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Home , Arachidonoyl serotonin, Cannabidivarin, Cannabigerol, Cannabinoidergic, JZL184

A Dissertation

Entitled

Uncovering Signaling in C. elegans: A New Platform to Study the Effects

of Medicinal

By

Mitchell Duane Oakes

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

Doctor of Philosophy Degree in

Biology

______Dr. Richard Komuniecki, Committee Chair

______Dr. Bruce Bamber, Committee Member

______Dr. Patricia Komuniecki, Committee Member

______Dr. Robert Steven, Committee Member

______Dr. Ajith Karunarathne, Committee Member

______Dr. Jianyang Du, Committee Member

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

The University of Toledo August 2018

Copyright 2018, Mitchell Duane Oakes

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

Uncovering Cannabinoid Signaling in C. elegans: A New Platform to Study the Effects

of

By

Mitchell Duane Oakes

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

The University of Toledo August 2018

Cannabis or marijuana, a popular recreational drug, alters sensory perception and exerts a range of medicinal benefits. The present study demonstrates that C. elegans exposed to (CBs) exhibit a “dazed and confused” phenotype, with delayed responses to aversive stimuli, decreased feeding rates, slowed locomotion and increased unproductive turning. These CB-dependent responses are mediated by distinct signaling pathways. For example, C. elegans synthesizes the endogenous CBs, 2- arachidonoylglycerol (2-AG) and (AEA), and contains a canonical CB signaling system, involving the human CB1 orthologue, NPR-19. The CB-dependent inhibition of feeding and nociception, as measured by pharyngeal pumping and the initiation of aversive responses to the noxious repellant, 1-octanol, respectively, are absent in npr-19 null animals and can be rescued by the expression of human CB1 driven by the npr-19 promoter. Similarly, inhibiting the breakdown of 2-AG or AEA mimics their addition and also inhibits feeding and nociception. Importantly, CBs directly activate NPR-19 after heterologous expression in Xenopus laevis oocytes with nM iii affinities similar to those human CB1, confirming NPR-19 as the canonical cannabinoid in C. elegans.

The CB-dependent inhibition of nociceptive responses also requires a D1-like receptor, DOP-1. Importantly, the CB-dependent inhibition of nociceptive responses is still present in cat-2 null animals that lack a tyrosine hydroxylase, a key required for dopamine (DA) , suggesting that CBs activate DOP-1 directly. At higher CB concentrations, the inhibition of nociceptive responses also requires the α2--like receptor, OCTR-1, and the 5-HT1-like receptor, SER-4, suggesting a complex interaction between octopaminergic and signaling. In fact, CBs activate OCTR-1 directly after heterologous expression in Xenopus laevis oocytes, but does not activate SER-4, suggesting that requirement for SER-4 to mediate CB-dependent inhibition of nociception is indirect.

In contrast, CBs also signal through NPR-19-independent pathways to inhibit locomotion and increase turning behavior, including reversals and omega turns. These

NPR-19-independent signaling pathways require multiple TRP channels and the release of both serotonin (5-HT) and DA from subsets of neurons. For example,

CB-dependent locomotory inhibition is dramatically reduced in tph-1 and cat-2 null animals that encode key required for 5-HT and DA biosynthesis, respectively, and in ser-4 and dop-4 null animals that encode a 5-HT1-like 5-HT receptor and a D1-like

DA receptor, respectively. CB-stimulated 5-HT release activates SER-4 in the two key

AIB interneurons, leading to “locomotory confusion” and transient paralysis. CB- dependent locomotory inhibition is also absent in cat-4 null animals that encode an orthologue of the human GTP cyclohydrolase 1 and contains reduced levels of both 5-HT iv and DA. CB-dependent locomotory inhibition is rescued in cat-4 null animals by incubation in either 5-HT or DA, suggesting that the overstimulation of either 5-HT or

DA signaling compensates for the absence of the other. Importantly, CBs also inhibit locomotion in remodeled, chimeric mutant animals designed to detect acute release of either 5-HT or DA. For example, the Gαo-coupled 5-HT (SER-4) and DA (DOP-3) receptors were expressed in the motor neurons of 5-HT receptor quintuple null animals (ser-5;ser-4;mod-1;ser-7 ser-1; 5-HT quints) and DA receptor quadruple null animals (dop-2; dop-4 dop-1 dop-3; DA quads) that lack most, if not all, 5-HT and

DA receptors, respectively. Theoretically, any CB-dependent monoamine release in these animals will activate its cognate receptor on the motorneurons, inhibit release onto the muscle and inhibit locomotion. As predicted, neither 5-HT nor DA have any effect on locomotory rate in the 5-HT quint and DA quad mutant, respectively, but rapidly inhibit locomotion following the selective expression of the appropriate inhibitory

Gαo-coupled monoamine receptor in the cholinergic motorneurons. CBs also increase reversal and the frequency of omega turn in an NPR-19-independent manner through a pathway requiring 5-HT, but not DA signaling.

Using a reverse genetics approach, our lab found that CB-dependent locomotory inhibition is absent in ocr-1, ocr-4, osm-9 and trp-4 null animals that lack functional TRP channel subunits. ocr-1, ocr-4, osm-9 encode TRPV-1 like channel subunits expressed in a number of sensory neurons and trp-4 the pore-forming subunit of a mechanosensitive TRPN (nompC) channel expressed in the neurons.

Similarly, 2-aminoethoxydiphenol (2-APB), a non-selective TRP channel blocker also inhibits CB-dependent locomotory inhibition. More importantly, 2-APB also inhibits CB-

v dependent locomotory inhibition in the monoamine receptor mutants expressing Go- coupled monoamine receptors in the motorneurons in a system designed to measure acute monoamine release, suggesting that CB-dependent TRP channel activation is required for monoamine release and locomotory inhibition. Interestingly, the temporal pattern of 2-

AG inhibition differs significantly in the 5-HT/TRPV-1 and DA/TRP4 -deficient signaling mutants. For example, in mutants with disrupted TRPV-1 or serotonergic signaling, 2-AG-dependent inhibition was initially delayed, but eventually these mutant animals began to slow (25% at 30 min). In contrast, in mutants with disrupted DA or

TRP-4 signaling, 2-AG never inhibited locomotion and, in fact, 2-AG rapidly (<30 sec) and significantly (>25%) stimulated locomotion in these mutants, suggesting the TRP-4 might be modulating DA release.

This study highlights the advantages of studying cannabinoid signaling in a genetically-tractable, whole-animal model and might also explain the proposed anthelmintic properties of Cannabis, given that the CB-dependent locomotory inhibition mimics the “locomotory confusion” phenotype previously proposed as a potential anthelmintic target by our lab. These studies provide insights into the potential effects of

Cannabis on monoaminergic signaling and suggest a role for CBs in activating the dopaminergic-mediated reward system and anxiety/depression in humans.

vi

I would like to dedicate the work in this study to my mentor Dr. Richard Komuniecki.

None of the work in this project would be possible if he had not allowed me to independently explore a topic I was interested in and turn it into a very successful project.

I do not think many, if any other PIs would trust a new graduate student to stop working on a project that had been on going in the lab for more than 15 years to start a new project on a controversial subject that was not even thought to exist in our model organism. Most importantly, I cannot thank Dr. Komuniecki enough for teaching me how to think critically, analyze carefully and to never stop asking questions. To me, learning to think critically is unequivocally the most important skill I have gained in my Ph. D and will undoubtedly help me become a successful researcher and veterinarian in the future.

v

Acknowledgements

A special thank you to my advisor Dr. Richard Komuniecki for transforming me from a naïve and unfocused undergraduate into the passionate, thinking scientist I am today. I cannot thank Dr. Komuniecki enough for the constant mentoring over the past 5 years, inside the lab and out. I am extremely grateful for his patience toward my countless idiosyncrasies and allowing me to explore my many strange hobbies including culturing tardigrades, growing plants and keeping a lot of fish in the lab.

A heartfelt thank you to my lab mate and now lifelong friend Dr. Wen Jing Law, who contributed greatly to the studies in this project. I am extremely grateful to him for performing the locomotory studies used in this study, teaching me everything I know about the art of keeping fish and most importantly, for being my ‘red pill’. I will be forever grateful to you for opening my eyes to the world and pulling me from the matrix.

I would also like to thank my former lab mate and now lifelong friend Dr. Tobias

Clark for putting up with my many strange quirks and many engaging debates and discussions throughout the years. A special thank you for taking the images for my first publication, helping to localize NPR-19 and teaching me basic confocal microscopy and image analysis. I hope to have many more collaborations in the future.

Lastly, I would like to thank Dr. Partricia Komuniecki, Dr. Bruce Bamber, Dr.

Robert Steven, Dr. Ajith Karunarathne and Dr. Jianyang Du for taking the time to be on my committee. vi

Table of Contents

Abstract ...... iii

Acknowledgements ...... vi

Table of Contents ...... vii

List of Figures ...... xi

List of Abbreviations ...... xiv

List of Symbols ...... xvii

Chapter 1. Introduction

1.1 The Endocannabinoid Signaling System ...... 1

1.2 Cannabis and Phytocannabinoids ...... 6

1.3 Therapeutic Potential of Cannabinoids ...... 9

1.4 Non-CB1/CB2-Dependent Cannabinoid Signaling ...... 10

1.5 Cannabinoid Interaction with TRP Channels ...... 13

1.6 -Dependent Activation of GIRK Channels ...... 14

1.7 and Adrenergic Interactions in Mammals ...... 15

1.8 Cannabinoidergic and Dopaminergic Interactions in Mammals ...... 17

1.9 Cannabinoidergic and Serotonergic Interactions in Mammals ...... 19

1.10 Cannabinoid Signaling in Nematodes: What Is Known? ...... 21

1.11 Nociception to 1-octanol ...... 24 vii

1.12 Neuronal Control of Pharyngeal Pumping in C. elegans ...... 28

1.13 Dopaminergic/Serotonergic Modulation of Locomotory Behavior in C. elegans ...... 30

Chapter 2. Materials and Methods

2.1 Nematode Strains ...... 33

2.2 Fusion PCR and Transgene Construction ...... 34

2.3 Octanol Avoidance Assay ...... 34

2.4 Pharyngeal Pumping Assay ...... 35

2.5 Feeding Assay ...... 36

2.6 Locomotion Assay ...... 36

2.7 Reversal Assay ...... 37

2.8 Heterologous Expression in Xenopus laevis ...... 37

2.8.1 Generation of Expression Vectors ...... 38

2.8.2 Two-electrode Voltage Clamp ...... 38

2.9 Confocal Imaging...... 39

2.10 C. elegans Microinjection ...... 40

2.11 Reagents and Supplies ...... 41

Chapter 3. Results

3.1 The endocannabinoids, 2-AG and AEA, inhibit aversive behavior...... 43

3.2 The 2-AG/JZL184-dependent inhibition of aversive responses requires is

absent in npr-19 null animals ...... 48

3.3 2-AG and AEA directly activate NPR-19 heterologously expressed in

viii

Xenopus laevis oocytes ...... 54

3.4 DOP-1 is required for the 2-AG-dependent inhibition of 5-HT stimulated

aversive responses ...... 57

3.5 Serotonin and octopamine receptors are required for the inhibition of

nociception at higher 2-AG concentrations ...... 59

3.6 Serotoninergic signaling is required for the cannabinoid-dependent

inhibition of aversive responses to 100% 1-octanol ...... 65

3.7 NPR-19 is expressed in a limited number of neurons and expression in the

URX sensory neurons is required for the cannabinoid-dependent inhibition

of aversive responses ...... 68

3.8 The cannabinoid-dependent inhibition of pharyngeal pumping and feeding

requires NPR-19 expression in the M3 pharyngeal neurons ...... 71

3.9 Cannabinoids modulate locomotory behavior through NPR-19-independent

signaling pathways ...... 77

3.10 The cannabinoid-dependent inhibition of locomotion requires the release

of 5-HT ...... 80

3.11 The cannabinoid-dependent inhibition of locomotion requires DA release

and cannabinoids stimulate locomotion in animals deficient in

dopaminergic signaling ...... 86

3.12 Cannabinoids stimulate the release of both 5-HT and DA in an assay

system developed to detect acute 5-HT and DA release...... 88

3.13 Identification of the neurons involved in the cannabinoid-dependent

5-HT and DA release ...... 91

ix

3.14 TRP channels are involved in the cannabinoid-dependent inhibition of

locomotion ...... 93

Chapter 4. Discussion

4.1 Overview ...... 102

4.2 Cannabinoid signaling through NPR-19-independent pathways stimulates

5-HT and DA release ...... 105

4.3 TRP channels are essential for the cannabinoid-dependent 5-HT and

DA release ...... 107

4.4 Cannabinoids modulate additional behaviors in nematodes ...... 108

4.5 Similarities in cannabinoid signaling between mammals and nematodes ..113

4.6 Cannabinoids as anthelmintics ...... 120

4.7 The cannabinoid-dependent inhibition of feeding as a potential

anthelmintic target ...... 121

4.8 Conclusion ...... 125

References ...... 121

x

List of Figures

1-1 Molecular structures of endocannabinoids 2-AG and AEA ...... 5

1-2 Molecular structures of phytocannabinoids THC and CBD ...... 8

3-1 BLAST of human cannabinoid receptor 1 (CB1) in C. elegans ...... 45

3-2 2-AG and AEA dramatically inhibit stimulated aversive responses to 1-octanol .46

