Views Have Catalogued a Host of Peptide-Regulated Processes and Peptide-Related Pathologies in Humans (Morgat Et Al., 2014, Russell Et Al., 2014

A Dissertation entitled

Opioid Signaling Contributes to the Complex, Monoaminergic Modulation of

Nociception in Caenorhabditis elegans

Holly J. Mills

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. Tomer Avidor-Reiss, Committee Member

______Dr. Bruce Bamber, Committee Member

______Dr. Scott Molitor, Committee Member

______Dr. Patricia Komuniecki, Committee Member

______Dr. Robert Steven, Committee Member

______Dr. Patricia R. Komuniecki, Dean College of Graduate Studies

The University of Toledo December 2014

This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the express permission of the author. An Abstract of

Opioid Signaling Contributes to the Complex, Monoaminergic Modulation of

Nociception in Caenorhabditis elegans

Holly J. Mills

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biology

The University of Toledo

December 2014

Opiates play a key role in the perception and modulation of pain. In the present study, we have identified and characterized an opiate-dependent neuropeptide signaling cascade in the nematode model system, Caenorhabditis elegans,that is involved in the modulation of nociception, on the assumption that the pathways modulating nociception in C. elegans might be comparable to those modulating pain in mammals. Indeed, mammalian opioid receptor agonists, such as morphine and salvinorin A, mimicked food or serotonin, and stimulated the initiation of aversive responses mediated by the two ASH sensory neurons, but paradoxically, induced animals to continue forward after the initial backward locomotion was complete, suggesting that, as in mammals, opiates might decrease the perception of noxious stimuli. Conversely, the mammalian opioid receptor antagonists, naloxone and norbinaltorphimine, abolished the serotonergic stimulation of aversive responses. Both morphine-and serotonin-dependent phenotypes required the expression of the neuropeptide receptor, NPR-17 and npr-17 null phenotypes could be rescued by the expression of either NPR-17 or the human kappa-opioid receptor, OPKR-

iii

1, in the two ASI sensory neurons. NPR-17 signaling mimicked that of human kappa- opioid receptor and appeared to involve both Gαo and a MAPK. NPR-17 functioned in a number of neurons to stimulate aversive responses and appeared to selectively inhibit the release of a subset of neuropeptides from the ASIs that were required for the tyraminergic-dependent inhibition of nociception mediated by the Gαq-coupled α- adrenergic-like receptor, TYRA-3. Together, these results strongly support the hypothesis that NPR-17 is a kappa opioid-like receptor and that C. elegans contains endogenous ligands for the human kappa opioid receptor. Indeed, the direct injection of an endogenous human kappa opioid receptor ligand, dynorphin A, mimicked serotonin and stimulated aversive responses off food.

To identify endogenous ligands for NPR-17, different predicted neuropeptide null animals were screened for aversive behavior. Aversive responses in either nlp-3 or nlp-24 neuropeptide null animals, mimicked npr-17 null animals, and were not stimulated by serotonin. Based on epistasis analyses, nlp-24 appeared to act upstream or in parallel to nlp-3 and both nlp-24 and nlp-3 appeared to act upstream of npr-17. Interestingly, only one of three nlp-3 neuropeptides, NLP-3.3, stimulated aversive responses, with NLP-3.1 antagonizing NLP-3.3-dependent stimulation. Indeed, the overexpression of a full length nlp-3 transgene, mimicked serotonin and stimulated the initiation of aversive responses off food, while the overexpression of a truncated transgene that included NLP-3.1, but not 3.3, abolished serotonin stimulation. Studies on nlp-3 signaling suggest that 1) nlp-3 encoded peptides can be released from a number of different sensory neurons, including the ASHs, to stimulate aversive responses, 2) nlp-3 encoded neuropeptides are released, in part, extra-synaptically and that local/humoral neuropeptide pools play a role in

iv modulation 3) behavioral responses to nlp-3 release are rapid and potentially short-lived,

4) nlp-3 overexpression phenotypes are not the result of developmental compensation and

5) individual neuropeptides encoded by the same gene have the potential to have antagonistic effects on individual behaviors.

Together, these studies have characterized a primitive opioid signaling pathway in

C. elegans that is involved in a complex, monoamine-dependent signaling cascade that modulates multiple components of the sensory-mediated locomotory circuit. This signaling cascade includes serotonin, -adrenergic-like and neuropeptide receptors and ultimately differentially modulates the release of a subset of ‘inhibitory” neuropeptides from the two ASIs to modulate aversive responses. Together, these results highlight the complex interactions of monoaminergic and peptidergic signaling in a “simple” organism, such as C. elegans, and the potential utility of the C. elegans model to untangle the complexities of opiate signaling in higher organisms.

Acknowledgements

The work presented in this thesis is due to the encouragement and support of many people, but most of all my advisor and mentor Dr. Richard Komuniecki. I would like to sincerely thank Rick for all his motivation and guidance throughout my graduate career and for creating a fun environment in which to do science. I would also thank my committee members, Dr. Bruce Bamber, Dr. Robert Steven, Dr. Tomer Avidor-Reiss, Dr.

Scott Molitor and Dr. Patricia Komuniecki for their time, suggestions and insights. I also thank all the lab members, past and present, in Komuniecki lab especially, Dr. Rachel

Wragg, Dr. Gareth Harris, Dr. Vera Hapiak, Philip Summers and Amanda Ortega for their contributes to my graduate work but also for their friendship. I also thank my friends who have helped make Toledo “home” during my time here, I will miss you all dearly.

Last and most importantly, I would like to sincerely thank my family: my wife, Rachel, my parents, Frederick and Carol, and my sister, Rosanne, for their love and support over the years.

Table of Contents

Abstract ...... iii

Acknowledgements ...... vi

Table of Contents ...... vii

List of Tables……………………………………………………………………………...x

List of Figures…………………………………………………………………………….xi

List of Abbreviations ...... xiii

List of Symbols ...... xvi

1 Significance …...... 1

1.1 Monoamines define behavioral state or “context” in C. elegans ...... 3

1.2 Neuropeptide signaling in C. elegans is essential for the modulation of

many behaviors…… ....……………………………………………………8

1.3 Opioid signaling in mammals ...... 16

1.4 Project focus……………………………………………………..…...21

2 Materials and Methods ...... 27

2.1 Materials…………………………………..…………………………27

2.2 Strains…………………………………………………..……………27

2.3 Generation of RNAi strains by bacterial feeding…………….………28

2.4 Molecular biology and transgenesis………………………………….29

2.5 Generation of cell selective RNAi constructs………….…………… 29 vii

2.6 Behavioral assays…………………………………………………….30

2.7 Peptide injections…………………………………………………….31

2.8 Microscopy and image analysis………………………………………31

3. Results……………………………………………………………………………34

3.1 Neuropeptides encoded by nlp-3 appeared to be released extra-

synaptically from an array of sensory neurons modulate ASH-mediated

aversive responses……………………………………………………...... 34

3.2 Peptides encoded by nlp-3 differentially modulate aversive

responses……...... 35

3.3 Peptides encoded by nlp-3 activate NPR-17 to stimulate aversive

responses………………………………………………………………...37

3.4 NPR-17 functions in multiple neurons including the ASI sensory

neurons to modulate aversive responses…………………………………41

3.5 NPR-17 is a kappa opioid-like receptor………………………………45

3.6 NPR-17 signaling requires Gαo in the ASIs…………………………..49

3.7 npr-17 null phenotypes could also be rescued by the expression of the

human kappa opioid receptor in the ASIs………………………………...49

3.8 NPR-17 functions in the ASI sensory neurons to inhibit a tyramine-

mediated peptidergic signaling cascade………………………………….54

3.9 Peptides encoded by nlp-24 are also essential for serotonergic

stimulation………………………………………………………………..57

3.10 Serotonin stimulates the release of nlp-24 encoded peptides from the

two ASI sensory neurons…………………………………………………61

viii

4. Discussion………………………………………………………………..………64

4.1 Neuropeptides encoded by nlp-3 appear to be released tonically from

multiple sensory neurons and function extra-synaptically to modulate

behavioral state…………………………………………………………...65

4.2 Neuropeptides encoded by the same gene can have antagonistic effects

on behavior…………………………………………………………….…69

4.3 The ASIs receive modulatory input from three different monoamine.71

4.4 Opioid signaling in other invertebrates………………………………76

4.5 Conclusions…………………………………………………………..77

5. References ...... 78

List of Tables

1.1 Deorphanized neuropeptide receptors in C. elegans ...... 23

2.1 Expression of cell selective promoters ...... 33

List of Figures

3-1 Peptides encoded by nlp-3 are expressed in multiple sensory neurons modulate

ASH-mediated aversive responses……………………………………………………….36

3-2 Peptides encoded by nlp-3 are conserved in distantly-related nematodes ...... 38

3-3 The direct injection of nlp-3 encoded peptides differentially modulates ASH- mediated aversive responses ...... 39

3-4 Aversive responses were not stimulated by serotonin in fourteen predicted neuropeptide receptor null mutants………………………………………………………42

3-5 The neuropeptide receptor encoded by npr-17 appears to be activated by nlp-3 encoded peptides…………………………………………………………………………43

3-6 Locomotory phenotypes in nlp-3 and npr-17 null animals are identical………….44

3-7 Fluorescence from an npr-17::gfp is observed in subsets of head and tail neurons…………………………………………………………………………………...47

3-8 The serotonin stimulation of aversive response requires the expression of npr-17 in multiple neurons...... 48

3-9 NPR-17 is conserved across nematode species and has limited identity to the human kappa opioid receptor…………………………………………………………….50

3-10 NPR-17 is required for the modulation of aversive response by a range of mammalian opioid receptor ligands……………………………………………………...51

3-11 Morphine mimics serotonin and modulates not only the indication of the aversive response but also a range of post-initiation locomotory behaviors………………………52

3-12 NPR-17 appears to be a Gαo-coupled receptor…………………………………..53

3-13 Expression of the human kappa opioid receptor rescues the serotonin and morphine stimulation of aversive responses in npr-17 null animals…………………….55

3-14 The injection of NLP-3.3 into npr-17 null animals expressing the human kappa opioid receptor or dynorphin, a mammalian kappa opioid receptor agonist, into wild-type animals stimulate aversive responses…………………………………………………….56

3-15 The tyramine inhibition of serotonin stimulated aversive responses is absent in wild-type animals overexpressing either NPR-17 or the human kappa opioid receptor in the ASIs sensory neurons………………………………………………………………...58

3-16 The octopamine inhibition of aversive responses to 100% is not affected in wild- type animals overexpressing either NPR-17 or the human kappa opioid receptor in the

ASIs sensory neurons ……………………………………………………………………59

3-17 Overexpression phenotype observed by nlp-24 overexpression are absent in nlp-3 null animals………………………………………………………………………………62

3-18 The serotonergic stimulation of aversive responses requires the expression of

SER-1 in the ASIs………………………………………………………………………..63

4-1 The two ASI sensory neurons function as a signaling hub to integrate monoaminergic and peptidergic signaling in the modulation of nociception…………... 66

xii

List of Abbreviations

ADF Serotonergic neuron AIB Interneuron AIM Serotonergic neuron AIY Interneuron ASG Serotonergic neuron ASH Sensory neuron ASI Sensory neuron ASK Sensory neuron AVB Forward command interneuron AWC Sensory neuron

BAG Sensory neuron

CAMKII Calmodulin-dependent protein kinase II CGRP Calcitonin gene released peptide CKR-2 Neuropeptide receptor

DAF-2 Neuropeptide receptor

EAT-4 Vesicular glutamate transporter EGL-21 Carboxypeptidase E EGL-3 Proprotein convertase EGL-6 Neuropeptide receptor ERK Extracellular signal-regulated kinase flp-10 Neuropeptide-encoding gene flp-17 Neuropeptide-encoding gene flp-21 Neuropeptide-encoding gene

GABA Gamma-aminobutyric acid GFP Green florescent protein GLR-1 AMPA-type ionotropic glutamate receptor GPCR G-protein coupled receptor GAR-3 G-protein-coupled muscarinic receptor

xiii

HSN Serotonergic neuron

INS-6 Neuropeptide K(ir)3 G-protein-gated inwardly rectifying potassium channel

LGC-55 Tyramine-gated chloride channel LPS Lipopolysaccharide

MAPK Mitogen-activated protein kinase MOD-1 Serotonin gated chloride channel MOD-5 Serotonin reuptake transporter

NEP-1 Neprilysin NEP-2 Neprilysin nlp-3 Neuropeptide-encoding gene nlp-7 Neuropeptide-encoding gene nlp-8 Neuropeptide-encoding gene nlp-9 Neuropeptide-encoding gene nlp-10 Neuropeptide-encoding gene nlp-12 Neuropeptide-encoding gene nlp-15 Neuropeptide-encoding gene nlp-24 Neuropeptide-encoding gene nlp-44 Neuropeptide-encoding gene NLP-3.1 Neuropeptide NLP-3.2 Neuropeptide NLP-3.3 Neuropeptide NMUR-1 Neuropeptide receptor NMUR-2 Neuropeptide receptor nBNI Norbinaltorphimine NPR-17 Neuropeptide receptor NSM Serotonergic neuron NTR-1 Neuropeptide receptor

OCTR-1 G-protein-coupled octopamine receptor OPKR-1 Human kappa opioid receptor p38 Mitogen-activated protein kinase PKA Protein kinase A PKC-1 Protein kinase C PMK-1 p38

RIA Interneuron RIH Serotonergic neuron

SER-1 G-protein-coupled serotonin receptor SER-2 G-protein-coupled tyramine receptor xiv

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 SER-7 G-protein-coupled serotonin receptor SNARE Soluble N-ethyl-maleimide-sensitive fusion protein attachment receptor SNET-1 Neuropeptide

TBH-1 Tyramine beta-hydroxylase TDC-1 Tyrosine decarboxylase TPH-1 Tryptophan hydroxylase TRPV Transient Receptor Potential Vanilloid TYRA-2 G-protein-coupled tyramine receptor TYRA-3 G-protein-coupled tyramine receptor

U50488 Kappa opioid receptor agonist UNC-104 Kinesin UNC-13 mUNC-13 UNC-31 Calcium-dependent activator protein for secretion

List of Symbols

α ...... Alpha β ...... Beta γ…………………….Gamma

xvi

Chapter 1

Significance

One of the primary goals of neuroscience is to understand how sensory inputs interact to define behavioral state and ultimately modulate perception and behavior. In fact, alterations in sensory integration are associated with a number of disease states, including autism, anxiety, depression, obesity and chronic pain. For example, the perception and response to painful stimuli is dependent upon context or behavioral state.

