosci, 11, 277.

doi:10.3389/fncel.2017.00277

Xia, X., Lessmann, V., & Martin, T. F. (2009). Imaging of evoked dense-core-vesicle

exocytosis in hippocampal neurons reveals long latencies and kiss-and-run fusion

events. J Cell Sci, 122(Pt 1), 75-82. doi:10.1242/jcs.034603

123 Xiao, H., Hapiak, V. M., Smith, K. A., Lin, L., Hobson, R. J., Plenefisch, J., &

Komuniecki, R. (2006). SER-1, a Caenorhabditis elegans 5-HT2-like receptor,

and a multi-PDZ domain containing protein (MPZ-1) interact in vulval muscle to

facilitate serotonin-stimulated egg-laying. Dev Biol, 298(2), 379-391.

doi:10.1016/j.ydbio.2006.06.044

Xu, Y. L., Reinscheid, R. K., Huitron-Resendiz, S., Clark, S. D., Wang, Z., Lin, S. H., . . .

Civelli, O. (2004). Neuropeptide S: a neuropeptide promoting arousal and

anxiolytic-like effects. Neuron, 43(4), 487-497. doi:10.1016/j.neuron.2004.08.005

Yamada, K., Hirotsu, T., Matsuki, M., Butcher, R. A., Tomioka, M., Ishihara, T., . . .

Iino, Y. (2010). Olfactory plasticity is regulated by pheromonal signaling in

Caenorhabditis elegans. Science, 329(5999), 1647-1650.

doi:10.1126/science.1192020

Yang, Z., Yu, Y., Zhang, V., Tian, Y., Qi, W., & Wang, L. (2015). Octopamine mediates

starvation-induced hyperactivity in adult Drosophila. Proc Natl Acad Sci U S A,

112(16), 5219-5224. doi:10.1073/pnas.1417838112

Yu, S., Avery, L., Baude, E., & Garbers, D. L. (1997). Guanylyl cyclase expression in

specific sensory neurons: a new family of chemosensory receptors. Proc Natl

Acad Sci U S A, 94(7), 3384-3387.

Zahratka, J. A., Williams, P. D., Summers, P. J., Komuniecki, R. W., & Bamber, B. A.

(2015). Serotonin differentially modulates Ca2+ transients and depolarization in a

C. elegans nociceptor. J Neurophysiol, 113(4), 1041-1050.

doi:10.1152/jn.00665.2014

124 Zang, K. E., Ho, E., & Ringstad, N. (2017). Inhibitory peptidergic modulation of C.

elegans serotonin neurons is gated by T-type calcium channels. Elife, 6.

doi:10.7554/eLife.22771

Zhang, B., Zhang, X. Y., Luo, P. F., Huang, W., Zhu, F. P., Liu, T., . . . Zhou, Z. (2012).

Action potential-triggered somatic exocytosis in mesencephalic trigeminal

nucleus neurons in rat brain slices. J Physiol, 590(4), 753-762.

doi:10.1113/jphysiol.2011.221051

Zhang, B. J., Yamashita, M., Fields, R., Kusano, K., & Gainer, H. (2005). EGFP-tagged

vasopressin precursor protein sorting into large dense core vesicles and secretion

from PC12 cells. Cell Mol Neurobiol, 25(3-4), 581-605. doi:10.1007/s10571-005-

3970-x

Zhang, C. F., Dhanvantari, S., Lou, H., & Loh, Y. P. (2003). Sorting of carboxypeptidase

E to the regulated secretory pathway requires interaction of its transmembrane

domain with lipid rafts. Biochem J, 369(Pt 3), 453-460. doi:10.1042/BJ20020827

Zhang, M., Schafer, W. R., & Breitling, R. (2010a). A circuit model of the temporal

pattern generator of Caenorhabditis egg-laying behavior. BMC Syst Biol, 4, 81.

doi:10.1186/1752-0509-4-81

Zhang, X., Bao, L., & Ma, G. Q. (2010b). Sorting of neuropeptides and neuropeptide

receptors into secretory pathways. Prog Neurobiol, 90(2), 276-283.

doi:10.1016/j.pneurobio.2009.10.011

Zhou, A., Webb, G., Zhu, X., & Steiner, D. F. (1999). Proteolytic processing in the

secretory pathway. J Biol Chem, 274(30), 20745-20748.

125 Zhou, K. M., Dong, Y. M., Ge, Q., Zhu, D., Zhou, W., Lin, X. G., . . . Xu, T. (2007).

PKA activation bypasses the requirement for UNC-31 in the docking of dense

core vesicles from C. elegans neurons. Neuron, 56(4), 657-669.

doi:10.1016/j.neuron.2007.09.015

126