3-3 Inhibiting the mammalian MAGL orthologue y97e10al.2 mimics 2-AG ...... 47

3-4 2-AG does not inhibit aversive responses in npr-19 null animals or following

npr-19 RNAi knockdown ...... 50

3-5 The MAGL inhibitor JZL184 does not inhibit aversive responses in npr-19 null

animals or following npr-19 RNAi knockdown ...... 51

3-6 npr-19 or CB1 expression driven by a minimal npr-19 promoter restores 2-AG

sensitivity in npr-19 null animals ...... 52

3-7 Comparison of CB1 and NPR-19 sequences ...... 53

3-8 2-AG and AEA activate NPR-19 after heterologous expression in Xenopus

laevis oocytes ...... 55

3-9 2-AG and AEA dose-response curves for NPR-19 heterologously expressed in

Xenopus laevis oocytes ...... 56

3-10 2-AG-dependent inhibition of 5-HT stimulated aversive responses requires the

D1-like receptor DOP-1...... 58 xi

3-11 OCTR-1 and SER-4 are required for inhibition of aversive responses at higher

2-AG concentrations ...... 60

3-12 octr-1 rescue of 2-AG-dependent inhibition of aversive responses in octr-1 null

animals ...... 61

3-13 2-AG dose-response curve for OCTR-1 heterologously expressed in Xenopus

laevis oocytes ...... 62

3-14 SER-4 expression in the AIB interneurons rescues the 2-AG-dependent inhibition

of aversive responses in ser-4 null animals ...... 63

3-15 2-AG has no effect on SER-4 heterologously expressed in Xenopus laevis

oocytes ...... 64

3-16 The 2-AG-dependent inhibition of aversive responses to 100% 1-octanol requires

serotonergic, but not dopaminergic signaling ...... 66

3-17 The 2-AG-dependent inhibition of aversive responses to 100% 1-octanol does not

require dopamine receptors ...... 67

3-18 NPR-19 is expressed in a limited number of neurons, including URX and M3 ....69

3-19 NPR-19 functions in the URX neurons to inhibit aversive responses to 100%

1-octanol ...... 70

3-20 Endocannabinoids 2-AG and AEA inhibit pharyngeal pumping in a

dose-dependent manner ...... 73

3-21 The 2-AG and AEA-dependent inhibition of pharyngeal pumping requires

NPR-19 ...... 74

3-22 NPR-19 functions in the M3 pharyngeal neurons to inhibit pharyngeal

pumping ...... 75

xii

3-23 The 2-AG and AEA-dependent inhibition of feeding requires NPR-19...... 76

3-24 2-AG inhibits locomotion in a dose-dependent manner ...... 78

3-25 The 2-AG-dependent, NPR-19-independent locomotory inhibition requires the

expression of SER-4 in the AIB interneurons ...... 79

3-26 2-AG-dependent locomotory inhibition requires 5-HT signaling ...... 83

3-27 Pre-incubation in 5-HT or DA restores 2-AG-dependent locomotory inhibition

in cat-4 null animals...... 84

3-28 The 2-AG-dependent stimulation of reversal frequency is dependent on

serotonergic signaling through MOD-1 and SER-1 ...... 85

3-29 2-AG-dependent locomotory inhibition requires DA signaling ...... 87

3-30 2-AG inhibits locomotion in remodeled C. elegans designed to detect acute 5-HT

or DA release ...... 90

3-31 2-AG-dependent inhibition of locomotion requires 5-HT and DA from the sensory

ADF and ADE neurons, respectively...... 92

3-32 Several TRP channel subunits are required for the 2-AG-dependent locomotory

inhibition ...... 97

3-33 The temporal pattern of 2-AG-dependent locomotory inhibition in osm-9 and

trp-4 null animals is similar to that of 5-HT and DA deficient animals ...... 98

3-34 At high concentrations, the TRP channel inhibitor, 2-APB, inhibits basal

locomotion in wild-type animals ...... 99

3-35 The TRP channel inhibitor, 2-APB, abolishes aversive responses to 100%

1-octanol in WT animals and the 2-AG dependent locomotory inhibition in

remodeled C. elegans designed to detect acute 5-HT release ...... 100

xiii

3-36 OSM-9 and TRP-4 function in the serotonergic and dopaminergic neurons,

respectively, to mediate 2-AG-dependent locomotory inhibition ...... 101

4-1 Comprehensive diagram of CB-dependent signaling in C. elegans ...... 112

List of Abbreviations

α2A-AR ...... α2A- Δ9-THC ...... Δ9- Δ9-THCV ...... Δ9-

2-AG ...... 2-arachidonoylglycerol 2-APB ...... 2- aminoethoxydiphenyl borate 5-HT ...... 5-hydroxytrptamine

AA ...... AA-5-HT ...... N- ACh ...... Acetylcholine AEA ...... N-arachidonoylethanolamine or Anandamide AMT ...... Anandamide Membrane Transporter ARS ...... Area Restricted Search

CB ...... Cannabinoid CB1 ...... Cannabinoid receptor type 1 CB2 ...... Cannabinoid receptor type 2 CBD ...... C. elegans ...... Caenorhabditis elegans C. indica ...... C. ruderalis ...... C. sativa ...... ChR2 ...... Channel rhodopsin 2 CNG ...... Cyclic Nucleotide-Gated CNS ...... Central Nervous System CRH ...... Corticotrophin Releasing Hormone Cu2+ ...... Copper

DA ...... Dopamine xiv

DAG ...... Diacylglycerol DAGL ...... Diacylglycerol Lipase DR ...... Dietary Restriction eCBs ...... Endocannabinoids ECS ...... Endocannabinoid Signaling System EPEA...... Eicosapentaenoyl ethanolamide

FAAH ...... Fatty Acid Amide Hydroxylase F0 ...... Primary or Parent generation F1 ...... First generation F2 ...... Second generation

GABA ...... gamma-Aminobutyric Acid GFP ...... Green Fluorescent Protein GIRK ...... G-protein Inwardly Rectifying Potassium Channel Glu...... Glutamate GPCR ...... G Protein-coupled Receptor GPR18 ...... Deorphanized G Protein-coupled Receptor 18 GPR35 ...... Deorphanized G Protein-coupled Receptor 35 GPR55 ...... Deorphanized G Protein-coupled Receptor 55 GPR119 ...... Deorphanized G Protein-coupled Receptor 119

HPA...... Hypothalamic-pituitary-adrenal axis

JZL184 ...... Selective MAGL inhibitor

L4 ...... C. elegans larval stage 4 LPA ......

MAGL ...... µM ...... Micromolar mM ...... Millimolar mPFC ...... Medial prefrontal cortex

N2 ...... Wild-type NAc ...... Nucleus accumbens NADA ...... N-arachidonoyl dopamine NAE ...... N-acylethanolamine NAGly ...... N-arachidonoyl NAPE ...... N-arachidonoyl NAPE-PLD ...... N-arachidonoyl phosphatidylethanolamine-phospholipase D NE ...... ng...... Nanogram NGM ...... Nematode Growth Media nM ...... Nanomolar xv nompC ...... No Mechanoreceptor Potential C NPR ...... Receptor

OA ...... Octopamine OEA ......

pCB ...... Phytocannabinoids PEA ...... Palmitoylethanolamine PGE2 ...... Prostaglandin PI3K ...... Phosphoinositide 3-kinase PLCβ ...... Protein Lipase C β PLD ...... Protein Lipase D PNS ...... Peripheral Nervous System PSI-BLAST ...... Position-Specific Iterative-BLAST PTX ...... Pertussis Toxin sCB ...... SYK...... Spleen Tyrosine Kinase

TA ...... TRP ...... Transient Receptor Potential Channel TRPN ...... Transient Receptor Potential nompC Channel TRPV ...... Transient Receptor Potential Vanilloid Receptor TRPV1 ...... Transient Receptor Potential Vanilloid Receptor type 1

URB597 ...... Selective FAAH Inhibitor

VMAT ...... Vesicular Monoamine Transporter

W/V ...... Weight/Volume

xvi

List of Symbols

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

xvii

Chapter 1

Introduction

1.1. The Endocannabinoid Signaling System

Collectively, cannabinoids (CBs) are lipophilic compounds that activate CB receptors and exist in two natural classes, phytocannabinoids (pCBs) and endocannabinoids (eCBs). Since the discovery of the endocannabinoid signaling system

(ECS), the interaction between pCBs and eCBs has been extensively studied; however, the increasing wealth of knowledge gained thus far has only produced more questions than answers. In fact, the literature contains many contradictory findings regarding receptor- interactions, downstream receptor signaling, and even the behavioral outcomes of CB treatment. One obvious difficulty in understanding the relationship between pCBs treatment and resulting behavioral phenotypes, is a lack of clear understanding regarding the complex ECS-mediated regulation of endogenous adrenergic, serotonergic and dopaminergic signaling systems, which are known to be modulated by pCBs.

Collectively, the ECS consists of eCBs, CB receptors and synthesis/degradation enzymes and is primarily responsible for retrograde modulation of excitatory and

1

inhibitory neurotransmission within the brain, central and peripheral nervous systems. eCBs are produced naturally within the brain and central nervous system (CNS). There are a number of eCBs, including arachidonic acid (AA), (PEA), oleoylethanolamide (OEA), N-arachidonoyldopamine (NADA); however, the two most common and well-studied eCBs are 2-arachidonoylglycerol (2-AG) and anandamide, or

N-arachidonoylethanolamine (AEA) (Figure 1-1). Although structurally different, 2-AG and AEA are functionally similar to Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol

(CBD), the two most common pCBs in Cannabis. In fact, 2-AG is considered the eCB mimic of CBD and AEA is the eCB mimic to THC. For example, 2-AG plays a key role in pre-synaptic neuromodulation and pain suppression. Conversely, AEA, also known as

‘the bliss molecule’ plays a minor role in pain suppression, but a major role in activating and enhancing the reward centers of the brain, and has recently been identified as the major responsible for exercise-induced euphoria, or “runner’s high”

(Fuss et al., 2015).

eCBs are synthesized ‘on demand’ following membrane depolarization, activation

2+ of Gαq-coupled receptor signaling, or increased intracellular [Ca ] (Hashimotodani et al.