Neuromodulators, such as noradrenaline, serotonin and a host of neuropeptides, including the endorphins, can alter both perception and response to painful stimuli within an unchanging anatomical context. Neuropeptides and monoamines are released both synaptically and extra-synaptically and alter activity patterns in the “hard-wired” circuitry at every level, from sensory inputs to motor outputs, by modulating both the intrinsic properties of the neurons and synaptic strength. To better understand the role of neuromodulators in the context-dependent perception of noxious stimuli, we have examined the role of opiates, extensively involved in the perception of pain in mammals, in the modulation of aversive behavior mediated by the two polymodal, nociceptive ASH sensory neurons in the nematode model system, Caenorhabditis elegans.

1 C. elegans contains only 302 neurons, but exhibits an array complex sensory- mediated behaviors, including chemotaxis, thermotaxis and mechanosensation that are modulated extensively by behavioral state. For example, upon the presentation of a noxious stimulus, such as 1-octanol, animals initiate a rapid, context-dependent reversal that is differentially modulated by an array of monoamines and neuropeptides (Chao et al., 2004, Wragg et al., 2007, Harris et al., 2010, Mills et al., 2012b). As outlined below, this 1-octanol dependent reversal is initiated by the two ASH sensory neurons.

Importantly, the ASHs and mammalian nociceptive neurons exhibit a number of similarities, highlighting the potential utility of the C. elegans model for studies of pain, even though in C. elegans the perception of “pain” concept is operationally-defined as a rapid reversal away from a noxious stimulus. For example, both C. elegans and mammalian nociceptors contain a G-protein coupled receptor mediated signaling cascade that activates orthologues of a TRPV4 calcium permeable, non-selective cation channel, both are glutamatergic/peptidergic, and both are modulated by serotonergic/adrenergic- like signaling (Salt and Hill, 1983, Otsuka and Yoshioka, 1993, Nathoo et al., 2001,

Mellem et al., 2002, Harris et al., 2010).

The two ASH sensory neurons respond to chemical repellents, high osmolarity and mechanical stimulation; all of which elicit a rapid avoidance response (Kaplan and

Horvitz, 1993, Hilliard et al., 2005). For example, the ASHs are necessary and sufficient for aversive responses to dilute (30% v:v) 1-octanol and the addition of dilute 1-octanol dramatically increases ASH somal calcium and initiates a rapid reversal (Wragg et al.,

2007, Harris et al., 2009, Mills et al., 2012b). In contrast, laser ablation of the ASHs or the suppression of ASH glutamatergic signaling by the ASH RNAi knockdown of the

2 EAT-4 vesicular glutamate transporter abolish responses to dilute 1-octanol (Chao et al.,

2004, Harris et al., 2010). Interestingly, this ASH-mediated aversive response is initiated more rapidly in the presence of food or serotonin and this food or serotonin stimulation requires three distinct serotonin receptors, SER-1 in the RIA interneurons, SER-5 in the

ASH sensory neurons and MOD-1 in the AIB and AIY interneurons, suggesting that these three serotonin receptors operate at different levels within the ASH-dependent locomotory circuit (Harris et al., 2009). These studies suggest that, as in mammals, the perception and response to noxious stimuli by C. elegans is also context-dependent.

1.1 Monoamines define behavioral state or “context” in C. elegans

In C. elegans, behavioral state or “context” is largely defined by food availability and changes in food availability are reflected by changes in locomotory behavior. For example, upon removal from food C. elegans initiate local search behavior, consisting of frequent reversals and omega turns (Gray et al., 2005). Similarly, when food-deprived animals encounter a bacterial lawn, locomotion decreases considerably; this “enhanced slowing response” is absent in animals lacking key enzymes for serotonin biosynthesis and can be rescued by exogenous serotonin (Sawin et al., 2000). Serotonin is thought to be the “food at hand” signal and exerts its effects though at least four G-protein coupled serotonin receptors that have significant identity to human serotonin receptors, the Gαq- coupled SER-1 (5-HT2-like) and SER-5 (5-HT6-like); Gαs-coupled SER-7 (5-HT7-like);

Gαo-coupled SER-4 (5-HT1-like) (Olde and McCombie, 1997, Hamdan et al., 1999,

Hobson et al., 2003, Hobson et al., 2006, Harris et al., 2010). In addition, C. elegans also expresses a unique serotonin-gated chloride channel, MOD-1 (Ranganathan et al., 2000).

3 Eleven serotonergic neurons (ADFs, AIMs, ASGs, HSNs, NSMs and RIH) have been identified in hermaphroditic C. elegans, either by a GFP gene fusion to tryptophan hydroxylase (tph-1), a key enzyme in serotonin biosynthesis, or direct antibody staining for serotonin (Horvitz et al., 1982, Sze et al., 2000, Duerr et al., 2001, Pocock and

Hobert, 2010). Most of these serotonergic neurons are capable of serotonin synthesis and reuptake, based on the co-expression of tph-1 and mod-5 that encodes a serotonin reuptake transporter, some are capable of reuptake, but not synthesis, such as the AIM and RIH interneurons, and some only express tph-1 under specific environmental conditions; for example, the expression of tph-1 in the ASG sensory neurons is strongly upregulated under low oxygen conditions (Sze et al., 2000, Kullyev et al., 2010, Pocock and Hobert, 2010, Jafari et al., 2011). Since many of the C. elegans serotonin receptors are not expressed on neurons that are directly innervated by the serotonergic neurons, it is assumed that the bulk of the serotonergic modulation occurs extra-synaptically and involves the local/humoral release of serotonin, with serotonin sometimes having opposing effects on behavior, initiating local signaling cascades that either stimulate or inhibit different microcircuits to coordinate different aspects of a behavioral response

(Hapiak et al., 2009, Harris et al., 2011). For example, in the presence of food, serotonin is released from the two neurosecretory motorneurons (NSMs) to stimulate ASH- mediated aversive behavior (Harris et al., 2011). In contrast, serotonin released from the two ADF sensory neurons appears to act antagonistically to NSM serotonin (Harris et al.,

2011). Similarly, NSM and ADF serotonin also play different roles in the food-activation of pharyngeal pumping. For example, ADF, but not NSM serotonin, is required for increased feeding following the recognition of familiar food (Song et al., 2013). This is

4 surprising given the neurosecretory nature and positioning of the NSMs in the pharynx.

In addition, serotonin secreted from the ADFs, but not the NSMs, has been implicated in aversive learning to pathogenic bacteria (Zhang et al., 2005). Both SER-1 and SER-7 are required for the serotonergic activation of pharyngeal pumping, but in a ser-1;ser-7 null background serotonin actually inhibits the food activation of pumping through SER-4 and

MOD-1. Finally, serotonin again stimulates pumping in a ser-1;ser-4;ser-7;mod-1 null background (Hobson et al., 2006), highlighting the complexity of serotonergic modulation and the potential for antagonistic modulatory serotonergic signaling, regardless of the ultimate effect of serotonin on the behavior. These observations highlight the potential complexities involved in understanding serotonergic modulation and also probably generalize to other less well studied C. elegans neuromodulators, such as the neuropeptides.

C. elegans does not synthesize noradrenaline or adrenaline, but instead synthesizes structurally-related invertebrate counterparts, tyramine and octopamine.

Tyramine is synthesized from tyrosine by a tyrosine decarboxylase (tdc-1), and a tyramine beta-hydroxylase (tbh-1) is required to convert tyramine to octopamine (Alkema et al., 2005). Tyramine and octopamine signal nutritional status and appear to increase with starvation, although this has never been measured experimentally. Tyramine and octopamine act antagonistically to food/serotonin to modulate a host of key behaviors, including pharyngeal pumping, egg-laying, locomotion and aversive responses (Horvitz et al., 1982, Wragg et al., 2007, Packham et al., 2010, Mills et al., 2012b, Hapiak et al.,

2013). In addition, tyramine functions similarly in a number of other invertebrate species, with tyraminergic signaling also modulating behavioral changes associated with

5 feeding, locomotion, olfactory avoidance (Kutsukake et al., 2000, Saraswati et al., 2004,

Nisimura et al., 2005). Three G-protein coupled tyramine receptors have been identified;

SER-2 and TYRA-2 appear to couple to Gαi/o and TYRA-3 to Gαq. In addition, C. elegans also encodes a tyramine-gated Cl- channel, LGC-55 (Rex and Komuniecki, 2002,

Rex et al., 2005, Wragg et al., 2007, Pirri et al., 2009). Exogenous tyramine causes locomotory paralysis and inhibits pharyngeal pumping and aversive responses (Alkema et al., 2005, Wragg et al., 2007, Pirri et al., 2009, Donnelly et al., 2013, Hapiak et al., 2013).

For example, tyramine inhibits forward locomotion and suppresses head oscillations by activating LGC-55 to inhibit the AVB forward command interneurons and neck muscle, respectively (Pirri et al., 2009). Similarly, tyramine coordinates different aspects of anterior touch avoidance responses; LGC-55 modulates the initial reversal from aversive stimuli, whereas SER-2 facilitates the completion of an omega turn and repositioning after initial reversal by modulating signaling from the inhibitory GABAergic (gamma - aminobutyric acid) motor neurons (Donnelly et al., 2013). Adding to the complexity of tyraminergic modulation, TYRA-3 appears to activate a global inhibitory signaling cascade, potentially stimulating the release of other inhibitory neurotransmitters, such as dopamine, octopamine, and an array of neuropeptides (Hapiak et al., 2013). For example, tyramine appears to stimulate the release of a subset of neuropeptides from the two ASI sensory neurons to inhibit the serotonin stimulation of aversive responses (Hapiak et al.,

2013). Similarly, TYRA-3 expression in the food-sensing ASK and CO2/O2-sensing BAG sensory neurons stimulate food-leaving behavior (Bendesky et al., 2011). Interestingly, only a two-fold change in TYRA-3 expression in these neurons is required for tyraminergic modulation (Bendesky et al., 2011). Together, these observations highlight

6 the complexity of tyraminergic modulation, the interaction between tyraminergic and neuropeptidergic signaling and the fact that only small changes in G-protein coupled receptor signaling can have dramatic effects on behavior.

Three octopamine receptors have been identified, SER-3, SER-6 and OCTR-1, that appear to couple to Gαq,Gαq and Gαi/o respectively, based on heterologous expression and genetic analyses, but unlike tyramine and serotonin, an octopamine-gated chloride channel has not yet been identified (Suo et al., 2006, Petrascheck et al., 2007, Wragg et al., 2007). All three C. elegans octopamine receptors are most similar to mammalian α- adrenergic receptors, with SER-6 most identical to mammalian α1-receptors and OCTR-1 mammalian α2-receptors (Mills et al., 2012a, Mills et al., 2012b). As noted above, octopamine, in concert with tyramine, functions antagonistically to serotonin and modulates most behaviors in C. elegans, with the octopaminergic modulation of ASH- mediated aversive behavior appearing to mimic the noradrenergic modulation of chronic pain in mammals (Mills et al., 2012a). For example, in mammals, Gαo-coupled α1- receptors presynaptically inhibit sensory input and Gαq-coupled α2-receptors activate more global inhibitory signaling cascades. Similarly, in C. elegans Gαo- coupled α1-like receptors (OCTR-1) presynaptically inhibit sensory input and Gαq-coupled α2-like receptors (SER-6) activate more global inhibitory neuropeptidergic signaling cascades

(Mills et al., 2012a, Mills et al., 2012b).

7 1.2 Neuropeptide signaling in C. elegans is essential for the modulation of many behaviors

Neuropeptides modulate most behaviors and in mammals defects in neuropeptidergic signaling have been associated with a variety of disease states, including mood disorders, such an anxiety and depression (Ebner and Singewald, 2006,

Aubry, 2013, Fraigne and Peever, 2013, Matsuda et al., 2013). Similarly, in C. elegans, neuropeptides are widely expressed throughout the nervous system and influence most, if not all, behaviors (Nathoo et al., 2001, Li and Kim, 2008). Neuropeptide genes can encode a solitary peptide, multiple distinct peptides, or multiple copies of a single peptide, adding to the complexity peptidergic signaling. In C. elegans, 113 putative neuropeptide-encoding genes have been identified that can be grouped into three families; 42 insulin-like- peptides, 31 FMRFamide and 42 neuropeptide-like proteins. In total, over 250 distinct neuropeptides are predicted, many of which have been confirmed directly by two dimensional liquid chromatography and mass spectrometry (Nathoo et al.,

2001, Husson and Schoofs, 2006, Husson et al., 2007a, Husson and Schoofs, 2007b).

Neuropeptides are synthesized de novo as inactive precursor polypeptides, packaged into dense core vesicles in the trans Golgi network alongside enzymes essential for peptide processing/maturation, unlike classical neurotransmitters, such as glutamate and GABA, that are recycled and repackaged into small, synaptic vesicles (Gondre-Lewis et al., 2012). Peptide maturation occurs in three steps within the dense core vesicle. First, neuropeptide preproteins undergo proteolytic cleavage, usually at dibasic cleavage sites, catalyzed by proprotein convertases; C. elegans encodes four kex-2/ subtilisin-like proprotein convertases, aex-5/kpc-3, bli-4/kpc-4, egl-3/kpc-2 and kpc-1. Second, basic

8 amino acids are removed from the carboxyl-terminus of the peptide intermediate by carboxypeptidase E encoded by egl-21. Third, neuropeptides typically undergo posttranslational modification that protect them from degradation, most often by C- terminal amidation (Kass et al., 2001, Jacob and Kaplan, 2003, Han et al., 2004).