2013). Post-synaptic activation of Gαq signaling leads to the production of diacylglycerol, or DAG, via activation of protein lipase Cβ (PLCβ). DAG is then converted to 2-AG by the membrane-bound enzyme diacylglycerol lipase α (DAGLα). AEA is synthesized from its membrane-bound precursor N-arachidonylphosphatidylethanolamine (NAPE) via a member of the phospholipase superfamily, phospholipase D (NAPE-PLD) (Liu et al.,

2006). Once released, by a yet uncharacterized mechanism, eCBs mediate retrograde inhibition of pre-synaptic neurotransmission in a CB receptor-dependent manner. In 2

general, eCB-mediated inhibition of neurotransmission occurs at both

(excitatory) and GABAergic (inhibitory) synapses, termed depolarization-induced suppression of excitation (DSE) and depolarization-induced suppression of inhibition

(DSI), respectively (Diana and Marty, 2004; Ohno-Shosaku and Kano, 2014). After the eCBs are released from the post-synaptic neuron, two membrane-bound pre-synaptic enzymes, monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH), degrade 2-AG and AEA, respectively, to terminate eCB signaling (Lichtman et al., 2004;

Long et al., 2009a and b; Deutsch, 2016). Although FAAH is capable of degrading both

AEA and 2-AG, MAGLs are more specific and are only capable of degrading 2-AG (Di

Marzo and Maccarrone, 2008).

As stated previously, eCBs mediate retrograde modulation of neurotransmitter release via activating the Gαo-coupled CB receptors 1 and 2 (CB1 and CB2) in primarily the CNS and immune system, respectively, although both receptors are broadly expressed. Within the brain and CNS, CB1 is localized primarily to pre-synaptic nerve terminals and mediates, in part, pre-synaptic inhibition of neurotransmission (Herkenham et al., 1990; Glass et al., 1997; Tsou et al., 1998; Ohno-Shosaku and Kano, 2014), while

CB2 receptors are expressed most abundantly in the immune system and immune effector cells, predominately macrophages and certain leukocytes, such as B-cells, T-cells, and monocytes (Munro et al., 1993). As a result fewer studies have been conducted to characterize the role of CB2 in CB-mediated effects on behavior. However, the exclusion of CB2 from the nervous system has become more controversial. Recent studies have suggested a functional role for CB2 in the peripheral nervous system, including the peripheral, sensory, nociceptive and enteric neurons. Additionally, a number of studies 3

have reported expression of CB2 in the CNS, albeit at much lower levels than CB1, but the function for CB2 in the CNS has yet to be determined (Griffin et al., 1997; Patel et al.,

2003; Belvisi et al., 2008; Nackley et al., 2004; Elmes et al., 2004; Sagar et al., 2005;

Storr et al., 2002; Duncan et al., 2008; Atwood and Mackie, 2010).

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Figure 1-1. Molecular structures of endocannabinoids 2- arachidonoylglycerol (2-AG) and anandamide (AEA).

Molecular structures of the two most common endocannabinoids 2-arachidonoylglycerol (2-AG) (top) and N-arachidonoylethanolamine (AEA) or anandamide (bottom). Although structurally very different from THC and CBD, 2-AG and AEA activate the same signaling pathways, as 2-AG and AEA are considered the endocannabinoid mimetics to CBD and THC, respectively. Molecular structures were modified from (Bisongo, 2008) using Microsoft PowerPoint.

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1.2. Cannabis and Phytocannabinoids

Cannabis or marijuana has been a popular recreational drug for thousands of years for its unique ability to alter sensory perception and cause euphoria. More importantly, marijuana also has the ability to exert a wide range of medicinal effects

(Pacher et al., 2006). The Cannabis genus contains three species of plants, each with a unique pharmacological profile of more than 100 bioactive compounds, or CBs. pCBs are derived from the plants of the Cannabis genus, with the two most studied being Δ9- tetrahydrocannabinol (Δ9-THC), the main psychoactive component and cannabidiol

(CBD), the main medicinal component (Figure 1-2). The Cannabis genus contains three species; C. sativa, C. indica and C. ruderalis. Among the three, C. sativa and C. indica have higher levels of Δ9-THC and moderate levels of CBD, and as a result, are the species of choice for recreational use.

C. ruderalis contains much lower levels of Δ9-THC than C. sativa and C. indica, but contains high levels of CBD and is rarely used for recreational purposes. However, C. ruderalis is used for medicinal purposes and is currently being used to treat disorders such as anxiety, epilepsy, sclerosis, loss of appetite, and even as a form of cancer treatment (Grotenhermen and Muller-Vahl, 2012).

THC, the main psychoactive component of Cannabis, exhibits its effects primarily through the classical CB signaling system, via activation of the CB receptors, CB1 and

CB2 with affinities in the low nanomolar (nM) range (Rinaldi-Carmona et al., 1994;

Felder et al., 1995; Bayewitch et al., 1996; Showalter et al., 1996; Rhee et al., 1998).

CBD is of particular interest in Cannabis research due to the lack of psychoactive activity, but has a significant and potentially untapped therapeutic potential. For example, 6

CBD is used as a neuroprotective agent and in the management of neuropathic pain associated with multiple sclerosis (MS), inflammation, and nausea/vomiting (Pertwee,

2005). Due to its therapeutic potential, it will be important to understand the complex mechanism of action and signaling pathways modulated by CBD. However, data is conflicting regarding the effects of CBD on CB receptors. Several reports suggest that

CBD displays weak agonist activity to CB1, while other reports identify CBD as an inverse agonist (Pertwee et al., 2002) and even a non-competitive antagonist (Thomas et al., 2007). In contrast, CBD acts as a potent CB2 antagonist (Thomas et al., 2007). In addition to CB1/CB2, CBD also interacts with other proteins within the ECS to amplify eCB signaling. For example, CBD increases AEA levels by inhibiting FAAH and the

AEA membrane transporter (AMT) (Di Marzo et al., 1994; Cravatt et al., 1996;

Watanabe et al., 1996; Rakhshan et al., 2000; Deutsch, 2016). In addition to modulating eCB signaling, CBD exerts a number of effects on proteins and receptors outside of the

ECS. For example, CBD is an agonist for the: 1) 5-HT1A-like 5-HT receptor (Russo et al.,

2005; Rock et al., 2011), 2) transient receptor potential vanilloid 1 receptor (TRPV1)

(Thomas et al., 2007; Iannotti et al., 2014), and 3) several receptors (Gonca and Darici, 2015; Carrier et al., 2005). CBD also interacts with the orphan G-protein coupled receptors GPR18 (Caldwell et al., 2013), GPR55 (Ryberg et al., 2007), and

GPR119 (Overton et al., 2006). Together, these studies demonstrate the complexity of

CB signaling. It is clear that pCBs activate and modulate the ECS, and thus, it will be especially important to understand the complexities of ECS modulation and interactions between the ECS and adrenergic, dopaminergic and serotonergic signaling.

7

Figure 1-2. Molecular structures of phytocannabinoids Δ9- tetrahydrocannabinol (Δ9-THC) and (CBD).

Molecular structures of the psychoactive component Δ9-THC (top) and the medicinal component CBD (bottom) of Cannabis satvia. THC and CBD have the same molecular formula (C21H30O2) and are structurally very similar, yet activate distinct signaling pathways. Molecular structures were modified from (Bisongo, 2008) using Microsoft PowerPoint.

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1.3. Therapeutic Potential of Cannabinoids

Perhaps the earliest documented study of CBs and pain was performed in 1899 by the pharmacologist, Walter Dixon, who demonstrated a suppression of canine reactions to pinpricks upon administration of CBs (Walker and Huang, 2002). The effects of CBs have been validated in several models of acute or physiological pain. CBs effectively suppress thermal (Buxbaum, 1972; Yaksh and Reddy, 1981; Lichtman and

Martin, 1991), mechanical (Sofia et al., 1973), and chemical pain (Sofia et al., 1973;

Formukong et al., 1988). However, CBs appear to suppress chronic pain, such as inflammatory and HIV-associated neuropathic, much more potently than acute pain

(Tsou et al., 1995; Herzberg et al., 1997). CB extracts and synthetic CB (sCB)-based medications relieve chronic pain associated with a number of diseases, including the spastic and neuropathic-associated pain in multiple sclerosis (Svendsen et al., 2004;

Wissel et al., 2006), HIV-associated sensory neuropathy (Abrams et al., 2007), rheumatoid arthritis (Blake et al., 2006), fibromyalgia (Skrabek et al., 2008) and even diabetic neuropathy (Selvarajah et al., 2008). However, no CB-based medications have been clinically approved specifically for the treatment of neuropathic pain.

In addition to its analgesic effects, Cannabis also exerts strong antiemetic, or anti- nausea/vomiting, effects and has long been used as an appetite stimulant for patients suffering from HIV/AIDS (Struwe et al., 1993; Haney et al., 2007), chemotherapy (Meiri et al., 2007; Duran et al., 2010), anorexia, including cancer-associated anorexia (Jatoi et al., 2002; Strasser et al., 2006; Brisbois et al., 2011) and even Alzheimer’s disease

(Volicer et al., 1997). In fact, CBs are just as, if not more, effective in treating chemotherapy-induced nausea/vomiting than standard antiemetics on the market today, 9

including the dopamine (DA) receptor antagonist and with fewer side effects (Colls et al., 1980; Gralla et al., 1984; Crawford et al., 1986; Cunningham et al.,

1988). pCB extracts, most commonly CBD (), and CB-based medications, such as sativex, and have been approved in many countries around the world to treat common conditions, including emesis. In the United States, dronabinol, or THC, was approved in 1985 for the treatment of chemotherapy-induced nausea and vomiting and again in 1992 for treating loss of appetite in HIV/AIDS patients with chronic weight loss and wasting. Fortunately, Cannabis and CB-based medications are being legalized and approved in more countries around the world, as a cheaper and less harmful alternative to common pharmaceutical drugs. Unfortunately however, given the limitations and direction of current funding patterns, researchers have been hampered in their ability to characterize the cellular and neuronal pathways mediating the therapeutic properties of CBs.

1.4. Non-CB1/CB2 –Dependent Cannabinoid Signaling

CBs also appear to activate a number of receptors/ion channels, in addition to

CB1/CB2, but their impact on CB-dependent behaviors is not clear. For example, THC and/or 2-AG induce analgesic effects such as catalepsy, hypothermia, tail flick analgesia, and hypolocomotion in mice, effects that are reversible using a potent CB1 antagonist,

SR141716A and absent in CB1 knockout mice. However, analgesia induced by AEA is still present in transgenic CB1 knockout mice and is still observed in the presence of

SR141716A, suggesting potential targets for AEA and CBD independent of CB1 and 10

CB2. These non-CB1/CB2 targets for AEA appear to involve other Gαi/o-coupled GPCRs, as the AEA-dependent effects are sensitive to pertussis toxin (PTX) (Sagan et al., 1999;

De Petrocellis and Di Marzo, 2009). Many of the recently identified non-CB1/CB2 receptors are members of the family generally known as orphan GPCRs, a number of which have recently been deorphanized by CBs, including the Gαi/o-coupled receptors

GPR18, GPR35, GPR55 and GPR119 (Begg et al., 2005; Mackie and Stella, 2006 ;

Brown, 2007).

In a study by AstraZeneca, several agonists, including 2-AG, AEA, and THC activated GPR55 expressed in HEK293 cells with an EC50s of 3, 18 and 8 nM, respectively (Ryberg et al., 2007). Interestingly, GPR55 activation was insensitive to pertussis or cholera toxins, suggesting GPR55 is able to bind a non Gi or Gs G-protein or signal through G-protein independent pathways. Similarly, the THC-dependent activation of GPR55 released from internal stores via the IP3R receptor on the endoplasmic reticulum. This activation was PTX-insensitive and dependent on Gq, G12, RhoA, and

PLC (Lauckner et al., 2008). In contrast, GPR55 was not involved in the calcium increases associated with other CBs such as 2-AG, PEA (an AEA homologue), CBD, and abn-CBD (a sCB structural isomer of CBD). Interestingly, GPR55 activation may involve interaction with other CB receptors. For example, AEA mobilized Ca2+ in human endothelial cells, only when GPR55 and CB1 are co-expressed (Waldeck-Weiermair et al., 2008). In addition, when CB1 and integrin receptors are clustered together, AEA activates CB1 and prevents GPR55 activation by inhibiting PI3K via activation of spleen tyrosine kinase (SYK). However, when CB1 and integrin receptors are clustered, CB1 dissociates from the integrins and no longer inhibits PI3K, resulting in GPR55 activation 11

and subsequent release of intracellular calcium (Waldeck-Weiermair et al., 2008).

Interestingly, CB1 and GPR55 co-localize within the rat striatum and when heterologously expressed in HEK293 cells, CB1 and GPR55 heterodimerize and antagonize the others downstream signaling (Martinez-Pinilla et al., 2014). Additionally,

CBD is not a GPR55 agonist, instead acts as an antagonist with an IC50 of 350 nM

(Ryberg et al., 2007). GPR55 antagonism by CBD may explain some of the reported analgesic properties of CBD, as GPR55 expression is upregulated in injury models of neuropathic pain (Malek et al., 2016). However the physiological role of GPR55 in the brain is unclear.

While much less abundant than GPR55, GPR18 has also recently been deorphanized by several CBs. GPR18 is a Gαi/o-coupled receptor involved in the modulation of microglial cell migration (McHugh et al., 2010). AEA does not activate

GPR18, in contrast to GPR55, but N-arachidonoyl glycine (NAGly), a product of AEA hydrolysis by FAAH, is a potent GPR18 agonist with an EC50 of 30 nM (Kohno et al.,

2006). The synthetic structural CBD isomer, abn-CBD is also a GPR18 agonist, although far less potent than NAGly, with an EC50 of 836 nM (McHugh et al., 2012). Lastly, the

THC activates heterologously expressed GPR18 in HEK293 cells with an EC50 of 960 nM (McHugh et al., 2012). Although not much is known about the clinical significance of GPR18 signaling, GPR18 expression, like GPR55, is upregulated in models of neuropathic pain in response to spinal cord injury. While the potent GPR55 agonist CBD displayed no activity for GPR18, the combined activation of GPR55 by CBD and GPR18 by THC may partially explain the strong association of these two pCBs with chronic pain relief. 12

Finally, GPR35 and GPR119 have also been recently deorphanized, but are expressed at much lower levels within the brain compared to GPR55 and GPR18. GPR35 is activated by 2A-LPA, a product of 2-AG hydrolysis by MAGL, and binds with an EC50 of 100 nM (Oka et al., 2010), while 2-AG had no effect. GPR119 is activated by the eCB oleoylethanolamide (OEA) with an EC50 of 3.2 µM when heterologously expressed in yeast cells (Overton et al., 2006). These are only a couple examples of non-CB1/CB2 CB receptors, but as the field of CB research expands, many more receptors are likely to emerge with agonist activity against pCBs and eCBs alike.

1.5. Cannabinoid Interaction with TRP Channels

Transient receptor potential (TRP) channels are predominately expressed in nociceptive sensory neurons and are activated by a variety of noxious mechanical, chemical, and thermal stimuli (Tominaga and Caterina, 2004). A number of CBs and/or eCBs activate at least five different TRP channels. When co-expressed, TRP channels and CB receptors are able to interact with one another, either directly or indirectly. For example, in addition to coupling to Gαi/o, several labs have reported CB1 coupling with

Gαq/11 proteins (De Petrocellis et al., 2007; McIntosh et al., 2007). When coupled to

Gαq/11, CB1 activation leads to activation of protein lipase C (PLC), the products of which are able to activate several TRP channels (Bandell et al., 2004). Further evidence supporting CB receptor and TRP channel interaction is the co-localization of TRP channels and several eCB degrading enzymes, such as FAAH, the primary enzyme responsible for degrading AEA. Inhibition of FAAH, which leads to the accumulation of 13

AEA and other fatty acid amides such as OEA and PEA, initiates the subsequent activation or inhibition of TRP channels. For example, AEA binds to the same intracellular binding site as , between the second and third trans-membrane domains of TRPV1 (De Petrocellis et al., 2001), most likely due to the structural similarities between capsaicin and AEA (Di Marzo et al., 1998). More importantly, AEA and N-arachidonoyldopamine (NADA) activate both CB1 and TRPV1 directly, as assessed by the ability of AEA to increase intracellular calcium in HEK cells heterologously expressing human TRPV1 (De Petrocellis et al., 2001). Interestingly, the

AEA-dependent activation of TRPV1 in HEK cells is partially dependent on AEA transport into the cell. For example, selective inhibition of the AEA membrane transporter (AMT) inhibited the AEA-dependent increase in intracellular calcium in

TRPV1 heterologously expressing HEK cells (De Petrocellis et al., 2001). Post-synaptic activation of TRPV1 by AEA can lead to termination of retrograde signaling at CB1 via inhibition of diacylglycerol lipase (DAGL), and subsequently the biosynthesis of 2-AG

(Di Marzo and Maccarrone, 2008; Maccarrone et al., 2008).

1.6. Cannabinoid Receptor-Dependent Activation of GIRK Channels

CB receptor signaling activates a class of ion channels known as the G-protein coupled inwardly rectifying potassium channels (GIRKs). For example, WIN 55,212-2, a potent CB1 agonist, activated an inwardly rectifying potassium current in AtT20 cells stably transfected with the rat CB1 receptor (Mackie et al., 1995). Similar effects were observed upon application of the AEA. The effects of WIN 55,212-2 and AEA were 14

blocked by PTX, validating the activation of inwardly rectifying potassium current by

Gi/o-proteins (Mackie et al., 1995). Interestingly, activation of the GIRKs is mediated via the Gβγ subunits of CB1. In Xenopus laevis oocytes, co-expression of GIRK1 and β1γ2 G- protein subunits resulted in an inwardly rectifying potassium current, as measured by two-electrode voltage clamp. Co-expression of GIRK1, β1γ2, and αi-2 caused an inwardly rectifying potassium current indistinguishable from GIRK1 and β1γ2 expressing oocytes. Conversely, expression of GIRK1 alone, or with αi-2, produced a significantly smaller inwardly rectifying current than with β1γ2 subunits present, indicating that

GIRK1 activation is mediated by βγ subunits more readily than α subunits alone

(Reuveny et al., 1994). GIRK channels expressed as heterotetramers in Xenopus laevis oocytes had more consistent expression and were more active than oocytes expressing

GIRK channel monomers. For example, compared to oocytes expressing CB1 and GIRK1 alone, expression of CB1 and GIRK1/4 as a heterotetramers enhanced WIN 55,212-2 dependent inwardly rectifying potassium currents 6-fold (McAllister et al., 1999). The

Gβγ-dependent activation of GIRK channels is an effective readout to examine the activation of CB receptors in heterologous expression systems (Hansen and Bräuner-

Osborne, 2009).

1.7. Cannabinoidergic and Adrenergic Interactions in Mammals

Recent animal studies have demonstrated a CB receptor-dependent increase in circulating corticosterone levels following administration of either pCB (Δ9-THC), eCB

(i.e. AEA) or sCB (i.e. HU-210), suggesting that CBs activate the hypothalamic-pituitary- 15

adrenal (HPA) axis. For example, the chronic administration of HU-210, a selective CB1 agonist, significantly increased the levels of circulating corticosterone nearly 7-fold, compared to saline-treated animals (McLaughlin et al., 2009). Co-administration of HU-

210 with either , a α1-adrenoceptor antagonist, or , a β- adrenoceptor antagonist, significantly reduced the HU-210-dependent increase in corticosterone levels.

These results suggest that the CB1-dependent increases in circulating corticosterone levels may be mediated via either direct or indirect stimulation of monoaminergic transmission (McLaughlin et al., 2009).

Additionally, CB1 activation is coupled to activation of the hypothalamic-pituitary- adrenal (HPA) axis, via adrenoceptor stimulation. For example, 2-AG, but not AEA, increases norepinephrine (NE) release (Kurihara et al., 2001). However, AEA also appear to activate the HPA axis, as increases in circulating corticosterone levels in mice were

AEA dependent, (McLaughlin et al., 2009). In addition, chronic administration of the sCB receptor agonist CP-55,940 increases the expression of corticotrophin releasing hormone (CRH) mRNA in the anterior pituitary (Corchero et al., 1999). These studies suggest that CBs activate the HPA axis and increase the release of various stress hormones that might contribute to the anti-stress effects of CBs.

The prostaglandin (PGE2) causes peripheral inflammation and hyperalgesia in mice and AEA alleviates PGE2-induced hyperalgesia, in a dose-dependent manner (Romero et al., 2013). The co-administration of AEA and AM25120, a selective CB1 receptor antagonist, blocked the anti-nociceptive effects of AEA, suggesting the direct involvement of CB receptors. In addition, , a nonselective α2 adrenoceptor antagonist inhibited the peripheral analgesic effects of AEA administration. These data 16

suggest that α2 adrenoceptors (α2A-AR) are required for the analgesic effects of CB receptor agonists on PGE2-induced hyperalgesia (Romero et al., 2013).

CB1 and α2A-AR co-localize in axon terminals of the medial prefrontal cortex

(mPFC), suggesting potential interactions between these two receptors. , a selective α2-AR agonist, increased excitability and input resistance in mPFC neuron.

WIN-55,212-2, a potent CB1 receptor agonist, directly depolarizes mPFC pyramidal neurons, and WIN-55,212-2 stimulates norepinephrine (NE) release in the mPFC. Acute or chronic treatment with WIN-55,212-2 abolished clonidine-dependent increases in mPFC neuron excitability and input resistance, but WIN-55,212-2 had no effect in the presence of the selective CB1 antagonist SR 141716A, suggesting the direct involvement of CB1. It has been suggested previously that the downregulation of α2-AR is a result of a decrease in free Gi protein following α2-AR stimulation. Since CB1 and α2-AR are both

Gi-coupled, activation of CB1 may sequester the available Gi-proteins, leading to down regulation of α2-AR signaling (Cathel et al., 2014).

1.8. Cannabinoidergic and Dopaminergic Interactions in Mammals

eCBs increase dopaminergic neuronal firing and DA release but CB1 is not expressed in mammalian dopaminergic neurons (Julian et al. 2003), suggesting CB1- mediated modulation of dopaminergic transmission is indirect, most probably by disinhibiting inhibitory GABAergic input to dopaminergic neurons via depolarization- induced suppression of inhibition (DSI) (Uchigashima et al. 2007; Wang et al. 2015).

Indeed, CB1 is expressed on GABAergic and glutamatergic neurons pre-synaptic to 17

dopaminergic neurons in the nucleus accumbens (Winters et al. 2012; Fitzgerald et al.

2012), suggesting that eCBs modulate DA neurotransmission by fine-tuning excitatory glutamatergic and inhibitory GABAergic inputs into the dopaminergic neurons. In fact,

2-AG self-administration stimulates DA release in the nucleus accumbens (NAc) in a dose-dependent manner, based on microdialysis studies in Sprague-Dawley rats (De Luca et al. 2014; Cheer et al. 2004; Cheer et al. 2005). 2-AG likely increases DA release via suppression of GABAergic inputs onto dopaminergic neurons. 2-AG may also increase

DA release directly, as 2-AG application increases the firing of action potential in cultured midbrain dopaminergic neurons by inhibiting A-type potassium channels, specifically Kv1.3 (Gantz and Bean, 2017).

While the 2-AG modulation of dopaminergic neuron function is primarily CB1- dependent, AEA-dependent modulation of DA signaling is primarily CB1-independent.