Classical neurotransmitters, such as glutamate, acetylcholine and GABA are typically released at micromolar concentrations from small clear vesicles at the presynaptic active zone to activate ionotropic receptors at adjacent synaptic sites only tens of nanometers away (Suudhof, 2008). In contrast, the release of neuropeptides from large dense core vesicles is not restricted to synaptic specializations, and may activate receptors extra-synaptically at a distance from the site of release (Xu and Xu, 2008,

Gondre-Lewis et al., 2012). C. elegans neurons are en passant without obvious postsynaptic specializations and, in the case of an interneuron, the single process may function as both an axon and dendrite (White et al., 1986). Neuropeptide release can potentially occur from the neuronal soma or anywhere along the length of the process, raising the question of how neuropeptide-containing dense-core vesicles are delivered to proximal and distal release sites. In Drosophila, a uniform supply of dense-core vesicles is achieved by anterograde and retrograde circulation; after packaging in the trans-golgi network, anterograde dense-core vesicles bypass proximal boutons and are trafficked to distal boutons (Wong et al., 2012). Excess dense-core vesicles then undergo dynactin- dependent retrograde transport back through the axon, delivering dense-core vesicles sporadically to more proximal boutons (Wong et al., 2012). Kinesin unc-104 is required for anterograde trafficking of dense-core vesicles in C. elegans, but a mechanism for

9 retrograde trafficking of vesicles has not yet been identified (Jacob and Kaplan, 2003,

Zahn et al., 2004).

Dense-core vesicle release appears to require higher frequency stimulation and is often coupled to the release of calcium from internal stores (Hartmann et al., 2001,

Matsuda et al., 2009, van de Bospoort et al., 2012). For example, in Drosophila motor neurons, sustained dense-core vesicle mobilization is achieved by calcium release from the endoplasmic reticulum, mediated by the ryanodine receptor, to activate calmodulin- dependent protein kinase II (CAMKII) (Shakiryanova et al., 2007). Likewise, octopamine stimulated dense core vesicle release in Drosophila motor neurons and required calcium release from intracellular stores (Shakiryanova et al., 2011). Similarly, the ryanodine receptor and CAMKII control dense-core vesicle exocytosis in C. elegans (Hoover et al.,

2014). For example, in CAMKII mutants dense core vesicles fail to enter axons and instead are prematurely secreted from the soma (Hoover et al., 2014). Interestingly, mobilization of intracellular calcium stores in the absence of soma electrical activity induces dense-core vesicle release from dendrites, but not nerve terminals of mammalian hypothalamic oxytocin neurons. Conversely, electrical activity in the soma can induce dense-core vesicle release from nerve terminals without release from dendrites (Ludwig et al., 2002). Clearly, much remains to be learned about the localization and release of dense-core vesicles.

Synaptic and dense-core release share a common machinery, but it appears that some components might be unique. For example, exocytosis of both classes of vesicles requires calcium, components of the SNARE (soluble N-ethyl-maleimide-sensitive fusion protein attachment receptor) complex, such as syntaxin, and are subject to regulation by

10 second messenger pathways. Traditionally, mUNC-13 and its C. elegans counterpart,

UNC-13, were believed to be essential for small clear vesicle, but not dense-core vesicle release; however, more recent work suggests that UNC-13 may be involved in both processes. For example, mUNC-13 primes dense core vesicles for release by accelerating the rate of transfer from an unprimed to a mature fusion-competent vesicle, possibly though its interaction with the SNARE protein syntaxin-1 in neuroendocrinergic chromaffin cells (Ashery et al., 2000, Stevens et al., 2005). In contrast, a much clearer role for UNC-31/CAPS, a calcium-dependent activator protein for secretion, has been described for dense-core vesicle release (Renden et al., 2001, Charlie et al., 2006,

Sadakata et al., 2007, Speese et al., 2007, Liu et al., 2008). In C. elegans, UNC-31 controls the number of docked and fusion-competent dense-core vesicles, but not small clear vesicles (Zhou et al., 2007, Hammarlund et al., 2008). For example, UNC-31 is essential for the regulated release of the ectopically expressed neuropeptide, ANF-GFP, from dense-core vesicles in cultured C. elegans neurons (Speese et al., 2007). Likewise, neuropeptide release was decreased in unc-31 null animals (Sieburth et al., 2007). Indeed, unc-31 null animals have a number of locomotory defects, most probably associated with alterations in neuropeptidergic signaling; for example locomotion in unc-31 mutants is wild-type off food, but these mutant animals are nearly paralyzed on food (Speese et al.,

2007). Interestingly, the activation of Gαs signaling and protein kinase A rescues locomotory phenotypes and defects in dense-core vesicle release in unc-31 null mutants, suggesting that a PKA substrate functions in parallel or downstream of UNC-31 (Charlie et al., 2006, Zhou et al., 2007). Similarly, protein kinase C (PKC-1) also promotes neuropeptide release, as pkc-1 null mutants had a 40% reduction in neuropeptide release

11 from the cholinergic motor neurons (Sieburth et al., 2007). Conversely, neuropeptide release was dramatically increased in pkc-1 gain of function mutants and this release was absent in unc-31 null animals (Sieburth et al., 2007). Tomosyn, a syntaxin-interacting protein also suppressed neuropeptidergic signaling defects associated with UNC-31, suggesting tomosyn negatively regulates UNC-31 dependent neuropeptide release

(Gracheva et al., 2007b, a). Given roles of both Gαs and Gαq in modulating dense core vesicle and neuropeptide release, it is clear that both processes can be extensively modulated by G-protein coupled receptor signaling driven by both monoamines and neuropeptides.

Neuropeptides are inactivated by a host of differentially localized, soluble and membrane-bound, synaptic and extra-synaptic, proteolytic enzymes. For example, in C. elegans the protease encoded by nep-2 is localized to the cell surface of muscle, glia-like cells and neurons and appears to degrade the neuropeptide, SNET-1, to modulate olfactory plasticity to several odorants (Yamada et al., 2010). Although C. elegans encodes over 250 distinct peptides, this is currently the only example pairing a peptide- degrading enzyme and a neuropeptide in behavioral modulation (Yamada et al., 2010).

For example, the protease encoded by nep-1 is associated with a number of phenotypes, including locomotion and pharyngeal pumping, although the actual neuropeptide substrates involved have yet to identified (Spanier et al., 2005). The role that peptide degradation plays in the modulation of neuropeptidergic signaling is a fruitful area for future research, given the ubiquitous role of peptidergic modulation in both C. elegans and mammals. For example, is the expression of individual peptide-degrading enzymes

12 differentially modulated or are peptide-degrading enzymes differentially localized to generate different pools of peptides?

Neuropeptidergic signaling modulates behavior in both C. elegans and mammals and a number of recent reviews have catalogued a host of peptide-regulated processes and peptide-related pathologies in humans (Morgat et al., 2014, Russell et al., 2014,

Sakurai, 2014, Steinhoff et al., 2014). For example, in C. elegans both EGL-3 and EGL-

21 that are required for peptide processing are expressed throughout the nervous system, and egl-3 and egl-21 null mutants have deficiencies in many behaviors, including pharyngeal pumping, egg-laying, locomotion, mechano-sensation and a host of aversive behaviors (Kass et al., 2001, Jacob and Kaplan, 2003, Harris et al., 2010). Behavioral defects in egl-3 and egl-21 null animals are thought to result from a failure to complete the peptide maturation process; indeed, recent direct analyses have indicated that the levels of processed peptides in these mutants are dramatically reduced (Husson et al.,

2007a, Husson and Schoofs, 2007a). Interestingly, the loss of egl-3 restores defects in the

ASH-mediated behaviors, such as nose touch and osmotic avoidance, caused by mutations in the ionotropic glutamate receptor mutant glr-1, suggesting that EGL-3 processes inhibitory neuropeptides that enhance glutamatergic signaling between the

ASHs and the command interneurons, either by decreasing glutamate release from the

ASHs or facilitating the uptake and removal of glutamate from the synaptic cleft (Kass et al., 2001, Mellem et al., 2002). Similarly, egl-3 mutants are also defective for the serotonergic stimulation of ASH-mediated avoidance of dilute 1-octanol and both egl-3 and egl-21 mutants have decreased basal acetylcholine release at neuromuscular

13 junctions, suggesting that they process neuropeptides that stimulate acetylcholine release

(Jacob and Kaplan, 2003, Harris et al., 2010).

Similar observations have been made on C. elegans mutants lacking individual peptide-encoding genes. A number of excellent reviews have appeared recently (Husson et al., 2007b, Beets et al., 2013, Peymen et al., 2014). For example, peptides encoded by nlp-12 activate the G-protein coupled neuropeptide receptor CKR-2 and provide proprioceptive feedback that couples muscle contraction to changes in presynaptic acetylcholine release (Janssen et al., 2008, Hu et al., 2011). Similarly, peptides encoded by flp-17 and flp-10 activate the Gαo-coupled neuropeptide receptor EGL-6 in the serotonergic HSNs to inhibit egg-laying and the insulin-like peptide, INS-6, activates the insulin-like receptor DAF-2 in the AWC sensory neurons to remodel the salt-sensing circuit when external salt concentrations are high (Ringstad and Horvitz, 2008, Leinwand and Chalasani, 2013). Importantly, the sensory neurons involved in the detection of 1- octanol appear to express a number of neuropeptides; for example nlp-3, nlp-15, flp-21, ins-1 in the ASHs, nlp-7, nlp-8, nlp-10 in the ADLs, and nlp-3, nlp-9 in the AWBs. In addition, a wide range of additional neuropeptides are expressed in other neurons in the

ASH-mediated locomotory circuit (Li et al., 1999, Nathoo et al., 2001, Harris et al., 2010,

Mills et al., 2012b). This large number of neuropeptides expressed within the ASH- mediated locomotory circuit suggests that the peptidergic modulation of even straight- forward aversive behaviors will be quite complex.

Neuropeptides typically bind to G-protein coupled receptors that belong to either

Class A, the rhodopsin-like receptors, or Class B, the secretin-like receptors. In contrast to monoamines that primarily bind to a binding pocket formed by residues in the

14 transmembrane helices, neuropeptides, such as oxytocin, vasopressin and opioids, typically bind to the extracellular N-terminus, causing a conformational change that allows secondary contacts within extracellular loop regions, leading to receptor activation

(Devi, 2005). C. elegans contains over 50 genes that encode putative peptide G-protein coupled receptors; however, only a few of these predicted neuropeptide receptors have been assayed for behavioral phenotypes and/or deorphanized by heterologous expression

(see Table 1). Interestingly, recent data suggests that individual peptides may activate multiple receptors; different peptides encoded by the same gene may activate different receptors, and different receptors may bind multiple peptides (Frooninckx et al., 2012).

Importantly, some peptides may promiscuously activate multiple receptors at high peptide concentrations (µM), while heterologous receptor expression may alter peptide binding. These observations highlight the potential complexity of peptidergic signaling, but also the potential artifacts in linking peptides with cognate receptors, stressing the importance of epigenetic analysis in parallel with heterologous expression to confirm functional ligand specificity.

Many C. elegans peptide receptors have putative mammalian orthologs. For example, ntr-1 encodes a putative vasopressin/oxytocin-like receptor that is activated by nematocin (NTC-1) (Beets et al., 2012). ntr-1 and ntc-1 nulls animals lack gustatory plasticity, as they fail to avoid NaCl after pre-exposure in the absence of food are defective ntr-1 null animals are defective in male-mating behaviors including, mate search and recognition (Beets et al., 2012, Garrison et al., 2012). Similarly, four predicted

C. elegans neuropeptide receptors appear to be structurally-related to mammalian neuromedin U receptors (NMUR 1-4), although little is known about potential orthology.

15 For example, nmur-1 null animals live longer when cultured on E. coli OP50 than

HT115, in contrast to wild-type animals and longevity in nmur-1 animals is dependent upon bacterial lipopolysaccharide (LPS) structure, as a short LPS shortens C. elegans lifespan (Maier et al., 2010). However, the receptor has not been deorphanized. In contrast, NMUR-2 has been deorphanized, but no phenotypes have been identified. For example, NMUR-2 is activated by peptides encoded by nlp-44 when expressed in HEK-

293 cells (Lindemans et al., 2009a). Finally, CKR-2 is a potential orthologue of the mammalian cholecystokinin (CCK) receptor. CKR-2 is expressed as two distinct isoforms (CKR-2a and CKR-2b) that differ at their carboxy-terminal. Both CKR-2 isoforms are activated by peptides encoded by nlp-12; with NLP-12 having a greater affinity for CKR-2a (Janssen et al., 2008). ckr-2 null animals are resistant to paralysis induced by the cholinesterase inhibitor, aldicarb. Wild-type behavior could be restored in ckr-2 null animals by the selective expression of ckr-2 in cholinergic motor neurons, suggesting that CKR-2 functions presynaptically to enhance cholinergic transmission (Hu et al., 2011). Together, these studies suggest that many aspects of neuropeptidergic signaling in C. elegans may mimic neuropeptidergic signaling in mammals and that given the ease with which behavior can be manipulated at the molecular level suggesting that

C. elegans might be a useful model to dissect the subtitles of neuropeptidergic signaling in mammals.

1.3 Opioid signaling in mammals

Opioid receptors have been attractive targets for pain management since morphine was first marketed as an analgesic in 1817 (Berger and Whistler, 2010). Opioid receptor

16 agonists induce analgesia by acting in several regains of the central nervous system.

Upon activation of peripheral nociceptors endogenous opioid neuropeptides are released by descending brainstem circuits and peripheral cells, and inhibit pain sensation by activating presynaptic G protein-coupled opioid receptors on central nociceptors to inhibit the release of excitatory neurotransmitters such as, glutamate, substance P and calcitonin gene released peptide (CGRP) (Stein, 2013, Lau and Vaughan, 2014). In addition, a more widespread role for opioids in the neuromodulation of other behaviors, such stress, learning and memory, mental illness and mood, renal and hepatic functions, cardiovascular responses, respiration and thermoregulation has become apparent, highlighting the need for additional studies focused on understanding how opioid signaling interacts with other neuromodulators (Stein, 2013, Bodnar, 2014, Galligan and

Akbarali, 2014).

Mammalian opioid receptors are activated by four classes of opioids: 1) endogenous neuropeptides, namely, the enkephalins, dynorphins and endrophins; 2) opium alkaloids such as morphine and codeine; 3) semi-synthetic opiates, including, heroin and oxycodone; 4) fully synthetic opiates, such as methadone. Endogenous opioid peptides differentially activate the four opioid receptor subtypes: mu, delta, kappa and nociceptin. However, the existence of only four opioid receptors is at odds with the many more receptor subtypes identified pharmacologically or with the plethora of signaling pathways identified both in vitro and in vivo. This discrepancy is most likely due to extensive alternative mRNA splicing, posttranslational modification, heterodimerization among the different opioid receptors subunits and the ability of individual ligands to

17 differentially activate downstream signaling pathways (Jordan and Devi, 1999, Devi,

2005).

The three classical opioid receptors, mu, delta and kappa, are about 65-70% identical, with the greatest identity observed in the transmembrane helices and the most divergence in the extracellular loops. These divergent extracellular loops are believed to act as “gates” conferring ligand-selectivity, allowing the different receptor subtypes to discriminate between different opioid peptides. The acute activation of kappa opioid receptors with morphine inhibits adenlyate cyclase and decreases intracellular cAMP and can be reversed by treatment with the antagonist naloxone (Sharma et al., 1975).

Similarly, pertussis toxin prevented inhibitory effects of opiate receptor activation following [3H]enkephalinamide application (Hsia et al., 1984). Together, these data suggest that kappa opioid receptor subtypes are primarily Gαi/o-coupled. In addition, kappa opioid receptors also couple to mitogen-activated protein kinase mediated signaling pathways, with opioid signaling modulating both calcium and potassium channels, typically leading to hyperpolarization and decreased neurotransmitter release, in addition to the traditional Gi/o –mediated negative regulation of adenlyate cyclase,

(Bruchas et al., 2007, Lemos et al., 2012, Galligan and Akbarali, 2014, Nagi and Pineyro,

2014). For example, kappa opioid receptors activate a G-protein-gated inwardly rectifying potassium channel, K(ir)3 to hyperpolarize and inhibit the excitability of serotonergic neurons in the dorsal raphe (Bruchas et al., 2007, Lemos et al., 2012).

Signaling from kappa opioid receptors is also coupled to 1) the activation of potassium channels in many cell types, 2) decreased calcium signaling in the myenteric plexus, inhibiting acetylcholine release and 3) the inhibition of oxytocin and vasopressin release

18 in the neurohypophysis (Cherubini and North, 1985, Kato et al., 1992, Russell et al.,

1993, Rusin et al., 1997, Galligan and Akbarali, 2014, Nagi and Pineyro, 2014).

Similarly, the opioid peptide, Met-Enkephaln inhibited presynaptic glutamate release and significantly attenuated excitatory postsynaptic currents in the solitary tract nucleus catecholamine neurons and reduced the frequency of miniature inhibitory postsynaptic currents without reduction in their mean amplitude in gamma-aminobutyric acid periaqueductal grey neurons, reducing the probability of transmitter release (Vaughan et al., 1997).

The MAPK pathway regulates diverse cellular programs, including apoptosis, cell proliferation, ion channel phosphorylation, protein-protein interactions and transcription factor regulation (Rubinfeld and Seger, 2005, Shaul and Seger, 2007). Opioid receptors differentially activate both extracellular signal-regulated kinases one and two (ERK 1/2) and p38-mediated signaling. For example, kappa opioid receptor signaling induced sustained stimulation of ERK 1/2 that persisted for several hours (Belcheva et al., 2005).

In contrast, mu opioid receptor activation induced only transient stimulation of ERK 1/2 that dissipates within 30 minutes (Belcheva et al., 2005). Kappa opioid receptor stimulation of ERK1/2 is biphasic, with a rapid initial β-arrestin independent peak (10 min) followed by a later sustained activation that is dependent upon β-arrestin (Gesty-

Palmer et al., 2006). Interestingly, kappa receptor mediated ERK1/2 stimulation is agonist and cell-specific. For example, rapid and sustained ERK 1/2 activity was induced by the kappa opioid receptor agonist U69593 in both primary and immortalized asterocytes (McLennan et al., 2008). In contrast, a different kappa receptor agonist, c(2)- methoxymethyl salvinorin B (MOM-sal-B), induced rapid ERK1/2 activation in

19 immortalized astrocytes, but both rapid and sustained ERK1/2 activity in primary astrocytes (McLennan et al., 2008).

The activation of p38 MAPK also plays a critical role in modulating chronic pain

(Watkins et al., 2001). Kappa opioid receptors activate p38 signaling pathways (Bruchas et al., 2007, Xu et al., 2007). Unlike the biphasic ERK1/2 activation by kappa opioid receptors that has a β-arrestin dependent and independent phase, the induction of p38 requires arrestin recruitment (Bruchas et al., 2006). Kappa opioid receptor activation induced p38-mediated increases in astrocyte proliferation following nerve injury and pharmacological blockade of p38 signaling attenuated nerve injury and induced hyperalegesia (Xu et al., 2007). Although the kappa opioid receptor activation of MAPK signaling has been well documented, there is little direct evidence of the downstream consequences.

Different kappa receptor agonists can have different effects on receptor phosphorylation, desensitization and internalization. For example, the kappa receptor agonist dynorphin A induces phosphorylation, desensitization and internalization, a phenomena that does not occur with other agonists, such as etorphine or levorphanol (Li et al., 2003). Similarly, the kappa receptor agonist, Salvinorin A was 40-fold less potent at receptor internalization and down-regulation, than U50488 another kappa receptor agonist (Wang et al., 2005). Interestingly, peptides encoded on the same proprotein can have different effects on kappa opioid receptors. For example, dynorphin A and dynorphin B cause significantly more receptor internalization that alpha-neoendrophin

(Chen et al., 2007). Together, these observations highlight the complexity of kappa opioid receptor signaling and the range of potential downstream targets.

20 1.4 Project focus

Opiates play a key role in the modulation of a host of behaviors including the perception of pain. In the present study, we have characterized the role of opiate- dependent neuropeptide signaling on the modulation of nociception mediated by the two

C. elegans ASH sensory neurons, on the assumption that the pathways modulating nociception in C. elegans might be comparable to those modulating pain in mammals.

Indeed, mammalian opioid receptor agonists, such as morphine and salvinorin A, mimicked food or serotonin, and stimulate the initiation of ASH-mediated aversive responses, but, paradoxically, induced animals to continue forward after the initial backward locomotion was complete, suggesting that, as in mammals, opiates decreased the perception of noxious stimuli or pain. Conversely, the opioid receptor antagonists, naloxone and norbinaltorphimine abolished the serotonin-stimulation of aversive responses. Both morphine and serotonin dependent phenotypes required the expression of the neuropeptide receptor, NPR-17, and npr-17 null phenotypes could be rescued by the expression of the human kappa-opioid receptor, OPKR-1. NPR-17 signaling mimicked that of human kappa-opioid receptor and appeared to involve both Gαo and a MAPK.

NPR-17 functioned in a number of neurons to stimulate aversive responses and appeared to selectively inhibit the release of a subset neuropeptides from the ASI sensory neurons involved in the tyraminergic-dependent inhibition of nociception mediated by the Gαq- coupled α-adrenergic-like receptor TYRA-3. Together, these results strongly support the hypothesis that NPR-17 is a kappa opioid-like receptor and that C. elegans contains endogenous ligands for the human kappa opioid receptor. Indeed, the direct injection of

21 an endogenous human kappa opioid receptor ligand, dynorphin A, mimics serotonin and stimulated aversive responses off food.

To identify endogenous ligands for NPR-17 different predicted neuropeptide null animals were screened for aversive behavior. Aversive responses in either nlp-3 or nlp-24 neuropeptide, mimicked npr-17 null animals, and were not stimulated by serotonin.

Based on epistasis analyses, nlp-24 appeared to act upstream or in parallel to nlp-3 and both nlp-24 and nlp-3 appeared to act upstream of npr-17. Interestingly, only one of three nlp-3 neuropeptides NLP-3.3 stimulated aversive responses, with NLP-3.1 antagonizing

NLP-3.3-dependent stimulation. Indeed, the overexpression of a full length nlp-3 transgene, mimicked serotonin and stimulates aversive responses off food, while the overexpression of a truncated transgene, including NLP-3.1, but not 3.3, abolished serotonin stimulation. Studies on nlp-3 signaling suggest that 1) nlp-3 encoded peptides can be released from a number of different sensory neurons, including the ASHs, to stimulate aversive responses, 2) nlp-3 encoded neuropeptides are released, in part, extra- synaptically and that local/humoral neuropeptide pools play a role in modulation 3) behavioral responses to nlp-3 release are rapid and potentially short-lived, 4) nlp-3 overexpression phenotypes are not the result of developmental compensation and 5) individual neuropeptides encoded by the same gene have the potential to have antagonistic effects on individual behaviors.

Together, these results demonstrate that C. elegans modulates nociception through a complex peptidergic signaling cascade, involving monoaminergic, adrenergic and opioid-like receptors and suggest that C. elegans will be an useful model to untangle the complexities of opiate modulation.

Table 1-1 Deorphanized neuropeptide receptors in C. elegans

Receptor Signaling Ligand(s) EC50 Method Reference NPR-1 Gαo FLP-21 - GLGPRPLRFa Xenopus (Coates and 215F oocytes de Bono, 2002, Rogers et al., 2003) NPR-1 Gαo FLP-18 - EIPGVLRFa Xenopus (Rogers et al., 215V FLP-21 - GLGPRPLRFa oocytes 2003) NPR-3 Gαo FLP-15 - GGPQGPLRFa 250.6nM CHO cells (Keating et FLP-15 - RGPSGPLRFa 162.4nM (GTPγS al., 2003, FLP-15 - GPSGPLRFa 598.6nM assay) Kubiak et al., 2003) NPR-4 Gαo FLP-18 - DVPGVLRFa Xenopus (Cohen et al., FLP-18 - KSVPGVLRFa oocytes 2009) FLP-18 - SEVPGVLRFa FLP-18 - SVPGVLRFa FLP-18 - DFDGAMPGVLRFa FLP-18 - EIPGVLRFa NPR-5 Gαq FLP-18 - DVPGVLRFa 32.3nM Xenopus (Cohen et al., FLP-18 - KSVPGVLRFa 28.3nM oocytes and 2009; Kubiak FLP-18 - SEVPGVLRFa 25.9nM CHO cells et al., 2007) FLP-18 - SVPGVLRFa 14.1nM (Fluo3 Ca2+ FLP-18 - DFDGAMPGVLRFa 117.2nM mobilization FLP-18 - EIPGVLRFa 13.3nM assay) NPR-6 FLP-18 - DVPGVLRFa CHO cells (Lowery et FLP-18 - KSVPGVLRFa 5µM (GTPγS al., 2003) FLP-18 - SVPGVLRFa assay)

23 NPR-10 FLP-18 - SVPGVLRFa 64nM CHO cells (Lowery et FLP-3 - SPLGTMRFa (GTPγS al., 2003; FLP-3 - SAEDFGTMRFa assay) Hapiak et al., FLP-3 - SADDSAPFGTMRFa 60- 2013) FLP-3 - EDGNAPFGTMRFa 330nM FLP-3 - EAEEPLGTMRFa

NPR-11 NLP-1 - MDANAFRMSFa N/A HEK-293 (Chalasani et cells (Fura-2- al., 2010) AM Ca2+ mobilization assay) NPR-22 FLP-7 - SPMERSAMVRFa 0.71µM HEK-293 (Mertens et FLP-1 - KPNFMRYa cells (Fluo-4- al., 2006) FLP-7 - TPMQRSSMVRFa AM Ca2+ FLP-7 – SPMDRSKMVRFa mobilization FLP-7 – SPMQRSSMVRFa assay) FLP-9 – KPSFVRFa FLP-11 - ASGGMRNALVRFa FLP-11 - AMRNAVLRFa FLP-11 - NGAPQPFVRFa FLP-13 - AADGAPLIRFa FLP-13 - ASPSAPLIRFa FLP-13 - ASSAPLIRFa FLP-13 - SAAAPLIRFa FLP-13 - SPSAVPLIRFa FLP-22 - SPSAKWMRFa CKR-2a Gαq NLP-12 - DYRPLQF 27.84nM CHO cells (Janssen et al., NLP-12 - DGYRPLQF 14.71nM (aequorin 2008) Ca2+

24 mobilization assay) CKR-2b NLP-12 - DYRPLQF 56.75nM CHO cells (Janssen et al., NLP-12 - DGYRPLQF 94.71nM (aequorin 2008) Ca2+ mobilization assay) EGL-6 Gαo FLP-10 - QPKARSGYIRFa 11nM Xenopus (Ringstad and FLP-17 - KSQYIRFa 1nM oocytes Horvitz, FLP-17 - KSAFVRFa 28nM 2008) FRPR-3 FLP-7 - TPMQRSSMVRFa 1.02µM HEK-293 (Mertens et FLP-11 - AMRNALVRFa 1.3µM cells (Fluo-4- al., 2004) AM Ca2+ mobilization assay) FRPR-18 Gαq FLP-2 - SPREPIRFa 53.1nM CHO cells (Mertens et FLP-2 - LRGEPIRFa (aequorin al., 2005) Ca2+ mobilization assay) GNRR-1 Gαq NLP-47 - QMTFTDQWTa 150nM HEK-293 (Lindemans et NLP-47 - QMTFTDQWTKa >970nM cells (Fluo-4- al., 2009b) AM Ca2+ mobilization assay) NMUR-2 NLP-44 - AFFYTPRIa 18nM HEK-293 (Lindemans et cells (Fluo-4- al., 2009a) AM Ca2+ mobilization assay)

25 NTR-1 Gαs/q NTC-1 CFLNSCPYRRYa 18.9nM CHO cells (Beets et al., (aequorin 2012) Ca2+ mobilization and CRE- luciferase assay) PDFR-1a Gαs PDF-1 - SNAELINGLIGMDLGKLSAVa N/A CHO cells (Janssen et al., PDF-1 -SNAELINGLLSMNLNKLSGAa (CRE- 2008) PDF-2 - luciferase NNAEVVNHILKNFGALDRLGDVa assay) PDFR-1b Gαs PDF-1 - SNAELINGLIGMDLGKLSAVa N/A CHO cells (Janssen et al., PDF-1 -SNAELINGLLSMNLNKLSGAa (CRE- 2008) PDF-2 - luciferase NNAEVVNHILKNFGALDRLGDVa assay) PDFR-1c Gαo PDF-1 - SNAELINGLIGMDLGKLSAVa N/A CHO cells (Janssen et al., PDF-1 -SNAELINGLLSMNLNKLSGAa (CRE- 2008) PDF-2 - luciferase NNAEVVNHILKNFGALDRLGDVa assay)

Chapter 2

Materials and Methods

2.1 Materials

Restriction enzymes were purchased from New England Biolabs (Beverly,

MA), NLP-3 neuropeptides from Neo-peptide (Cambridge, MA) and salvinorin A from

Tocris (Minneapolis, MN). A plasmid containing the human kappa opioid receptor cDNA was purchased from Open Bioscience (Lafayette, CO). All other chemicals were purchased from Sigma Aldrich (St Louis, MO), including morphine sulfate salt pentahydrate, naloxone, Norbinaltorphimine, Dynorphin A, tyramine and octopamine hydrochloride, serotonin creatine sulfate complex and 1-octanol. cDNA pools were constructed from mixed stage C. elegans mRNA using standard techniques. Green fluorescent protein (GFP) reporter constructs were obtained from Andy Fire (Carnegie

Institute of Washington, Washington, DC).