AEA is a high-affinity agonist for TRPV1 (Starowicz et al. 2007) and TRPV1 is expressed in glutamate-, GABA-, and DA-containing neurons (Mezey et al. 2000), suggesting AEA-dependent DA modulation can be direct or indirect, as well as location dependent. For example, in the striatum, dopaminergic neuronal activity was reduced following AEA-dependent TRPV1 activation on upstream GABAergic neurons (de Lago et al. 2004), whereas TRPV1 activation in the substantia nigra pars compacta stimulated

DA release (Marinelli et al. 2003). Early studies merely suggested that 2-AG activates

TRPV1directly, but more recently, the 2-AG dependent TRPV1 activation was recorded after heterologous expression in either HEK293 and CHO cells (Zygmunt et al., 2013) suggesting 2-AG and AEA may have either antagonistic or synergistic effects on dopaminergic modulation. 18

1.9. Cannabinoidergic and Serotonergic Interactions in Mammals

In recent years, a number of studies have demonstrated positive interactions between pCBs and serotonergic signaling in mammals. For example, both endogenous and synthetic CB receptor agonists dose-dependently inhibit 5-HT reuptake (Steffens and

Feuerstein, 2004), and chronic Δ9-tetrahydrocannabinol (THC) administration increases endogenous 5-HT levels in rat prefrontal cortex (Sagredo et al. 2006). In addition, the pCB Δ9-tetrahydrocannabivarin (THCV) functions as a positive allosteric modulator of 5-

HT1A, increasing the efficacy (Emax) of 8-OH-DPAT, a potent 5-HT1A agonist (Cascio et al. 2015). Similarly, the sCB receptor agonist, CP 55,940, enhances 5-HT2A receptor activity, and upregulates 5-HT2A receptor mRNA and protein expression in rat prefrontal cortex (Franklin and Carrasco, 2015). In addition, chronic THC exposure increased the expression of the 5-HT1A receptor (Moranta et al., 2009; Zavitsanou et al., 2010).

Together, these studies suggest that CBs modulate serotonergic signaling at multiple levels.

The serotonergic and eCB signaling systems exhibit significant cross-talk. For example, the eCB-mediated modulation of behaviors such as, body temperature, feeding behavior, sleep and emotional processing require serotonergic signaling (Malone and

Taylor, 2001; Marco et al., 2004). Interestingly, eCBs appear to inhibit 5-HT release, in contrast to their stimulatory effects on DA release (Nakazi et al., 2000; Egashira et al.,

2002). In fact, inhibition of CB receptors increases basal 5-HT release (Darmani et al.,

2003; Tzavara et al., 2003; Aso et al., 2009). However, eCBs have a regulatory effect on 19

the expression and activation of a number of 5-HT receptors, including 5-HT1A and 5-

HT2A. For example, after CB1 knockout, 5-HT1A and 5-HT2A receptor expression was unaffected but their ability to stimulate [35S] GTPγS binding was significantly enhanced, suggesting an increase in 5-HT1A and 5-HT2A receptor function (Mato et al., 2007; Aso et al., 2009). However, whether this increase in GTPγS binding resulted from the direct effect of the knockout or compensatory responses to serotonergic signaling are unclear.

Interestingly, the basal activation of serotonergic neurons was reduced in CB1 knock-out mice, suggesting that CBs function to modulate the activity of serotonergic neurons (Aso et al., 2009). Indeed, several electrophysiological studies report the eCB-dependent modulation of 5-HT neuronal excitability (Gobbi et al., 2005; Bambico et al., 2007; Haj-

Dahmane and Shen, 2005, 2009). Specifically, increasing AEA levels by inhibiting the

AEA hydrolytic enzyme FAAH increased 5-HT neuronal firing in a CB1-dependent manner (Gobbi et al., 2005). Interestingly, serotonergic neurons synthesize and release eCBs in an activity-dependent manner, even though they do not express CB receptors

(Haj-Dahmane and Shen, 2005; 2009). eCB synthesis and release from 5-HT neurons can be stimulated in two ways, either via post-synaptic membrane depolarization and

2+ subsequent increases in intracellular calcium (Ca ) or activation of Gαq/11-coupled receptors that stimulates phospholipase C (PLC) and diacylglycerol lipase (DAGL), leading to eCB synthesis and release (Haj-Dahmane and Shen, 2005; 2009). In conclusion, CBs extensively modulate the serotonergic signaling system in mammals and may explain the anti-anxiety and anti-depression effects of cannabinoids. However, the behavioral consequences of CB-dependent modulation of serotonergic signaling have yet to be fully characterized. 20

1.10. Cannabinoid Signaling in Nematodes: What is known?

Do worms contain CB receptors? A BLAST search using human CB1 suggests that

C. elegans do not contain any receptors with significant sequence identity. However, the nematicidal reported in the literature would suggest otherwise

(McPartland and Glass, 2001; Kayani et al., 2012; Mukhtar et al., 2013).

(Cannabis sativa L.) is rarely infested with nematodes and mixing dried Cannabis sativa leaves and flowers in with soil protects the surrounding plants and repels pathogenic nematodes, bacteria, fungus and insects. Similarly, C. sativa is also used as a companion crop to help keep nearby crops healthy and ward off nematode infections. The molecular targets of these pCBs have never been identified in nematodes, despite the wealth of anecdotal evidence in the literature. In fact, a mammalian CB receptor orthologue has never been identified in nematodes (McPartland and Glass, 2001).

In 2001, nematode CB1 orthologues in C. elegans were potentially identified by

BLAST search, including the D1-like DA receptor, DOP-1, a predicted neuropeptide receptor, NPR-19 and a 5-HT1A-like 5-HT receptor, SER-4. However, because all three of these proteins exhibit less than 30% sequence identity to the human CB receptor 1

(CB1), they were considered non-homologous to CB1 (McPartland and Glass, 2001). In particular, NPR-19 was labeled as a misidentified putative CB receptor due to several substitutions of amino acid residues that are important for CB1 receptor-ligand binding and affinity. Based on these observations, no molecular studies were conducted.

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N-acylethanolamines (NAEs), including both 2-AG and AEA have been identified in C. elegans using mass-spectrometry (Lehtonen et al., 2008; 2011), but a functional role for these NAEs has not been determined. However, NAEs and NAE signaling appear to mediate dauer formation and the effect of diet on lifespan extension in C. elegans

(Lucanic et al., 2011). For example, the over-expression of fatty acid amide hydroxylase

(FAAH), the enzyme responsible for NAE hydrolysis, resulted in a developmental delay, suggesting that NAEs may play a role in promoting larval development. During normal larval development and growth, NAE levels peak during larval stage 2 (L2), the developmental stage in which the animal is committed to reproductive growth rather than entry into dauer. In fact, NAE levels are highest at the early pre-dauer L2 (L2d) stage and rapidly decline in the late L2d and subsequent dauer stages (Lucanic et al., 2011). These data suggest that NAEs may provide a signal of nutrient availability and modulate dauer entry. Indeed, exogenous application of NAEs such as eicosapentaenoyl ethanolamide

(EPEA) and AEA prevented dauer entry and stimulated reproductive growth in a number of dauer-constitutive mutants, including daf-2. However, EPEA had a more pronounced effect on dauer entry than AEA. Similarly, the CB receptor antagonist AM251 suppressed dauer entry and promotes reproductive growth in daf-2 mutant animals (Reis-Rodrigues et al., 2016). The ability of NAEs to prevent dauer entry is likely due to the NAE- dependent mobilization of cholesterol from internal stores. For example, 2-AG and AEA inhibit dauer entry in dauer constitutive daf-7 animals during cholesterol depletion, suggesting that ECBs facilitate the mobilization and utilization of sterols to prevent dauer arrest (Galles et al., 2018).

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In addition, endogenous NAE levels appear to be regulated by food availability and feeding habits. For example, starved larval stage 1 (L1) animals showed significantly reduced NAE levels compared to well-fed L1 animals. NAE levels in these starved L1 animals dramatically increased following feeding. Dietary restriction (DR) via means of starvation or deficiencies in pharyngeal pumping significantly increase life-span in adult

C. elegans (Klass, 1977; Hosono et al., 1989; Avery, 1993; Lakowski and Hekimi, 1998;

Vanfleteren and Braeckman, 1999; Finch and Ruvkun, 2001; McKay et al., 2004).

Decreasing endogenous NAE levels via faah-1 over-expression, especially in the pharynx, mimicked DR and increased adult lifespan. Similarly, fat-4 null animals with significantly reduced NAE levels also exhibit an extended lifespan. Conversely, exogenous EPEA application significantly reduced DR-mediated lifespan increases in wild-type animals. Taken together, NAEs appear to play a role in nutrient sensing and act as a “food availability” signal that controls dauer entry and determines lifespan (Lucanic et al., 2011).

eCBs also modulate axon regeneration following neuronal injury in C. elegans

(Pastuhov et al., 2012). In wild-type animals, laser severed axons begin regeneration within 24 hours and make a full recovery. However, faah-1 null animals that have increased levels of endogenous EPEA and AEA exhibited a significantly reduced frequency of axon regeneration and failed to recover days after surgery, suggesting that endogenous EPEA and AEA inhibit axon regeneration. Similarly, the over-expression of the AEA synthesis enzyme, NAPE-specific phospholipase D or NAPE-1, mimicked loss of faah-1 and significantly inhibited axon regeneration. As predicted, exogenous treatment with EPEA or AEA significantly reduced axon regeneration in wild-type 23

animals. In contrast to mediating dauer formation in which EPEA played a more significant role, AEA had a much more pronounced inhibitory effect on axon regeneration than EPEA. Also, the AEA-dependent inhibition of axon regeneration was absent in animals lacking the Gαo subunit encoded by goa-1, suggesting that AEA inhibits axon regeneration via a Gαo-coupled receptor (Pastuhov et al., 2012).

Two predicted neuropeptide receptors, NPR-19 and NPR-32, were identified as potential mediators of the AEA-dependent inhibition of axon regeneration, based on the ability of npr-19;faah-1 and npr-32;faah-1 double mutants to rescue the axon regeneration deficiency of faah-1 single mutants. However, npr-19 and npr-32 null animals do not have axon regeneration deficiencies similar to faah-1 (tm5011), and therefore, were proposed to function redundantly in inhibiting axon regeneration. Unfortunately, axon regeneration phenotypes were not rescued in either npr-19 or npr-32 null animals and axon regeneration was not measured in npr-19;npr-32 double mutants (Pastuhov et al.,

2016). Without functional pharmacological characterization, NPR-19 and NPR-32 still remain as only candidate orthologues of mammalian CB receptors.

1.11. Nociception to 1-octanol

Compared to the human brain that contains roughly 1011 (100 billion) neurons, the

C. elegans nervous system is significantly smaller with only 302 neurons. Amazingly, each neuron in the human brain appears to be connected to about 10,000 other neurons, resulting in upwards of 1015 (1 quadrillion) synapses. In contrast, the 302 neurons of C.

24

elegans form only 7,000 chemical synapses and the entire neuronal connectivity has been anatomically mapped, forming a complete “wiring diagram” (White et al., 1983). This wiring diagram is a useful tool to dissect the neuronal circuits of specific behavioral outputs. The C. elegans nervous system also shares a number of similarities with the mammalian nervous system. For example, both cyclic nucleotide-gated (CNG) and TRP channels are the most common effectors of GPCR activation in chemosensory neurons, both are glutamatergic/peptidergic, and both are modulated by serotonergic/adrenergic- like signaling pathways. Most importantly, C. elegans and mammals alike initiate analgesic behavior to noxious stimuli that in C. elegans can be operationally defined as the perception of pain.

C. elegans detect the noxious stimulus 1-octanol using three pairs of amphid sensory neurons, the ASH, ADL and AWB. The two polymodal ASH sensory neurons are solely responsible for detecting dilute (30%) 1-octanol, while detection of 100% 1- octanol requires the ASH, ADL, and AWB sensory neurons (Chao et al., 2004; Wragg et al., 2007). Detection of 1-octanol at any concentration is dependent on glutamatergic signaling and TRP channels, as animals defective in the vesicular glutamate transporter eat-4 or the TRPV1 channel subunit orthologues osm-9 and ocr-2, fail to respond to 1- octanol (Hart et al., 1999).

Behavioral responses to 1-octanol can be quantified using the well-established octanol avoidance assay; originally published by Anne Hart and co-workers (Chao et al.,

2004) and extensively modified by our lab (Wragg et al., 2007; Harris et al., 2009; 2010;

2011; Mills et al., 2012; Hapiak et al., 2013; Summers et al., 2015; Oakes et al., 2017).