2.2 Strains

General techniques for the culture and handling of worms have been described

(Brenner, 1974). The Bristol N2 strain was used as wild-type. Strains obtained from the

Caenorhabditis Genetics Center (University of Minnesota, Mn) include dop-5 (ok568)

27 dyf-7 (m539), eri-1(kp3948), frpr-18 (ok2698), gnrr-1(ok238), nmur-1 (ok1387), nmur-2

(ok3502), nmur-3 (ok2295), nmur-4 (ok1381) npr-1 (ok1447), npr-5 (ok1583), npr-7

(ok527), npr-10 (ok1442), npr-11 (ok594), npr-14 (ok2375), npr-15 (ok1026), npr-16

(diDf6), npr-18 (ok1387), npr-19 (ok 2068), npr-20 (ok2575), npr-25 (ok2008),npr-32

(ok2541), npr-33 (gk963311), npr-35 (ok3258), ntr-1 (ok2780), tkr-1 (ok2886), tkr-2

(ok1620), tkr-3 (ok381), t11f9.1(ok2284) and ador-1 (tm3971), ckr-1 (ok2502), ckr-2

(tm3082) nlp-3 (tm2302), nlp-24 (tm2105), npr-4 (tm1782), npr-6 (tm1497), npr-8

(tm1553), npr-9 (tm1652), npr-12 (tm1498), npr-13 (tm1504), npr-17 (tm3210) were received from the National Bio-Resources Project (Tokyo Women’s Medical University,

Tokyo, Japan). The double mutant nlp-3 (tm2302);npr-17 (tm3210) was constructed using standard genetic techniques. All mutant animals were backcrossed with the N2

Bristol strain at least 4X before use in assays or crosses.

2.3 Generation of RNAi strains by bacterial feeding

RNA interference was performed as previously described (Kamath and Ahringer,

2003). eri-1(kp3948) animals were grown on nematode growth medium plates containing

25µg Ampicillin, 1 mM isopropyl-β-D-thiogalactopyranosidase (IPTG) and seeded with

HT115(DE3) bacteria containing either RNAi or empty vector. All animals were cultured at 16°C. Synchronized second generation larval stage four were picked 24 hours pre- assay and examined for 1-octanol sensitivity. The following RNAi animals were generated through feeding: frpr-3, frpr-5, frpr-7, frpr-10, frpr-14, frpr-15, npr-21, npr-

22, npr-26, npr-27, npr-28 and ntr-2.

28 2.4 Molecular biology and transgenesis

cDNA or genomic regions corresponding to the entire coding sequences of nlp-3, nlp-24, npr-17 and oprk-1 were amplified by PCR and expressed under neuron-selective promoters where indicated (for promoter list see table 2). Neuron-selective rescue constructs were created by overlap fusion PCR (Hobert, 2002). Transcriptional and translation fusion constructs for receptor::gfp localization were created by PCR fusion.

PCR products were pooled from at least three separate PCR reactions and co-injected with a selectable marker (myo-3::gfp, unc-122::rfp or F25B3.3::gfp) and carrier DNA into gonads of C. elegans wild-type or mutant animals by standard techniques (Kramer et al., 1990, Mello and Fire, 1995).

2.5 Generation of cell selective RNAi constructs

Neuron selective RNAi constructs were created as previously described (Esposito et al., 2007, Harris et al., 2010, Mills et al., 2012b). A neuron-selective promoter was fused to exon rich regions of the target gene. Exon rich regions were amplified using a forward and reverse primer to create template A by PCR-fusion. Neuron-selective promoters were amplified using a forward primer with reverse (sense) or reverse

(antisense) primers to create templates B and C, respectively. Templates A and B were then fused to create the sense construct (Product D). Templates A and C were fused to create the antisense construct (Product E). PCR products were pooled from at least three separate PCR reactions (25-100 ng/µL) and co-injected with a selectable marker (myo-

3::gfp or unc-122::rfp). Neuron-selective RNAi were injected into either wild-type or mutant animals. Multiple transgenic lines were examined for each construct.

2.6 Behavioral assays

Octanol avoidance

All experiments used age-matched, well-fed young adults grown at 20°C on agar plates containing standard nematode growth medium (NGM), seeded with E. coli strain

OP50. 1-octanol avoidance was assayed as described previously (Chao et al., 2004,

Wragg et al., 2007, Harris et al., 2009). Briefly, fourth-stage larvae were picked 24 hours before testing and maintained at 20°C. Nematode growth medium plates were prepared the morning of the experiment by adding either serotonin, tyramine, or octopamine (final concentration, 4 mM) to liquid nematode growth medium before pouring. Morphine

(320µM), salvinorin A (320µM), naloxone (320µM) or nor-BNI (320µM) were spread on top of test plates immediately prior to incubation. Dilute (30%) 1-octanol was prepared daily using 100% ethanol (vol/vol). For assay, animals were placed on a transfer plate for

10 minutes to minimize bacteria carry over, then transferred to fresh test plates and incubated for 10 minutes (Off food), 20 min (Morphine, Salvinorin A) or 30 min

(serotonin, naloxone, octopamine, tyramine) prior to assay. Five animals were placed on each plate and the blunt end of a hair (Loew-Cornell series 8 paintbrush) dipped in 1- octanol was placed in front of a forward moving animal after it just reversed. The time taken to initiate backward locomotion during a 20 second exposure to 1-octanol was recorded, as wild-type C. elegans spontaneously reverse on average every 20 seconds. In addition, range of post-initiation behaviors were also recorded, including the length of the backward locomotion and the decision to move forward or reverse with or without an

30 omega turn after the initial backward locomotion was complete, as described by Harris et al. (2011).

Spontaneous reversal

The frequency of spontaneous reversal was assayed as described previously (Tsalik and Hobert, 2003). Well-fed animals were transferred to agar plates containing nematode growth medium for 30 seconds, then transferred to assay plates for 1 minute and assayed.

Reversal frequency was scored as the number of times an animal reversed within 3 minutes

(Pierce-Shimomura et al., 1999).

2.7 Peptide injections

NLP-3.1,2,3 peptides and DYN-A were diluted in either water or external buffer

(150 mM NaCl, 5 mM KCl, 5 mM CaCl2, 1 mM MgCl2, 10 mM glucose, 15 mM

HEPES, pH 7.30, 327-333 mOsm) from a 0.001 M stock solution and were injected into test animals close to the nerve ring at concentrations speficied. Peptide dilution in either water or external buffer yielded similar results. As a control, animals were injected with

Texas red fluorescent dye and assayed for aversive responses. The dye diffused rapidly throughout the animals. Immediately after injection, animals were placed on agar plates containing nematode growth medium off food and allowed to recover for 10 minutes prior to assay.

2.8 Microscopy and image analysis

Transcriptional and translational transgenes for npr-17::gfp and nlp-24::gfp were generated by PCR fusion (Hobert, 2002). PCR products were pooled from at least three

31 separate PCR reactions and co-injected with a selectable marker (unc-122::rfp or rol-6) by standard techniques. Neuronal identification was aided by fluorescence from of 1,1’- dioctadecyl-3,3,3’,3’-tetramethylindodicarbocyanine (DiD) was used to identify a subset of six pairs of amphid sensory neurons capable of dye uptake (Herman and Hedgecock,

1990). Briefly, a stock solution (1mM) of DiD (Molecular Probes/Invitrogen Labeling and Detection) was diluted 1:200 in M9 buffer. Larval stage four (L4) animals were incubated in 100 l of diluted DiD for one hour at room temperature, transferred to agar plate containing fresh nematode growth medium seeded with OP50, allowed to crawl on the bacterial lawn for one to two hours to destain and then were placed on 2.5% agarose pads with 2µl sodium azide (10 mM) to immobilize the animals for visualization. At least three transformed lines were analyzed for gfp fluorescence and DiD staining using an

Olympus IX81 confocal microscope.

Table 2-1. Expression of cell selective promoters

Promoter Expression ceh-2 NSM, I3, M3, M4 flp-5 ASE, PVT, RMG, I4 flp-8 AUA, PVM, URX gcy-33 BAG glr-1 AIB, AWB, AVA, AVD, AVE, AVG, PVG, RMD, SMD, PVQ, URY gpa-4 ASI gpa-9 ASJ, PHB, PVQ npr-9 AIB odr-2b AIB, AIZ, ASG, AVG, IL2, PVP, RIV, SIAV, opt-3 AVA sra-6 ASH, ASI srg-47 ASI str-1 AWB tdc-1 RIC, RIM

33 Chapter 3

Results

3.1 Neuropeptides encoded by nlp-3 appeared to be released extra-synaptically from an array of sensory neurons to modulate ASH-mediated aversive responses

As we have demonstrated previously, the ASH expression of peptides encoded by nlp-3 is essential for the serotonergic stimulation of aversive responses (Harris et al.,

2010). In addition to the ASHs, nlp-3::gfp expression is observed in a number of additional sensory neurons (Nathoo et al., 2001). Therefore, to better understand nlp-3 mediated peptidergic signaling, nlp-3 was selectively rescued and overexpressed in individual pairs of sensory neurons endogenously expressing nlp-3 (Figure 1).

Surprisingly, the serotonergic stimulation of 1-octanol dependent aversive responses could be restored in nlp-3 null animals by selective nlp-3 expression in the two AWBs,

BAGs or NSMs (Figure 1; top panel). In contrast, selective nlp-3 expression in the ASJ or ASE neurons did not restore serotonergic stimulation (Figure 1; top panel).

Conversely, selective nlp-3 overexpression in the two ASEs, ASJs, AWBs, BAGs or

NSMs of wild-type animals stimulated the initiation of aversive responses to levels observed on serotonin, i.e., from about 10 to 5 seconds (Figure 1; bottom panel). Perhaps, the low volume release of nlp-3 peptides from the ASJs and ASEs was insufficient to

34 restore serotonin-stimulated aversive responses, whereas nlp-3 selective overexpression, coupled to endogenous release from other neurons, resulted in high volume release capable of more robust diffusion and/or the saturation of extracellular peptidases.

Together, these data suggest that locally/humorally released nlp-3 encoded neuropeptides sensitize the ASH-mediated aversive circuit.

3.2 Peptides encoded by nlp-3 differentially modulate aversive responses

Three different peptides are encoded by nlp-3; two are closely related, NLP-3.1

(AINPFLDSMG) and NLP-3.2 (AVNPFLDSIG), and one is distinct, NLP-3.3

(YFDSLAGQSLG). Each peptide has been isolated directly from C. elegans and characterized by Q-TOF MS/MS (Nathoo et al., 2001, Husson and Schoofs, 2007b).

Importantly, these nlp-3 peptides are conserved among nematodes, with NLP-3.3 nearly identical in nematodes as diverse as C. elegans, Ascaris suum and filarial parasites, such as Loa loa (Figure 2). No NLP-3 orthologues have been identified outside the phylum, although structure is probably more highly conserved than amino acid sequence in these small molecules. To determine which of the nlp-3 encoded peptides was required for the serotonergic stimulation of ASH-mediated aversive responses, each peptide was injected individually into wild-type animals and aversive responses assayed after 10 minutes.

Each peptide was purchased commercially and modified by C-terminal amidation as observed physiologically (Eipper et al., 1992). Mock-injection or the injection of NLP-

3.1 or NLP-3.2 alone had no effect on aversive responses off food (Figure 3A). In contrast, the injection of NLP-3.3 mimicked exogenous serotonin or nlp-3 and stimulated

Figure 1. Peptides encoded by nlp-3 are expressed in multiple sensory neurons modulate ASH-mediated aversive responses. Transgenes composed of a neuron- selective selective promoter fused to nlp-3 cDNA were injected into either wild-type or nlp-3 null animals. Bolded neurons indicate neurons where nlp-3::GFP has previously been reported (Nathoo et al., 2001). Black bars represent wild-type or null animals; green bars animals expressing a nlp-3::nlp-3 transgene. Aversive responses to 30% 1-octanol were assayed with or without 4 mM serotonin. Data presented as mean +/- SE and analyzed by two-tailed student t-test. *p<0.001, significantly different from wild-type animals under identical conditions.

36 aversive responses off food (Figure 3A). This NLP-3.3 stimulation was short-lived and absent 20 minutes after injection, suggesting either that the peptide was rapidly degraded or the receptor desensitized/downregulated (Figure 3A). Interestingly, the more rapid aversive responses observed after NLP-3.3 injection were absent when NLP-3.3 was co- injected with NLP-3.1, suggesting that NLP-3.1 might antagonize the action of NLP-3.3

(Figure 3A). This apparent “inhibition” did not appear to result from increased amounts of peptide injected, as it was also apparent when the concentrations of both peptides were halved (Figure 3A). To further define a role for these potentially inhibitory nlp-3 encoded peptides, an nlp-3 transgene was constructed that included NLP-3.1 and 3.2, but not

NLP-3.3. As predicted, the expression of this truncated transgene had no effect on aversive responses off food, but abolished serotonergic stimulation (Figure 3B).

Together, this data suggest that the effects of nlp-3 expression are rapid, short-lived, not the result of developmental compensation and that individual peptides encoded by the same gene have the potential to have antagonistic effects on individual behaviors.

3.3 Peptides encoded by nlp-3 activate NPR-17 to stimulate aversive responses

G-protein coupled receptors potentially activated by nlp-3-encoded peptides were identified by screening predicted peptide receptor-null mutants for phenotypes identical to those observed in nlp-3 animals, i.e., an absence of serotonin-stimulated aversive responses. The C. elegans genome contains more than 50 genes that encode putative neuropeptide G-protein coupled receptors, some of which have been identified previously based on limited identity to mammalian peptide receptors. For example,

Figure 2. Peptides encoded by nlp-3 are conserved in distantly-related nematodes.

Annotated NLP-3 protein sequences were obtained using NCBI blast and were aligned using Clustal Omega. Red background indicates identical amino acids; blue potential dibasic cleavage sites.

Figure 3. The direct injection of nlp-3 encoded peptides differentially modulates

ASH-mediated aversive responses. Aversive responses to 30% 1-octanol were assayed with or without 4 mM serotonin. Peptides were injected directly into the pseudocoelom and injected animals were incubated off food for 10 min prior to assay of aversive responses unless otherwise indicated. Black bars represent wild-type or null animals; green bars represent animals expressing a transgene; blue bars represent peptide injected animals. Data presented as mean +/- SE and analyzed by two-tailed student t-test

*p<0.001, significantly different from wild-type animals under identical conditions;

**p<0.01, significantly different from wild-type animals injected with NLP-3.3 alone.

39 putative neuropeptide Y (NPY)-like receptors have been identified using this approach

(Cohen et al., 2009). We used a bioinformatics approach to identify predicted neuropeptide receptors (Harris et al., 2010). This analysis used predicted amino acid sequences from truncated receptors lacking potential hypervariable regions, including most of the predicted N and C termini and third intracellular loops; an approach used previously to identify C. elegans monoamine receptors (Smith et al., 2007). Indeed, most of the neuropeptide Y-like receptors identified previously clustered using this approach

(Harris et al., 2010). Putative null alleles were available for many of these predicted neuropeptide receptors; others were examined using RNAi in an eri-1 null background that is more sensitive to neuronal RNAi knockdown (Kennedy et al., 2004). Importantly, eri-1 null animals exhibited wild-type responses to dilute 1-octanol (data not shown).

Surprisingly, serotonin did not stimulate the initiation of aversive responses to dilute 1- octanol in fourteen different predicted receptor null animals or after RNAi knockdown: dop-5, dyf-7, ckr-1, frpr-15, frpr-18, nmur-2, nmur-3, npr-4, npr-5, npr-10, npr-17, npr-

25, ntr-1 and t11f9.1 (Figure 4). This relatively large number of receptors was surprising, even though nlp-3 encodes at least three different neuropeptides, and suggests that interactions between monoaminergic and peptidergic signaling might be more complex that previously appreciated. However, it is important to note that each of these putative null animals also contains a number of additional mutations, even after extensive outcrossing, so that until they are rescued by the expression of the corresponding genomic transgene these results should be considered tentative.

To identify the specific G-protein coupled receptors activated by nlp-

3 peptides, nlp-3 was overexpressed in the receptor null animals that were not stimulated

40 by serotonin. Of the mutants examined, only npr-17-null animals failed to decrease aversive responses off food after nlp-3 overexpression (Figure 5). As predicted, the expression of npr-17 (npr-17p::npr-17) in npr-17-null animals rescued serotonin stimulation (Figure 6A). In addition, the overexpression of npr-17 in wild-type animals mimicked nlp-3 overexpression and stimulated aversive responses off food (Figure 6A).

To determine if NPR-17 is required for NLP-3.3 dependent stimulation of aversive responses, the NLP-3.3 peptide was injected directly into npr-17 null animals. As predicted, the injection of NLP-3.3 into npr-17 null animals did not stimulate aversive responses (Figure 5). npr-17 null animals share additional phenotypes with nlp-3 animals.

For example, upon removal from food the frequency of spontaneous reversal increases significantly in wild-type animals, but not in nlp-3 and npr-17 animals (Figure 6B). Taken together, these data support our hypothesis of NPR-17 is a receptor for nlp-3 encoded peptides, although it is also possible that NPR-17 acts downstream or in parallel to a signaling cascade that includes nlp-3.

3.4 NPR-17 functions in multiple neurons including the ASI sensory neurons to modulate aversive responses

An npr-17::gfp transcriptional transgene appears to be expressed in the AVG interneuron, faintly in the two ASI and AUA sensory neurons and the PVPs, PVQs, and

PQR in the tail (Figure 7A-D). To determine where NPR-17 is functionally expressed to modulate aversive responses to dilute 1-octanol, npr-17 was selectively knocked down using RNAi in neurons expressing npr-17::gfp, as well as a selection of neurons within the ASH-mediated locomotory circuit. The serotonin-stimulation of aversive response

Figure 4. Aversive responses were not stimulated by serotonin in fourteen predicted neuropeptide receptor null mutants. Putative neuropeptide receptor-null mutants were examined for serotonin dependent increases in aversive responses to 30% 1-octanol. Data presented as mean +/- SE and analyzed by two-tailed student t-test *p<0.001, significantly different from wild-type animals under identical conditions.

Figure 5. The neuropeptide receptor encoded by npr-17 appears to be activated by nlp-3-encoded peptides. Green bars represent putative neuropeptide receptor-null mutants overexpressing nlp-3::nlp-3 were examined for aversive responses to 30% 1- octanol with or without 4mM serotonin. Blue bars represent NLP-3.3 peptide injected into wild-type and npr-17 null animals. Data presented as mean +/- SE and analyzed by two-tailed student t-test *p<0.001, significantly different from wild-type animals under identical conditions.

Figure 6. Locomotory phenotypes in nlp-3 and npr-17 null animals are identical.

A. Wild-type and mutant animals were assayed for aversive responses to 30%1-octanol with or without 4 mM serotonin. Green bars represent animals expressing a npr-17::npr-

17 transgene; red bars transgenic animals expressing npr-17 RNAi; black bars wild-type or null animals. B. Animals were assayed for spontaneous reversals immediately off food, as described in methods. Data presented as mean +/- SE and analyzed by two-tailed student t-test *p<0.001, significantly different from wild-type animals under identical conditions.

44 was significantly attenuated or abolished by npr-17 knock-down in the AUAs (pflp-8),

ASIs (pgpa-4 or psrg-47) and probably PVPs (podr-2b), validating our previous identification of these neurons (Figure 8). Conversely, aversive responses were restored in npr-17 null animals when npr-17 was expressed selectively in each of these neurons. For example, npr-17 expression in the ASIs alone was sufficient to rescue serotonin stimulation in npr-17 null animals (Figure 8). These data raise a question that may have broad application for the interpretation of neuron‐selective RNAi and rescue; that is, how can one get an RNAi knockdown phenotype in one pair of neurons in wild-type animals and yet get rescue in a different pair of neurons in null animals. This observation is perplexing, but we have also made similar observations in other signaling pathways, after having rigorously ruled out potential RNAi spreading (Esposito et al., 2007, Harris et al., 2010,

Mills et al., 2012b). These conflicting observations may result from receptor overexpression in the rescued animals, suggesting that experiments focused on the expression of single copy genes are warranted. Together, these data suggest that NPR-17 is functionally expressed in the ASIs, AVGs and PVPs to modulate aversive responses to dilute 1-octanol.

3.5 NPR-17 is a kappa opioid-like receptor

As noted above, nlp-3 encoded peptides and the neuropeptide receptor, NPR-17 are essential for the food or serotonergic stimulation of ASH-mediated aversive responses. NPR-17 is conserved among nematodes and, not surprisingly, is most closely related to the C. brenneri CBN-NPR-17 (89% identity; E value 0.0) and a predicted opioid-like receptor in the distantly-related nematode Brugia malayi (69% identity; E

45 value 2x10-170). In addition, NPR-17 is more distantly related to the human kappa opioid receptor (24% identity; E value 1x10-12), with increased identity in the transmembrane domains (30%) (Figure 9).

To date, four major mammalian opioid receptor subtypes have been isolated, mu

(MOR), delta (DOR), kappa (KOR) and nociceptin; each with their own unique repertoire of endogenous ligands (Waldhoer et al., 2004). To determine if C. elegans has an endogenous opioid signaling system, wild-type animals were incubated with morphine, a broad-spectrum opioid receptor agonist and salvinorin A, a selective kappa opioid receptor agonist. Both morphine and salvinorin A stimulated the initiation of an aversive response to levels observed on serotonin or after nlp-3 overexpression (Figure 10).

Conversely, naloxone a high affinity mu antagonist that also antagonizes kappa and delta opioid receptors and norbinaltorphimine (nBNI), a selective kappa antagonist, inhibited serotonin-stimulated aversive responses (Figure 10). As predicted, aversive responses in npr-17 null animals were not stimulated by morphine or salvinorin A and these responses could be rescued by the expression of an ASI selective npr-17 transgene in npr-17 null animals (Figure 10). Interestingly, although morphine decreased the time taken to initiate withdrawal form a noxious stimulus, morphine also mimicked serotonin and induced the animals to move forward, instead of initiating an omega turn and reversing after the initial backward locomotion was complete, as observed in wild-type animals off food, in keeping with an overall hypothesis of morphine-induced suppression of responses to noxious stimuli of pain (Figure 11).

Figure 7. Fluorescence from an npr-17::gfp is observed in subsets of head and tail neurons. The npr-17 transcriptional transgene includes 5 kb upstream of the start ATG and the first npr-17 exon and intron fused to sequence encoding for GFP. Construct made by Holly Mills, image taken by Vera Hapiak. Animals were viewed by confocal microscopy. A-D. Florescence from wild-type animals expressing an npr-17::gfp transcriptional reporter merged with DiD staining by confocal microscopy. B. z-Section from inset in A.

Figure 8. The serotonin stimulation of aversive response requires the expression of npr-17 in multiple neurons. Animals were assayed for aversive responses to dilute 1- octanol. Green bars represent animals expressing a neuron selective npr-17 transgene; red bars transgenic animals expressing npr-17 RNAi; black bars wild-type or null animals.

Data presented as mean +/- SE and analyzed by two-tailed student t-test *p<0.001, significantly different from wild-type animals under identical conditions. **p<0.001 significantly different from npr-17 null animals under identical conditions.

48 3.6 NPR-17 signaling requires Gαo in the ASIs

Signaling from mammalian kappa opioid receptors is complex, with both G o/i and MAP kinases involved (Prather et al., 1995). To date, only one ortholog of the mammalian G o/i subunit, encoded by goa-1, has been identified in C. elegans (Mendel et al., 1995, Segalat et al., 1995). To determine if NPR-17 is potentially Gαo/i coupled, goa-1 was selectively knocked down using RNAi. The RNAi knockdown of goa-1 in all

NPR-17 expressing neurons, using the npr-17 promoter, or specifically in the ASIs, using the srg-47 or gpa-4 promoters, had no effect on basal aversive responses off food, but abolished morphine or serotonin stimulation (Figure 12). Similarly, morphine or serotonin stimulation was also absent in p38 MAPK null animals (pmk-1) (Figure 12).

Together, these data suggest that, like the human kappa opioid receptors, NPR-17 is also potentially G o/i coupled and may also involve a MAPK to inhibit ASI signaling.

3.7 npr-17 null phenotypes could also be rescued by the expression of the human kappa opioid receptor in the ASIs

Morphine or serotonin-stimulated aversive responses could be rescued in npr-17 null animals by the ASI expression of not only npr-17, but also the human kappa opioid receptor, even though the human kappa opioid receptor exhibits less than 25% amino acid sequence identity to NPR-17 (Figure 13). These data suggest that C. elegans contains endogenous peptides that also activate the human kappa opioid receptor or that the human receptor exhibits constitutive activity. Indeed, many GPCRs exhibit constitutive activity when overexpressed (Tubio et al., 2010). To address this possibility directly the human kappa opioid receptor was expressed in the ASIs of nlp-3;npr-17 null animals.

Figure 9. NPR-17 is conserved across nematode species and has limited identity to the human kappa opioid receptor. Annotated protein sequences were obtained from

NCBI blast and aligned using Clustal Omega. Black shading with white letters indicates identical amino acid sequence. Trans-membrane domains have been highlighted.

Figure 10. NPR-17 is required for the modulation of aversive response by a range of mammalian opioid receptor ligands. Aversive responses to 30% 1-octanol were assayed in the presence of either 320 uM Morphine or Salvinorin A and either 4 mM serotonin alone or in combination with Naloxone or nBNI. Data presented as mean +/-

SE and analyzed by two-tailed student t-test *p<0.001, significantly different from wild- type animals under identical conditions.

Figure 11. Morphine mimics serotonin and modulates not only the indication of the aversive response but also a range of post-initiation locomotory behaviors. Post- initiation responses were assayed off food or in the presence of 4 mM serotonin or

320µM morphine as previously described (Harris et al., 2011). A. Two components of an aversive response were measured. B. Number of body bends during backward locomotion. C. Direction the animals turned following backward locomotion, with or without an omega turn. Data presented as mean +/- SE and analyzed by two-tailed student t-test *p<0.001, significantly different from wild-type animals off food.

Figure 12. NPR-17 appears to be a Gαo-coupled receptor. Aversive responses to 30%

1-octanol were assayed in the presence of either 320 uM Morphine or 4 mM serotonin.

Black bars represent wild-type or null animals and red bars transgenic animals expressing neuron selective RNAi. Data presented as mean +/- SE and analyzed by two-tailed student t-test *p<0.001, significantly different from wild-type animals under identical conditions.

53 Morphine still stimulated the initiation of aversive responses in these transgenic nlp-

3;npr-17 null animals expressing the human kappa opioid receptor (Figure 13). In contrast, serotonin had no effect on aversive responses (Figure 13). Similarly, the direct injection of NLP-3.3 into nlp-3;npr-17 null animals expressing either NPR-17 or the human kappa opioid receptor in the ASIs stimulated the initiation of aversive responses off food (Figure 14). Together, these observations support the hypothesis that C. elegans contains endogenous ligands for the human kappa opioid receptor. The human kappa opioid receptor is activated in vivo by endogenous dynorphin (DYN) peptides, such as

DYN-A. Therefore, DYN-A was injected into the pseudocoelum of either wild-type or npr-17 null animals. As predicted, aversive responses off food were stimulated by the injection of DYN-A into wild-type, but not npr-17 null animals (Figure 14). Together, these data strongly support the hypothesis that NPR-17 is a kappa opioid-like receptor and that C. elegans contains endogenous kappa opioid receptor ligands.