In the context of aversive behavior, increasing concentrations of 1-octanol produces a ‘bi- 25

phasic’ behavioral response. For example, upon presentation of a hair dipped in dilute

(30%) 1-octanol, wild-type animals initiate backward locomotion in about 10 seconds

(Chao et al., 2004; Wragg et al., 2007; Harris et al., 2009; 2010), while wild-type animals presented with 100% 1-octanol initiate backward locomotion in 5 seconds (Wragg et al.,

2007; Harris et al., 2011; Mills et al., 2012). However, exogenous food (bacteria) or 5-

HT modulates aversive responses to dilute 1-octanol and decrease the time taken to initiate backward locomotion. For example, in the presence of food or 5-HT, wild-type animals presented with dilute 1-octanol decrease the time taken to initiate backward locomotion from 10 to 5 seconds (Harris et al., 2009; 2010; 2011).

The food stimulation of aversive responses is mediated by 5-HT release from the neurosecretory NSM sensory neuron and three distinct 5-HT receptors, MOD-1, SER-1 and SER-5, operating at different levels within the ASH-mediated locomotory circuit. For example, food-stimulated 5-HT release from the NSM activates the 5-HT-gated chloride channel, MOD-1, on the AIBs and AIYs, the 5-HT6-like receptor, SER-5, on the ASHs and the 5-HT2-like receptor, SER-1, on the RIAs to increase responsiveness to dilute 1- octanol (Harris et al., 2009). To add an additional layer of complexity, 5-HT release from the ADFs antagonizes food-stimulated NSM 5-HT release via activation of SER-1 in the

RIC (Harris et al., 2011). Interestingly, the interaction of additional monoaminergic signaling plays a critical role in modulating responsiveness to 1-octanol. For example,

DA, octopamine (OA) and tyramine (TA) antagonize the food and 5-HT stimulation of aversive responses, but have no effect on basal aversive responses to dilute 1-octanol

(Wragg et al., 2007). Monoamines also modulate the cross-inhibition between the ASH and ASI sensory neurons that modulates nociception and avoidance to copper (Cu2+) 26

(Guo et al., 2015). Cu2+ activates the ASHs and the octopaminergic RIC interneurons via gap junctions between the ASH and RICs. Subsequent RIC activation stimulates OA release to inhibit the ASI via activation of the OA receptor SER-3 on the ASIs.

Conversely the Cu2+ evoked reciprocal inhibition of the ASHs by the ASIs is more complex. Although the ASIs synapse directly on the ASH, the Cu2+-evoked ASI- dependent inhibition of the ASH requires 5-HT from the ADF neurons and subsequent activation of the 5-HT receptor SER-5 (Guo et al., 2015).

These studies highlight the complex modulation of nociceptive behaviors in C. elegans that share many similarities with modulation of mammalian nociceptive neurons

(Mills et al., 2016). The suppression of chronic pain in mammals involves the modulation of signaling to pain receptors via the Gαq-coupled α1 and Gαo-coupled α2 AR-dependent regulation of neurotransmitter and neuropeptide release (Pertovaara, 2006). In C. elegans, a similar system functions to mediate nociceptive behaviors, with the Gαq and Gαo- coupled α-adrenergic like receptors TYRA-3 and OCTR-1, respectively, modulating and fine-tuning the release of monoamines and from the ASH and ASI sensory neurons to elicit escape behaviors to noxious stimulus (Harris et al., 2009;

Hapiak et al., 2013; Mills et al., 2016; Oakes et al., 2017; Clark and Hapiak et al., 2018).

Given these similarities, the signaling mechanisms mediating nociceptive responses in C. elegans can be functionally translated to help decode the more complex signaling mechanisms mediating nociceptive behavior in mammals. Therefore, since CBs have many proposed anti-nociceptive effects and suppress pain, we can use the well- established avoidance behavior to the noxious stimulus 1-octanol, as a functional readout

27

of pain in C. elegans and characterize the receptors and neuronal pathways involved in

CB-dependent modulation of behavioral responses to pain.

1.12. Neuronal Control of Pharyngeal Pumping in C. elegans

The C. elegans pharynx is a tubular structure whose rhythmic muscular contraction and relaxation function to pull food from the environment into the worm, where it is concentrated and ground up for digestion. The pharyngeal nervous system contains 20 different neurons that interact to control and fine tune the rate of pharyngeal pumping, isthmus peristalsis and grinding. Of these 20 pharyngeal neurons, only 3 are absolutely necessary for feeding and normal development, the MC, M3 and M4. Animals with all pharyngeal neurons ablated except the MC, M3 and M4 still develop and grow normally and only shows subtle feeding deficiencies (Avery and You, 2012). The coordinated contraction and relaxation cycles of pharyngeal muscle are controlled by the excitatory MC and inhibitory M3 pharyngeal neurons, both of which signal directly onto pharyngeal muscle. In general, pharyngeal pumping is controlled via 2 different sets of neurons. The timing and rate of pumping are modulated by the MC, an excitatory cholinergic neuron that upon stimulation depolarizes pharyngeal muscle and causes contraction via activation of nicotinic acetylcholine receptors. The inhibitory glutamatergic M3 neurons synapse onto the metacorpus and isthmus and function to hyperpolarize and relax pharyngeal muscle following contraction. To stimulate contraction, the MC releases acetylcholine (ACh) which activates the nicotinic acetylcholine receptor EAT-2 to initiate pharyngeal muscle action potentials and muscle 28

contraction. Immediately following muscle contraction, the M3 neuron releases glutamate onto the muscle to activate the glutamate gated chloride channel AVR-15 to terminate muscle cell excitation and initiate muscular relaxation (Avery, 1993a and b;

Dent et al., 1997; Avery and You, 2012). avr-15 encodes a subunit of the glutamate-gated chloride channel that responds directly to glutamate transmission from M3. AVR-15 is expressed in the muscles of the metacorpus (pm4) and isthmus (pm5), and functions to hyperpolarize, and therefore, relax the pharyngeal muscle. Animals with mutations in avr-15 are resistant to M3 stimulation and therefore, exhibit prolonged pharyngeal muscle contractions. The single M4 pharyngeal neuron synapses onto the isthmus muscle and controls isthmus peristalsis which effectively transports ingested bacteria to the grinder to be crushed for further ingestion. 5-HT increases both the rate of pumping and isthmus peristalsis via activation of the 5-HT7-like receptor SER-7 expressed in both the

MC and M4 pharyngeal neurons (Hobson et al., 2006; Song and Avery, 2012). Since the proper coordination between pharyngeal muscle excitation by the MCs and pharyngeal muscle inhibition is essential for successful pumping, 5-HT must also stimulate the inhibitory M3 neurons to coordinate with the increased excitatory signals from the MC.

However, the excitatory receptor mediating stimulation of M3s during 5-HT stimulated pumping has yet to be determined. 5-HT also stimulates isthmus peristalsis via SER-7, coupled to Gα12 signaling instead of Gαs, however, isthmus peristalsis does not occur after every pharyngeal pump in order to maintain efficient feeding (Song and Avery,

2012). In conclusion, the timing and control of pharyngeal pumping is absolutely dependent on the coordination between the MC and M3 pharyngeal neurons and surprisingly little is known about the modulation of the M3s. 29

1.13. Dopaminergic and Serotonergic Modulation of Locomotory

Behavior in C. elegans

C. elegans has long been used as a model for genetic and physiological studies on locomotion due to their relatively simple nervous system and availability of a complete anatomical wiring diagram. C. elegans locomotion can be divided into two main categories; crawling and swimming, depending on the medium presented. In liquid medium C. elegans exhibit swimming or “thrashing” behavior characterized by alternating C-shape conformations that ultimately propel the worm forward. When presented with a solid medium, such as agar, C. elegans exhibit the classical crawling behavior characterized by sinusoidal movement that propels the worm forward and is interrupted by periodic reversals. Swimming and crawling are both highly coordinated by the nervous system and are modulated by monoaminergic signaling, primarily dopaminergic and serotonergic, which promote crawling and swimming behaviors, respectively (Vidal-Gadea and Pierce-Shimomura, 2012).

DA modulates crawling behavior via two antagonistic DA receptors, a Gαs-coupled

D1-like receptor DOP-1 and a Gαo-coupled D2-like receptor DOP-3 co-expressed in the cholinergic motor neurons (Chase et al., 2004; Allen et al., 2011). The most common

DA-mediated locomotory behavior is basal slowing, characterized by dramatically reduced forward locomotion upon encountering food. Interestingly, basal slowing is

30

mediated by DOP-3, as dop-3, but not dop-1, mutant animals displayed severe defects in basal slowing and dop-1;dop-3 double mutants exhibited wild-type basal slowing behavior, highlighting the antagonistic relationship between the two receptors (Chase et al., 2004). However, the transition from swimming to crawling was dependent on DOP-1 and not DOP-3, as dop-1 null animals failed to transition to crawling and collapsed upon exiting liquid medium (Vidal-Gadea and Pierce-Shimomura, 2012). Additionally, DA is involved in area-restricted search (ARS) behavior, characterized by a decrease in forward locomotion and an increase in turning and reversal frequency upon encountering food in order to maximize time spent in the nutrient rich environment (Hills et al., 2004). For example, wild-type animals with ablated dopaminergic neurons or cat-2 null animals which lack DA biosynthesis are deficient in ARS behavior, suggesting that DA functions to increase turning and reversal frequency. Indeed, exogenous DA increases turning rate in a dose-dependent manner and the DA receptor antagonists , raclopride or promote forward movement and decreases turning and reversal frequency

(Hills et al., 2004; Donohoe et al., 2008). In addition, dopaminergic neuron photostimulation via channel rhodopsin 2 (ChR2) significantly reduces the rate of forward locomotion (Ezcurra et al., 2011), mimicking exogenous DA application and in agreement with DA receptor antagonism studies. Together, these studies suggest that DA mediates ARS after encountering nutrient dense environments and functions to inhibit forward locomotion while increasing the turning and reversal frequency.

In contrast, when removed from a food source, C. elegans exhibit search behaviors to locate food again, termed local search and dispersal behaviors. Local search behaviors

31

occur during acute starvation, within the first 30 minutes following removal from food, and are characterized by a decrease in short reversals, with a corresponding increase in long reversals and omega turns. Prolonged starvation (>30 min) causes a switch from local search to dispersal behavior in which reversals and omega turns are rare and forward locomotion predominates (Gray et al., 2005). Local search behavior during acute starvation does not require DA signaling as cat-2 null animals that lack DA biosynthesis exhibited little defects in local search behavior, however, tph-1 null animals that lack 5-

HT biosynthesis were completely deficient in local search behavior (Gray et al., 2005), suggesting that 5-HT functions to inhibit reversals during acute starvation. Indeed tph-1, ser-1 and mod-1 null animals that lack 5-HT biosynthesis, a 5-HT2-like receptor and 5-

HT-gated chloride channel, respectively, exhibit an increased reversal frequency and the

5-HT receptor antagonists and mimic this phenotype (Donohoe et al., 2008; Harris et al., 2009). Indeed, tph-1 null animals exhibit a decreased rate of forward locomotion during chronic starvation, suggesting that endogenous 5-HT promotes forward locomotion during dispersal behavior or chronic starvation, and therefore, decreases reversal frequency. In contrast, reversals were decreased in ser-1 and mod-1 null animals on food (Harris et al., 2009). Together these studies highlight the complex antagonist relationship between DA and 5-HT in the modulation of locomotory rate and reversal frequency as well as the food-dependent effects of 5-HT on reversal frequency, with 5-HT promoting reversals on food and inhibiting reversals off food.

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Chapter 2

Materials and Methods

2.1. Nematode Strains

Strains were grown and maintained at 16°C and room temperature (22°C) on nematode growth media (NGM) agar plates with OP50 E. coli as a food source (Brenner,

1974). All strains were purchased from the Caenorhabditis Genetics Center (CGC) at the

University of Minnesota and the National Bioresource Project (NBRP) at Tokyo’s

Women’s Medical University in Tokyo Japan. The following strains were used: N2

(Bristol), N2 (ancestral), cat-1 (ok411), cat-2 (n4547), cat-4 (ok342), che-2 (e1033), ckr-2

(tm3082), dat-1 (ok157), dop-1 (ok298), dop-2 (vs105), dop-3 (ok295), dop-4 (tm1392), DA

Quadruple (DA quad) null (dop-2 (vs105) V; dop-4 (ok1321) dop-1 (vs100) dop-3 (vs106)

X), dop-5 (ok568), dop-6 (ok2090), lgc-53 (n4330), mod-5 (n3314), npr-3 (tm1583), npr-5

(ok1583), npr-19 (ok2068), npr-24 (ok3192), octr-1 (ok371), ocr-1 (ok132), ocr-2 (ak47), ocr-3 (ok1559), ocr-4 (vs137), osm-6 (p811), osm-9 (ok1677), osm-9 (ky10), ser-2

(pk1357), ser-4 (ok512), and tph-1 (n4622), trp-4 (ok1605), trp-4 (sy695), and 5-HT

Quintuple(5-HT quint) null (ser-5 (tm2654);ser-4 (ok512);mod-1 (ok103);ser-7 (tm1325) ser-1 (ok345)).