3.8 NPR-17 functions in the ASI sensory neurons to inhibit a tyramine-mediated peptidergic signaling cascade

The monoaminergic inhibition of ASH-mediated aversive responses is focused on the two ASIs (Mills et al., 2012b, Hapiak et al., 2013). For example, the tyraminergic inhibition of the food or serotonin stimulation of responses to 30% 1-octanol requires the

ASI expression of the Gαq-coupled tyramine receptor, TYRA-3, and neuropeptides- encoded by nlp-1, -14, -18. Similarly, and the octopamine inhibition of aversive responses to 100% 1-octanol requires the ASI expression of the Gαq-coupled octopamine receptor, SER-6, and the neuropeptides-encoded by nlp-6, -7, -9 in the ASIs

Figure 13. Expression of the human kappa opioid receptor rescues the serotonin and morphine stimulation of aversive responses in npr-17 null animals. Aversive responses to 30% 1-octanol were assayed with either 320 uM Morphine or 4 mM serotonin. Black bars represent wild-type or null animals; green bars represent animals expressing neuron selective transgenes. Data presented as mean +/- SE and analyzed by two-tailed student t-test *p<0.001, significantly different from wild-type animals under identical conditions.

Figure 14. The injection of NLP-3.3 into npr-17 animals expressing the human kappa opioid receptor or dynorphin, a mammalian kappa opioid receptor agonist, into wild-type animals stimulate aversive responses. Aversive responses to 30% 1- octanol were assayed off food 10 min post-injection. Black bars represent wild-type or mock-injected animals; blue bars peptide-injected animals. Data presented as mean +/-

SE and analyzed by two-tailed student t-test *p<0.01, significantly different from mock injected wild-type animals under identical conditions; **p<0.001, significantly different from DYN-A injected wild-type animals.

56 (Mills et al., 2012b, Hapiak et al., 2013). We hypothesized that ASI NPR-17 mediated

Gαo-signaling might inhibit the release of inhibitory neuropeptides from the ASIs.

Therefore, NPR-17 and the human kappa opioid receptor OPRK-1 were selectively overexpressed in the ASIs of wild-type animals. Surprisingly, the ASI overexpression of either NPR-17 or OPKR-1 abolished the tyramine, but not octopamine, inhibition of aversive responses, but had no effect on aversive responses off food (Figure 15-16).

Conversely, tyramine, abolished the morphine-stimulation of aversive responses (Figure

15). Interestingly, in contrast to the overexpression of NPR-17 in the ASIs, the overexpression of a full length npr-17 genomic transgene stimulated aversive responses, suggesting that additional NPR-17 expressing neurons were involved, as outlined above.

Together, these data further support the identification of NPR-17 as a kappa opioid-like receptor and, more importantly, suggest that ASI NPR-17 signaling might selectively inhibit the tyramine, but not the octopamine-dependent release of ASI neuropeptides.

3.9 Peptides encoded by nlp-24 are also essential for serotonergic stimulation

Interestingly, none of the peptides encoded by nlp-3 appear to exhibit any sequence identity to mammalian dynorphin A or other kappa opioid receptors ligands, although structure is probably more highly conserved than sequence in these small molecules.

Opioid peptides share an N-terminal motif Tyr-Gly-Gly-Phe-(Met or Leu). Therefore, we examined the sequences of predicted C. elegans neuropeptides to identify any with identity to human opioid peptides. Using this approach, we identified nlp-24 that encodes six different peptides with limited sequence identity to the dynorphins, with each peptide

Figure 15. The tyramine inhibition of serotonin stimulated aversive responses is absent in wild-type animals overexpressing either NPR-17 or the human kappa opioid receptor in the ASIs sensory neurons. Aversive responses to 30% 1-octanol were assayed off food or in the presence of the various ligands. Black bars represent wild-type animals and green bars animals expressing an ASI-selective transgene. Data presented as mean +/- SE and analyzed by two-tailed student t-test *p<0.001, significantly different from wild-type animals under identical conditions.

Figure 16. The octopamine inhibition of aversive responses to 100% is not affected in wild-type animals overexpressing either NPR-17 or the human kappa opioid receptor in the ASIs sensory neurons. Aversive responses to 100% 1-octanol were assayed off food or with 4mM octopamine. Black bars represent wild-type animals; green bars animals expressing an ASI selective transgene. Data presented as mean +/- SE and analyzed by two-tailed student t-test *p<0.001, significantly different from wild-type animals under identical conditions.

59 containing a Tyr-Gly-Gly motif. Interestingly, aversive responses in nlp-24 null animals mimicked those of nlp-3 null animals, i.e., they were insensitive to serotonin. As predicted, serotonin stimulation could be restored in nlp-24 null animals by theexpression a full length nlp-24 transgene (Figure 17A). Conversely, nlp-24 overexpression in wild- type animals mimicked serotonin and nlp-3 overexpression phenotypes and stimulated the initiation of aversive responses off food and, more importantly, nlp-24 overexpression phenotypes were absent in npr-17 null animals (Figure 17B). As predicted, morphine stimulated the initiation of aversive responses in nlp-24 null animals (Figure 17A), suggesting that both nlp-24 and nlp-3 acted upstream npr-17.

Together, given the identity of some of the nlp-24 peptides with dynorphin, these results suggested that nlp-24 peptides might be endogenous ligands for NPR-17. Since

NPR-17 is highly conserved among the nematodes, this observation suggests that nlp-24 would also be conserved. However, this is not the case, with nlp-24, in contrast to nlp-3 and npr-17, only exhibiting identity to a gene in the very closely related nematode, C. briggsae, and no other nematode. In addition, although nlp-24 peptides exhibited limited identity to mammalian dynorphin, nlp-24 appeared to act upstream of nlp-3. For example, nlp-3 overexpression phenotypes, i.e., a more rapid initiation of aversive responses than wild-type animals off food, were still present in an nlp-24 null background, but nlp-24 overexpression phenotypes were absent in an nlp-3 null background (Figure 17B).

Together, this data suggests that nlp-24 and nlp-3 operate in a signaling cascade with nlp-

24 acting upstream of both nlp-3 and npr-17 and since all three genes are required for serotonergic stimulation also suggests that serotonin acts upstream or in parallel to nlp-

24.

3.10 Serotonin stimulates the release of nlp-24 encoded peptides from the two ASI sensory neurons

An nlp-24::gfp transgene is expressed in the spermatheca and the two ASI sensory neurons (Nathoo et al., 2001). As predicted, the ASI::RNAi knockdown of nlp-24 abolished the serotonin-stimulation of aversive responses. As noted above, serotonin appears to act upstream of nlp-24; therefore, the Gαs and Gαq-coupled serotonin receptors, SER-7, SER-1 and SER-5 were selectively knocked down in the ASIs of wild- type animals, on the assumption that serotonin stimulated the release of nlp-24 encoded peptides. The ASI RNAi knockdown of ser-5 or ser-7 had no effect on serotonin- stimulation (Figure 18). This result confirms that the RNAi did not spread significantly from the ASIs, as ser-5 null animals fail to respond to serotonin in aversive assays (Harris et al., 2009, Harris et al., 2010). In contrast, the ASI RNAi knockdown of ser-1 mimicked the knockdown of nlp-24 and abolished serotonin-stimulation (Figure 18). Together, these results suggest that serotonin might activate SER-1 on the ASIs and stimulate the release of nlp-24 encoded peptides.

Figure 17. Overexpression phenotypes observed by nlp-24 overexpression are absent in nlp-3 null animals. Aversive responses to 30% 1-octanol were assayed with either 320 uM morphine or 4 mM serotonin. Black bars represent wild-type animals and green bars animals expressing a transgene. Data presented as mean +/- SE and analyzed by two-tailed student t-test *p<0.001, significantly different from wild-type animals under identical conditions.

Figure 18. The serotonergic stimulation of aversive responses requires the expression of SER-1 in the ASIs. Aversive responses to 30% 1-octanol were assayed with 4 mM serotonin. Black bars represent wild-type animals; red bars represent transgenic animals expressing RNAi. Data presented as mean +/- SE and analyzed by two-tailed student t-test *p<0.001, significantly different from wild-type animals under identical conditions.

Chapter 4

Discussion

Opiate analgesics are used extensively to treat chronic pain and their effects are mediated through three distinct classes of opiate receptors that respond to an array of endogenous opioid ligands, including the enkephalins, endorphins and dynorphins. The present study demonstrates that a primitive opioid signaling pathway mediated by the kappa opioid-like receptor, NPR-17 also modulates nociception in the model nematode,

C. elegans. For example, morphine, a broad-spectrum opioid receptor agonist, and salvinorin A, a selective kappa opioid receptor agonist, both stimulate the initiation of withdrawal from a noxious stimulus in C. elegans through a complex pathway requiring

NPR-17. In addition, both morphine and salvinorin A induce animals to move forward after the initial withdrawal response is complete, demonstrating that even in a simple nervous system responses to morphine are complex. Indeed, forward movement toward a noxious stimulus could be interpreted as a suppression of a response to pain, although clearly in this system pain is only operationally-defined.

Somewhat surprisingly, C. elegans npr-17 null phenotypes can be rescued by the expression of not only NPR-17 but also by the human kappa opioid receptor, OPKR-1, even the NPR-17 and OPKR-1 exhibit less than 25% amino acid sequence identity,

64 supporting the observation that NPR-17 is a kappa opioid-like receptor and that C. elegans also contains endogenous opioid-like ligands. Indeed, a neuropeptide encoded by nlp-3, NLP-3.3, when either overexpressed or injected directly, also stimulates this withdrawal reflex in wild-type animals and npr-17 null animals expressing either NPR-17 or OPKR-1, but not in npr-17 null animals. These results suggest that serotonin stimulates the initiation of the aversive response, in part, by modulating a complex, interactive, multicomponent, monoaminergic/ peptideric signaling cascade that results in the differential NPR-17 mediated inhibition of the release of subset of “inhibitory” neuropeptides from the ASI sensory neurons (Figure 18). This level of complexity, in a

“simple” model organism, was unexpected and highlights the potential utility of C. elegans for modeling the complex interactions between monoaminergic and peptidergic signaling involved in the opiate-dependent modulation of nociception.

4.1 Neuropeptides encoded by nlp-3 appear to be released tonically from multiple sensory neurons and function extra-synaptically to modulate behavioral state

As noted in the present study, neuropeptides encoded by nlp-3 are expressed in a number of different sensory neurons, based on fluorescence from nlp-3 promoter::gfp fusions and the observation that genomic nlp-3 rescue and overexpression phenotypes can be mimicked by selective nlp-3 expression in individual pairs of many of the nlp-3 expressing neurons (Nathoo et al., 2001, Harris et al., 2010). These results suggest

Figure 18. The two ASI sensory neurons function as a signaling hub to integrate monoaminergic and peptidergic signaling in the modulation of nociception.

Serotonin stimulates ASH-mediated aversive responses by activating at least three distinct serotonin receptors, one of which SER-1, is required in the two ASI sensory neurons. ASI SER-1 signaling appears to stimulate the release of peptides encoded by nlp-24. These nlp-24 encoded peptides activate an, as of yet, unidentified receptor that stimulates the local/humoral release of nlp-3 encoded peptides from a second set of neurons, that in turn, activate the extra-synaptic opioid-like receptor NPR-17 on the ASIs to selectively inhibit the release of a third group of peptides that inhibit serotonin- stimulation. The release of these inhibitory ASI peptides, encoded by nlp-1, - 14 and 18 is mediated by the Gαq-coupled alpha adrenergic-like tyramine receptor, TYRA-3 (Hapiak et al., 2013). Importantly, the receptors for these ASI peptides appear to be distributed on both sensory and interneurons throughout the sensory-mediated locomotory circuit

(Chalasani et al., 2010, Hapiak et al., 2013).

66 that nlp-3 encoded neuropeptides may be released humorally or more locally onto the nerve ring to define behavioral state. It will be important to determine if the release of nlp-3 encoded neuropeptides is tonic and/or acute and subject to modulation and to define the specific stimuli required for the release. It is important to recognize that many individual behaviors are probably modulated by a delicate balance of extra-synaptically released, synergistic/antagonistic monoamines and neuropeptides. Indeed, the wiring diagram only presents a static picture of a dynamic situation and, at any one time, subsets of synapses can be selected by context-dependent changes in internal state mediated by an array of neuromodulators, with subtle changes in monoaminergic or peptidergic signaling resulting in significant behavioral phenotypes. For example, a host of peptide and peptide receptor overexpression phenotypes have been identified in C. elegans.

Importantly, only two-fold changes in the expression of the tyramine receptor encoded by tyra-3 were sufficient to dramatically alter individual nutritionally-dependent locomotory behaviors, highlighting the subtlety of peptidergic modulation. (Bendesky et al., 2011). A similar delicate balance is also apparent in other organisms. For example, the overexpression of NPY in mammals or the NPY-like receptor, NPFR1 in Drosophila lead to the increased intake of noxious food or obesity, respectively. Similarly, increasing the ratio of CRF1/CRF2 receptors that differentially bind corticotrophin-releasing factor, in the bed nucleus of stria terminalis increased fear responses in rats (Elharrar et al., 2013,

Zheng et al., 2013). These observations suggest we may need to rethink our approach to examining behavioral modulation, since most published studies have focused on “all or none” changes in receptor expression, when the real key to understanding behavior may

67 lie in our ability to comprehend the effects of more discrete, subtle variations in signaling interactions.

Humorally/locally released nlp-3-encoded peptides appear to function extra- synaptically to activate the G o-coupled neuropeptide receptor, NPR-17, in a limited number of neurons, including, the AUAs, AVG, PVPs and the ASIs (Harris et al., 2010).

Indeed, NPR-17 appears to selectively inhibit peptidergic signaling in the ASIs. Although nlp-3 is expressed in a number of sensory neurons, none of these neurons synapse directly on the NPR-17 expressing neurons, suggesting that the release of nlp-3 encoded peptides may be largely extra-synaptic. Indeed, the release of neuropeptides from dense- core vesicles is not restricted to synaptic specializations. For example, oxytocin is released from the dendrites in hypothalamic neurons, dynorphin and vasopressin are co- released from dendrites, axon terminals and soma of vasopressin neurons and gonadotropin-releasing hormone neurons can release GnRH-1 from either their soma or dendrites (Ludwig et al., 2002, Ludwig and Pittman, 2003, Fuenzalida et al., 2011). In fact, neuropeptides can diffuse long distances, with peptidergic fibers and receptors often found in spatially distinct regions of the brain, aided by their relatively long half-life compared to classical transmitters, with this long distance peptidergic signaling aided by high affinity receptors and post-translational modifications designed to prevent degradation (Merkler, 1994, Ludwig and Leng, 2006, Nassel, 2009). Most studies in the mammalian brain have focused on synaptic peptidergic signaling, but it becoming clear that peptides also signal extra-synaptically. Indeed, examples of an anatomical asynchrony between neuropeptide release sites and receptor localization are beginning to be identified. For example, the simultaneous firing of oxytocin neurons raises the

68 extracellular concentration of oxytocin by 100-fold in the supraoptic nucleus, and oxytocin receptors are abundant in the amygdala that is sparsely innervated by oxytocin fibers (Huber et al., 2005, Terenzi and Ingram, 2005, Ludwig and Leng, 2006). Similarly, substance P releasing neurons are abundant in the substantia nigra, despite the absence of tachykinin receptors (Maeno et al., 1993). Conversely, tachykinin receptors are at their highest density in the caudal cerebellum of gymnotiform fish, despite the absence of substance P fibers (Zupanc et al., 1991, Zupanc et al., 1994). Together, these studies highlight the complexities of peptidergic modulation and its role in defining behavioral state.

4.2 Neuropeptides encoded by the same gene can have antagonistic effects on behavior

As noted above, nlp-3 overexpression stimulates aversive responses by activating the opioid-like receptor, NPR-17. The nlp-3 gene encodes three distinct neuropeptides, but the injection of only NLP-3.3-3 mimics nlp-3 overexpression and stimulates aversive responses. In fact, NLP-3.1 appears to abolish both serotonin and NLP-3.3 stimulation, whether co-injected with NLP-3.3 or overexpressed from an nlp-3 transgene lacking sequence for nlp-3.3. The opposing activities of the two nlp-3 peptides could be mediated by the same receptor, with NLP-3.1 and NLP-3.3 competing for a common NPR-17 binding site, or, alternatively, NLP-3.1 could activate a second receptor that is co- expressed in the ASIs or downstream in the locomotory circuitry. In fact, most peptide- encoding genes encode multiple peptides, each with potentially different properties. For example, the dynorphin opioid peptides, dynorphin A, dynorphin B and α-neoendorphin,

69 are encoded by the same gene and although all are potent agonists of kappa opioid receptor, dynorphin A and dynorphin B cause significantly more kappa opioid receptor internalization and down-regulation than α-neoendorphin (Chen et al., 2007). Similarly, each of the C. elegans nlp and flp peptide-encoding genes encode from a single to up to nine different neuropeptides (Li et al., 1999, Nathoo et al., 2001). In addition, it is not uncommon for peptides encoded by the same gene to bind to different receptors. For example, in C. elegans neuropeptides encoded by flp-18 differentially activate NPR-1,

NPR-14 and NPR-5 in the modulation of social feeding, foraging and metabolism

(Rogers et al., 2003, Cohen et al., 2009). Similarly, in mammals, the proopiomelanocortin precursor encodes the opioid peptide β-endorphin, a potent but non-selective, agonist for mu- and delta opioid receptors and the non-opioid peptide hormones, ACTH and α/β/γ- melanocyte-stimulating that activate different melanocortin receptor subtypes (Chang and

Chang, 1983, Raynor et al., 1994, Cortes et al., 2014). Since these nlp-3 encoded peptides are released extra-synaptically and probably degraded rapidly, based on the short half-life of NLP-3.3 stimulation after injection, this arrangement could provide additional levels of modulation, with distinct local/humoral pools with different NLP-3.1/3.3 ratios, dependent on the relative placement and specificity of the different peptide-degrading peptidases. Indeed, C. elegans expresses a wide array of extracellular peptide-degrading enzymes, but little is known about their localization (Turner et al., 2001, Spanier et al.,

2005). For example the neprilysin, NEP-2, is localized to the cell surface of muscle, glia- like cells and neurons and degrades SNET-1 to modulate olfactory plasticity (Yamada et al., 2010). NEP-2 is currently the only example of pairing a peptide-degrading enzyme and a neuropeptide in C. elegans (Yamada et al., 2010). These observations raise

70 intriguing questions about the constraints that limit the transmission and precision of peptidergic signaling and highlight its potential complexity, with multiple potentially antagonistic peptides encoded by the same gene, differently degraded by different spatially-localized peptidases and binding to different receptors. Indeed, it is even possible the neuropeptides encoded by the same or different genes may heterodimerize, as has been observed for G-protein coupled receptors (Jordan et al., 2001, Pfeiffer et al.,

2003, Lee et al., 2004, Somvanshi and Kumar, 2014).

4.3 The ASIs receive modulatory input from three different monoamines

Food stimulates the initiation of ASH-mediated aversive responses by stimulating the local/humoral release of serotonin from the two serotonergic NSM neurons that, in turn, activates three different serotonin receptors throughout the locomotory circuit

(Harris et al., 2009, Harris et al., 2011). In wild-type animals, each of these three serotonin receptors is essential for food or serotonin stimulation, but in various null backgrounds the absence of one receptor can be compensated for by the overexpression of another, suggesting that a complex interactive cascade of serotonergic signaling modulates the locomotory machinery to initiate backward locomotion in response to aversive an stimulus (Harris et al., 2009, Harris et al., 2010, Harris et al., 2011). In the present study, we have dissected a portion of this serotonergic cascade and have presented convincing genetic evidence demonstrating that serotonin activates a Gαq- coupled serotonin receptor, SER-1, on the ASIs to stimulate the release of peptides encoded by nlp-24. These nlp-24 encoded peptides activate an, as yet, unidentified receptor that stimulates the local/humoral release of nlp-3 encoded peptides from a

71 second set of neurons that, in turn, activate NPR-17 on the ASIs to selectively inhibit the release of a third group of peptides from the ASIs that inhibit serotonin stimulation. The release of these inhibitory ASI peptides, encoded by nlp-1, -14 and -18 is mediated the

Gαq-coupled alpha adrenergic-like tyramine receptor, TYRA-3 (Hapiak et al., 2013).

Importantly, the receptors for these ASI peptides appear be distributed on both sensory and interneurons throughout the sensory-mediated locomotory circuit (Chalasani et al.,

2010, Hapiak et al., 2013). Similarly, at higher levels of ASH stimulation, octopamine inhibits aversive responses by the activation of an additional ASI Gαq-coupled receptor,

SER-6, that appears to stimulate the release of a completely different group of ASI peptides, encoded by nlp-6, nlp-7 and nlp-9 that again activate receptors throughout the sensory mediated locomotory circuit (Mills et al., 2012b). These observations suggest that the two ASIs function as “master neurons,” modulating many aspects of behavior in a way not dissimilar from that of the mammalian anterior pituitary that functions as a

“master gland” in modulating an array of physiological processes and behaviors through the hypothalamic/hypophyseal axis (Jacobson, 2005, Nader et al., 2010). Indeed, the secretion of the six anterior pituitary hormones stimulates the release of array of additional hormones from peripheral endocrine glands, such as the thyroid, adrenal cortex and gonads, with its own secretion selectively modulated by expansive peptidergic inputs from the hypothalamus conveyed by the hypothalamic/hypophyseal portal system

(Herman et al., 2003, Jacobson, 2005, Fekete and Lechan, 2014).

The present results also suggest that at least three distinct Gαq-coupled monoamine receptors are expressed on the ASIs that stimulate the release of distinct subsets of neuropeptides. Indeed, although each of these three monoamine receptors

72 couples to Gαq in heterologous assays, they could differently couple to additional G- protein dependent or independent signaling pathways in vivo to account for the differential release. Numerous examples of both selective G-protein-dependent and independent signaling have been described that range from G-protein coupled receptor switching G subunits under different conditions to the differential G-protein independent interaction of C-terminal PDZ motifs with different PDZ-domain containing binding partners. For example, β2-adrenergic receptors switch coupling from G s to G i the protein kinase A-mediated phosphorylation and also bind a Na+/H+ exchanger regulatory factor through a PDZ-domain-mediated interaction that facilitates Na+/H+ exchange (Daaka et al., 1997, Hall et al., 1998) . Similarly, G-protein βγ subunits, upon disassociation of the GTP-bound α-subunit, can independently inhibit Ca2+ and activate

K+ channels and modulate an array of additional signaling pathways (Huang, 1995, De

Waard et al., 2005).

Mammalian monoamine and opioid receptors also can exist as heterodimers and heterodimerization can alter the pharmacological properties of given G-protein coupled receptors to regulate receptor-activated signaling. For example, the serotonin 5-HT1A and

5-HT7 receptors form heterodimers that decrease 5-HT1A-mediated activation of Gαi and a G-protein-gated inwardly rectifying potassium channel, while enhancing activation of

MAPK pathways (Renner et al., 2012). Similarly, D1 and D2 dopamine receptors heterodimerize, resulting in a switch in G-protein signaling from Gαs and Gαo, respectively, to Gαq (Lee et al., 2004). Kappa and delta opioid receptors form a heterodimer that has no significant affinity for either kappa or delta receptor selective agonists or antagonists, but strong affinity for partially-selective ligands (Jordan and

73 Devi, 1999). Additionally, opioid receptors can also form heterodimers with non-opioid receptors. For example, delta opioid receptors form heterodimers with somatostatin receptor 4, leading to greater inhibition of cAMP and modulation of ERK 1/2 than either receptor alone (Somvanshi and Kumar, 2014). Similarly, mu opioid receptors heterodimerize with Substance P receptors (NK1) to dramatically modify receptor internalization (Pfeiffer et al., 2003). In addition, G-protein coupled receptors that couple to different G-proteins can form heterodimers. For example, β2-adrenergic receptors that couple to Gαs can heterodimerize with the Gαo-coupled delta and kappa opioid receptors, with heterodimerization significantly modifying agonist-mediated internalization (Jordan et al., 2001). Finally, opioid receptors not only activate classical G-protein signaling pathways, but also MAPKs to exert their regulatory effects (Belcheva et al., 2005,

Bruchas et al., 2007). Indeed, these observations suggest that even though these three ASI expressed monoamine receptors might signal through G q in heterologous systems, there is still a significant potential for differential signaling in vivo.

However, in spite of potential differences in signaling from these three ASI monoamine receptors, it is also likely that neuropeptide release in the ASIs is compartmentalized, with different peptides localized to different dense-core vesicles and/or receptors localized to different sites within the neurons. Indeed, although C. elegans neurons appear to be isopotential, with the G-protein coupled receptor-mediated activation of TRPV-channels sufficient to initiate neurotransmitter release, increases in internal calcium would probably be required for dense-core vesicle release that would be dependent on the relative position of L-type voltage-gated calcium channels, such as

EGL-19, and the release of calcium from internal stores, modulated at least in part by

74 localized G-protein coupled receptor signaling (Simmons et al., 1995, Sabatier et al.,

1997, Ludwig et al., 2002). Indeed, localized calcium signaling has been observed in the two C. elegans RIA interneurons, with GAR-3, a G-protein coupled muscarinic acetylcholine receptor, independently mediating calcium events in the ventral and dorsal segments of its axon (Hendricks et al., 2012). The compartmentalization of calcium signaling was also observed in the C. elegans AIY interneurons, where calcium increased in the neurite, but not the soma, in response to odorant removal (Chalasani et al., 2007).

Similarly, calcium increases were reported in AIY axon varicosities adjacent the nerve ring, but not in the soma when animals were exposed to temperature oscillations (Clark et al., 2006). Spatially-restricted calcium signals induced by local calcium stores have also been observed in higher organisms. For example, the peptide bradykinin induces the release of internal calcium at junctional microdomains leading to the activation of a calcium-activated chloride channel in nociceptive sensory neurons in the dorsal root ganglion (Jin et al., 2013). Local calcium signals could also result from calcium entry from differentially localized calcium channels. For example, a spatially restricted dendritic calcium signal mediated by nicotinic acetylcholine receptors was observed in blowfly tangential neurons that were not present in other branches of the dendrite or axon

(Oertner et al., 2001). Although compartmentalized calcium signals have only been reported in a limited number of C. elegans neurons, genetic data from this study and others suggest that local calcium signaling within a single neuron is probably more widespread, occurring in most, if not all C. elegans neurons.

75 4.4 Opioid signaling in other invertebrates

The role of opioid signaling in modulating nociception in higher organisms has been extensively studied. However, less is known about its role in invertebrates.

Interestingly, morphine and its active metabolite, M6G, have been isolated directly from the parasitic nematodes, Ascaris suum and Dracunculus medinesis (Pryor et al., 2005).

Although the neuroanatomies of C. elegans and its distant relative, A. suum are nearly identical, neither endogenous morphine nor a morphine metabolite have been identified in C. elegans or other free living nematodes. The production of endogenous morphine may be required to suppress the hosts’ immune response in the parasites, since morphine can act as an immunosuppressant, perhaps limiting its role to the parasites. Exogenous morphine modulates thermal aversion in both C. elegans and A. suum. Interestingly, morphine had no effect on the initiation of “rapid reflex withdrawal” to heat, but dramatically altered the post-withdrawal response by inducing animals to backup less and continue moving forward, mimicking exactly the C. elegans aversive response to 1- octanol on serotonin or morphine observed in the preset study (Nieto-Fernandez et al.,

2009). It appears that primitive endogenous opioid signaling may have been directly primarily at enhancing the withdrawal away from noxious stimuli, but in higher organisms was expanded to also suppress the psychological perception of pain by down regulating the signaling pathways associated with pain perception, as foreshadowed in C. elegans by the ability of serotonin or morphine to suppress reversal after the initiation of the aversive response.

76 4.5 Conclusions

The results of the present study have defined a primitive opioid signaling pathway in C. elegans that is involved in a complex, serotonin-dependent signaling cascade that modulates multiple components of the sensory-mediated locomotory circuit. This signaling cascade includes serotonin, -adrenergic-like and neuropeptide receptors and ultimately differentially modulates the release of a subset of ‘inhibitory” neuropeptides from the two ASI sensory neurons to modulate aversive responses. Together, these results highlight the complex interactions of monoaminergic and peptidergic signaling in a

“simple” organism, such as C. elegans and the potential utility of the C. elegans model to untangle the complexities of opiate signaling in higher organisms.

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