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2.2. Fusion PCR and Transgene Construction

RNAi transgenes were generated by PCR fusion as described in Esposito et al.

(Esposito et al., 2007), and co-injected with f25b3.3::gfp (to 100 ng) as a transfection marker. The octr-1 (+), npr-19 (+) full-length genomic and npr-19::npr-19::gfp transcriptional transgenes were generated by PCR fusion and co-injected with f25b3.3::gfp

(to 50 ng).The npr-19::npr-19 full-length genomic transgene was generated by PCR amplification. The npr-19::gfp transcriptional transgene was constructed by PCR fusion of

1.5 kb npr-19 promoter including the first intron fused to gfp::unc-54 3”UTR and co- injected with rol-6 (su1106) (to 50 ng). The npr-19::CNR1::gfp transgene was generated by 3-piece PCR fusion of the npr-19 promoter including the first intron, full-length human

CNR1 cDNA, and gfp::unc-54 3”UTR and were co-injected with unc-122::rfp (to 50 ng) . unc-17β-driven transgenes were generated by PCR fusion of unc-17β promoter (562 bp) to

GPCR cDNA and gfp::unc-54 3”UTR and co-injected with unc-122::rfp (to 50 ng). npr-

9::ser-4::gfp transgene was generated by PCR fusion using native npr-9 promoter and co- injected with unc-122::rfp (to 50 ng) . PCR fusions were created by overlap fusion PCR as previously described (Hobert, 2002).

2.3. Octanol Avoidance Assay

All 1-octanol avoidance assays were performed as described in Chao et al. (Chao et al., 2004), as modified by Harris et al. (Harris et al., 2011). All 1-octanol avoidance assays were conducted on freshly poured NGM plates. For all behavioral assays, L4 stage animals

34

were picked 24 hours before assaying. 2-AG and AEA plates were prepared 10 min before assay by spreading 60 µl of 2-AG or AEA (in H2O) on fresh NGM plates. To measure aversive responses to 1-octanol, the end of a hair was briefly dipped in 1-octanol and placed in front of a forward moving worm and the time taken to initiate backward locomotion is recorded. Animals were first transferred to intermediate (non-seeded) plates, left for 30 sec, transferred to assay plates and tested after 10 min. In all assays, 20-25 worms were examined for each strain and condition and each assay was performed at least

3 times. Statistical analysis was performed using mean ± S.E. and Student’s t-test.

2.4. Pharyngeal Pumping Assay

Pharyngeal pumping was assayed on NGM plates as described by Hobson et al.

(Hobson et al., 2006). 2-AG plates were prepared 10 min before assay by spreading 60 µl of 320 μM 2-AG (in H2O) on fresh, pre-dried NGM plates. For all pumping assays, L4 animals were picked 24 hours before assay. Animals were moved from food plates to either a non-seeded NGM plates containing either vehicle (control) or 2-AG, and were incubated for 10 min. During assay, pharyngeal pumps were recorded using a SONY Exwave HAD color-video digital camera for 2 min. Videos were played back in slow-motion and the number of pharyngeal pumps per min was counted. Statistical analysis was performed using mean ± S.E. and Student’s t-test.

2.5. Feeding Assay 35

The uptake of fluorescently-labeled latex beads was performed as described in

Kiyama et al., 2012). Fluoresbrite® YG Microspheres 0.75 µm were purchased from

Polysciences Inc. (Catalog No. 17154-10), diluted in ethanol and stored at 4°C. Feeding plates were made by spreading 150 µl of M9 bead solution (1x108 microspheres/plate) and drying for 30 min. Wild-type and npr-19 null animals were incubated for 10 min on plates containing 2-AG, AEA, or no drug. Animals were transferred to bead plates ± 2-AG or

AEA, allowed to feed for 30 min at RT and then removed, washed with M9 to remove excess beads, and immobilized on agarose pads with 20 mM Na azide for imaging using an

Olympus IX81 inverted confocal microscope. Images were analyzed using ImageJ and statistical analysis was performed using mean ± S.E. and Student’s t-test.

2.6. Locomotion Assay

Locomotion was assayed as described by Law et al. (Law et al., 2015). Freshly poured agar plates (non-NGM) containing either 320 µM 2AG/AEA were used for assay.

Well-fed, young adult hermaphrodite animals are picked prior to assay and maintained on

NGM plates with E. coli OP50. During assay, seven animals were transferred to the assay plate. Motility was assessed as number of body bend/20 sec at 5 min intervals for 30 min, starting as soon as animals are transferred. Each strain was assayed at least 3 times with 7 animals/assay. Statistical analysis was performed using mean ± S.E. and Student’s t-test.

2.7. Reversal Assay

36

Reversal assays were performed as described in WormBook

(www.wormbook.org), using freshly poured NGM plates containing either control

(vehicle) or 2-AG (320 µM). Well-fed, young adult hermaphrodites are picked prior to assay and maintained on NGM plates with E. coli OP50 overnight. During assay, seven animals were transferred to the assay plate containing vehicle or 2-AG. The motility of each animal was assessed as number of spontaneous reversals per 3 min following 10 min incubation in control (vehicle) or 2-AG (320 µM). Each strain was assayed at least 3 times with at least 7 animals/assay. Statistical analysis was performed using mean ± S.E. and

Student’s t-test.

2.8. Heterologous Expression in Xenopus laevis

Xenopus laevis oocytes were obtained from Xenopus One Inc. (Dexter, MI) and Nasco

(Fort Atkinson) and prepared as described by Hansen and Bräuner-Osborne (Hansen and

Bräuner-Osborne, 2009). Oocytes were mechanically separated prior to incubation in ND-

2+ 96 (Ca free) medium (96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 5 mM HEPES, pH 7.6) containing 1 mg/ml of collagenase type 1A (Sigma Aldrich, Product No. C9891) for 30 min. De-folliculated oocytes were separated and incubated in modified Barth’s medium

(Barth’s medium with 1 mM Na Pyruvate, 0.01 mg/ml Gentamicin, and 1x Antibiotic- antimycotic; Gibco, Invitrogen, Carlsbad, CA, USA) at 16°C overnight. Receptor cRNAs were injected at 50 ng/50 nL and GIRK1 and GIRK2 channel cRNAs were injected at 0.5 ng/50 nl. Oocytes were incubated at 16°C for 48-72 hrs post-injection and then transferred to 4°C.

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2.8.1. Generation of Expression Vectors

The human CB1 (CNR1), α2A-adrenergic (ADRA2A), 5-HT1A (HTR1A), GIRK1,

GIRK2 and C. elegans npr-19, octr-1, and ser-4 cDNAs were cloned between Not1 and

Age1 restriction enzyme sites into a Xenopus expression vector containing a T7 promoter and the Xenopus 5’ and 3’ β-globin untranslated regions, to generate pxGIRK1, pxGIRK2, pxCNR1, pxADRA2A, pxHTR1A, pxser-4, pxoctr-1 and pxnpr-19, respectively. Linearized plasmids were transcribed using an Ambion mMessage mMachine T7 kit (ThermoFisher,

Catalog No. AM1344) cRNA synthesis kit. ADRA2A (Plasmid No. 66216), CNR1

(Plasmid No. 66254) and GIRK2 (Plasmid No. 20675) cDNAs were purchased from

Addgene. GIRK1 (Clone ID: OHu01264) was purchased from GenScript and HTR1A cDNA was purchased from GE Healthcare Dharmacon Inc.

2.8.2. Two-Electrode Voltage Clamp

Two-electrode Voltage-Clamp (TEVC) recordings were performed 72 hours post- injection using an Axon Gene Clamp 500 Amplifier (Molecular Devices, Sunnyvale, CA) as described previously (Stuhmer, 1998; Bamber et al., 2003; Hansen and Bräuner-

Osborne, 2009). For TEVC recordings, standard Low K+ Ringers solution (115 mM NaCl,

+ 2.5 mM KCl, 1.8 mM CaCl2, 10 mM HEPES, pH 7.2) and a High K Ringers solution (96 mM KCl, 2 mM NaCl, 1.8 mM CaCl2, 10 mM HEPES, pH 7.2) were applied by gravity

38

perfusion. Ligands were applied by gravity perfusion; initially at 1 µM. Oocytes co- expressing GIRK1/2 and GPCRs were perfused with intervals of increasing concentrations of 2-AG and AEA to determine ligand specificity and EC50. 2-AG and AEA dose-response

(log EC50 – [agonist]) x n) curves were fitted with the equation: I-Imax /(1+10 , where I is current at a given 2-AG or AEA concentration, Imax is current at saturation, EC50 is the 2-AG and AEA concentration required to elicit half-maximal current and n is the slope coefficient. Curve fitting was performed using GraphPad Prism software (San Diego).

2.9. Confocal Imaging

To localize NPR-19 expression, a transcriptional npr-19::gfp transgene was generated using 1.5 kb upstream of the predicted npr-19 start site, including the first intron.

To identify a subset of amphid sensory neurons, animals were incubated in 5 µM 1,1′- dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine (DiD, Molecular Probes) for one hour and then transferred to a standard NGM plate seeded with OP50 for one hr to de-stain, as described by Schultz and Gumienny (Schultz and Gumienny, 2012). For neuronal identification, npr-19::gfp was co-injected with either tph-1::rfp, tdc-1::rfp, flp-8::rfp, flp-

18::rfp, or ceh-36::rfp and rol-6 (SU1106) (to 50 ng) was used as a selection marker. All imaging was performed on an Olympus IX81 inverted confocal microscope. Animals expressing the npr-19::gfp transgene were immobilized on 3% agarose pads with 20 mM sodium azide and imaged for GFP/RFP/DiD fluorescence.

To assess potential 2-AG-dependent changes in cat-2p::mCherry fluorescence, a cat-2p::mCherry transcriptional transgene was generated by overlap PCR fusion (Hobert, 39

2002) of 1.5 Kb of cat-2 promoter to mCherry and was co-injected at 50 ng with rol-6

(su1106) (50 ng) into wild-type (N2) animals. For all cat-2 transcriptional fluorescence analysis, well-fed L4 animals were picked 24 hrs prior to the assay. For assay, wild-type animals expressing cat-2p::mCherry were picked from plates containing OP50 onto a fresh

NGM plate without food to allow any residual bacteria to be removed. Animals were then picked from the transfer plates onto fresh NGM plates (10 animals/plate) containing either vehicle (water) as a control or 300 µM 2-AG for one hr. Following treatment, animals were immobilized on 3% agarose pads with 20 mM sodium azide and imaged using an Olympus

IX81 inverted confocal microscope. The resulting images were analyzed using ImageJ, in which each ADE and CEP neuron was isolated and mCherry fluorescence within each neuron was quantified independently using ImageJ and Microsoft Excel.

2.10. C. elegans Microinjection

Microinjections were performed as described in Wormbook using a Leica DM IRB

Inverted Research Microscope. Healthy, non-starved, young adult (L4) animals were picked 24 hours prior to microinjection. For injection, animals were picked from the food plate and placed on a transfer plate without food to remove any residual bacteria. Then, worms were picked from the transfer plate and placed into halocarbon oil on a glass coverslip with a 2% (w/v) agarose pad. Animals were allowed to settle and crawl about until they stuck to the agarose pad. Following injection, animals were removed from the agarose pad and placed into a drop of M9 on a fresh, seeded NGM plate to recover. For each construct, 10 to 15 animals were injected. Animals were allowed to recover on food 40

overnight. The next morning, surviving healthy animals were moved to new, freshly seeded

NGM plates with one animal per plate (parent, F0, plates). Over the next few days, the parent plates were screened for F1 animals carrying the chosen selection marker. Any F1 animals carrying the selection maker were picked onto their own seeded NGM plate and allowed to grow to adult and lay eggs (F1 plates). After the F1 generation has produced eggs and the eggs start to hatch, the F1 plates were screened for F2 animals carrying the selection marker. F2 animals carrying the selection marker were picked onto their own plates and maintained as an independent line. In general, rol-6 (su1106) (to 50 ng) was used as a selection marker for transgenic strains to be used only for confocal imaging, pan-neuronal f25b3.3::gfp (to 100 ng) was used as a selection maker for RNAi strains and coelomocyte marker unc-122::rfp (to 50 ng) was used as a selection marker for strains carrying transgenes containing gfp::unc-54 3”UTR.

2.11. Reagents

2-arachidonoylglycerol (2-AG) (Catalog No. 1298), 2-aminoethoxydiphenylborane

(2-APB) (Catalog No. 1224), Anandamide (AEA) (Catalog No. 1339), Nicotinamide

(NAM) (Catalog No. 4106), JZL184 (Catalog No. 3836), and URB597 (Catalog No.

4612) were all purchased from Tocris Bioscience and stock solutions are in DMSO or

ethanol at 100 mM and are stored at -80°C. For PCR purification: Qiagen QIAquick PCR

Purification Kit (Catalog No. 28104). For octanol avoidance assay: 1-octanol (Sigma

Aldrich, Product No. 95446). For Pharyngeal pumping: Fluoresbrite® YG Microspheres

0.75 µm were obtained from Polysciences Inc. (Catalog No. 17154-10), diluted in ethanol 41

and stored at 4°C. For microinjection: Halocarbon Oil Series HC-700 (Sigma Aldrich,

Product No. H8898), Kwik-Fil Borosilicate Glass Capillaries (1.0mm, World Precision

Instruments, Inc) needles, and a Leica DM IRB Inverted Research Microscope. For

Xenopus laevis heterologous expression: Age1-HF (NEB, Catalog No. R3552S), BstEII-

HF (NEB, Catalog No. R3162S) Not1-HF (NEB, Catalog No. R3189S) restriction enzymes were used for cloning, Kpn1-HF (NEB, Catalog No. R3142S) was used for plasmid linearization. Qiagen QIAquick Gel Extraction Kit (Catalog No. 28704) was used to extract and isolate linearized plasmids prior to cRNA synthesis. Ambion mMessage mMachine T7 cRNA synthesis kit (ThermoFisher, Catalog No. AM1344) for cRNA synthesis.

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Chapter 3

Results

3.1. The endocannabinoids, 2-AG and AEA, inhibit aversive behavior

2-AG and AEA have recently been identified in C. elegans extracts by mass spectrometry (Lehtonen et al., 2008; 2011), but a simple BLAST search using the human

CB receptor, CB1, failed to identify any C. elegans receptors with significant identity

(>30%) to CB1, consistent with previous reports that C. elegans lacks clear mammalian

CB receptor orthologues (Figure 3-1) (McPartland and Glass, 2001; Pastuhov et al.,

2016) . In mammals, 2-AG and AEA exert anti-nociceptive action in models of acute inflammatory and neuropathic pain; therefore, we examined their effects on aversive responses to 1-octanol in C. elegans (Iskedjian et al., 2007; Clapper et al., 2010). This aversive decision-making circuit is mediated primarily by the two ASH sensory neurons and has been extensively characterized (Wragg et al., 2007; Harris et al., 2011; Mills et al., 2012). Neither 2-AG nor AEA had any effect on the initiation of aversive responses to dilute (30%) 1-octanol. In contrast, 2-AG and AEA completely inhibited the 5-HT stimulation of aversive responses to 30% 1-octanol (Figure 3-2A). 5-HT functions as the

“food is at hand” signal in C. elegans and food or exogenous 5-HT stimulates the

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initiation of aversive responses to 30% 1-octanol to levels reported for the more rapid responses observed for 100% 1-octanol, through a complex signaling pathway requiring multiple monoamine and neuropeptide receptors (Chao et al., 2004; Harris et al., 2011).

2-AG and AEA also inhibited the more rapid initiation of aversive responses to 100% 1- octanol (Figure 3-2A), with 2-AG exhibiting an EC50 of 1.2 µM (Figure 3-2B). These relatively high CB concentrations were probably necessary to overcome the relative impermeability of the nematode cuticle.

In mammals, the degradation of 2-AG and AEA and the termination of CB signaling are initiated by a membrane-bound monoacylglycerol lipase (MAGL) and fatty acid amide hydroxylase (FAAH), respectively (Long et al., 2009b). The predicted C. elegans proteins, Y97E10AL.2 and FAAH-1, exhibit significant amino acid sequence identity to human MAGL (39%), and FAAH (38%), respectively, and selective inhibitors are available for both mammalian enzymes (Piomelli et al., 2006; Long et al., 2009b). As anticipated, the inhibition of either MAGL (Y97E10AL.2) with JZL184, or FAAH with

URB597, predicted to inhibit the degradation of 2-AG or AEA, respectively, mimicked

2-AG or AEA addition and inhibit aversive responses to 100% 1-octanol (Figure 3-3).

Similarly, the RNAi knockdown of the MAGL (Y97E10AL.2), driven by 1.6 kb of the y97e10al.2 promoter, abolished JZL184-dependent, but not URB597-dependent, inhibition of aversive responses to 100% 1-octanol (Figure 3-3). Together, these results suggest that C. elegans contains an endogenous CB signaling system.

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Figure 3-1. BLAST of human cannabinoid receptor 1 (CB1) in C. elegans.

A BLAST search using the human cannabinoid 1 receptor (CB1) amino acid sequence against the C. elegans genome identifies a number of monoamine and neuropeptide receptors with limited sequence identity to CB1, including the D1-like DA receptor DOP- 1, the α2-adrenergic-like OA receptor OCTR-1 and the 5-HT1-like 5-HT receptor SER-4. We searched for non-redundant protein sequences (nr) using a protein-protein BLAST (BLASTP), specifically a position-specific iterated BLAST (PSI-BLAST) was performed with all additional criteria and parameters set at default.

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Figure 3-2. 2-AG and AEA inhibit aversive responses to 1-octanol.

A) 2-AG and AEA inhibit basal aversive responses to 100% 1-octanol and 5-HT stimulated aversive responses to 30% 1-octanol in wild-type (N2) animals. B) 2-AG dose-response curve for wild-type animals with an EC50 of 1.2 μM. Aversive responses to 1-octanol in wild-type animals were examined as described by Harris et al. (Harris et al., 2009). For aversive assays using 100% 1-octanol, worms were incubated in vehicle (control), 2-AG (30μM) or AEA (30μM) for 10 minutes prior assay. For 5-HT stimulated aversive assays using 30% 1-octanol, animals were incubated in vehicle (control + 4mM 5-HT), 2-AG (30μM + 4mM 5-HT) or AEA (30μM + 4mM 5-HT) for 30 minutes prior assay. 2-AG dose-response curve was generated in GraphPad Prism. * indicates a significant difference from wild-type animals in the absence of 2-AG (p value of ≤ 0.05). Data are presented as a mean ± SE (n) and analyzed by two-tailed Student t test.

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Figure 3-3. The inhibition of endogenous 2-AG and AEA degradation inhibits basal aversive responses to 100% 1-octanol.

Treatment with the monoacylglycerol lipase (MAGL) inhibitor JZL184 or the fatty acid amide hydroxylase (FAAH) inhibitor URB597 mimic exogenous 2-AG and AEA application, respectively, and inhibit aversive responses. JZL184-dependent, but not URB597-dependent, inhibition of aversive responses is absent following RNAi knockdown of the mammalian MAGL orthologue y97e10al.2. Aversive responses to 1- octanol in wild-type (N2) and transgenic animals were examined as described by Harris et al. (Harris et al., 2009). Animals were incubated in vehicle (control), JZL184 (300 μM) or URB597 (300μM) for 10 minutes prior to assay. The y97e10al.2 RNAi construct was generated by PCR fusion as described in Esposito et al. (Esposito et al., 2007). * indicates a significant difference from wild-type animals in the absence of inhibitor (p value of ≤ 0.05). Data are presented as a mean ± SE (n) and analyzed by two-tailed Student t test.

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3.2. The 2-AG/JZL184-dependent inhibition of aversive responses is absent in npr-19 null animals

To identify potential C. elegans CB receptors, we re-examined protein BLAST data using human CB1 and identified a number of previously characterized C. elegans monoamine receptors and predicted neuropeptide receptors with limited identity to CB1

(Figure 3-1) (McPartland and Glass, 2001). To determine if any of these receptors were required for the CB-mediated inhibition of aversive responses, we screened the appropriate null animals for loss of JZL184 or 2-AG-dependent inhibition of aversive responses (Figures 3-4 and 3-5).

2-AG or JZL184 still inhibited aversive responses in ckr-2, dop-1, npr-3, octr-1, ser-2, and ser-4 null animals. In contrast, the 2-AG or JZL184-dependent inhibition of aversive responses was dramatically reduced in npr-19 null animals (Figure 3.2.1 and

3.2.2) (Oakes et al., 2017). Similarly, 2-AG or JZL184-dependent inhibition was absent after npr-19 RNAi knockdown in wild-type animals, using a predicted 1.5 kb npr-19 promoter (Figure 3-4 and 3-5) (Oakes et al., 2017). 2-AG inhibition could be rescued in npr-19 null animals by expression of a full-length npr-19 transgene driven by the predicted 1.5 kb npr-19 promoter, including 1 kb of the npr-19 3’UTR (Figure 3-6)

(Oakes et al., 2017). In addition, wild-type animals overexpressing this npr-19 transgene mimicked the addition of 2-AG, and initiated aversive responses more slowly than wild- type animals in the absence of 2-AG (Figure 3-6) (Oakes et al., 2017). Importantly, 2-AG sensitivity in npr-19 null animals could also be rescued by the expression of the human

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CB1-encoding cDNA driven by the npr-19 promoter described above, confirming the potential orthology of the two receptors (Figure 3-6) (Oakes et al., 2017).

As predicted, although NPR-19 and human CB1 exhibited only 23% sequence identity, many key amino acids involved in AEA binding appear to be conserved (Figure

3-7). The residues delimiting the AEA-binding pocket are largely hydrophobic, based on both modeling and site-directed mutagenesis (Reggio, 2010) and include F189, L193, F379 and S383. All four residues were conserved in NPR-19 (Figure 3-8) (Oakes et al., 2017).

F189 interacts with the AEA amide oxygen and an F189A mutation in CB1 decreases AEA binding 6-fold (McAllister et al., 2004). The AEA amide oxygen also interacts with a charged residue at position 192 (K in CB1, D in NPR-19), and the AEA hydroxyl forms a hydrogen bond with S383 (McAllister et al., 2003). Interestingly, a BLAST of NPR-19 in

C. elegans reveals that DOP-1 exhibits the closest homology to NPR-19. These data highlight the effective coupling of a human G-protein-coupled receptor to endogenous C. elegans G-proteins and strongly support the hypothesis that NPR-19 is a mammalian CB receptor orthologue.

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Figure 3-4. 2-AG does not inhibit aversive responses in npr-19 null animals or following npr-19 RNAi knockdown.

A screen of the potential cannabinoid receptor null animals, ckr-2 (tm3082), dop-1 (ok398), npr-3 (tm1583), npr-19 (ok2068), octr-1 (ok371) and ser-2 (pk1357) for loss of 2-AG-dependent inhibition of aversive responses. Aversive responses to 1-octanol in wild-type (N2) and transgenic animals were examined as described by Harris et al. (Harris et al., 2009). Animals were incubated in vehicle (control) or 2-AG (3μM) for 10 minutes prior to assay. * indicates a significant difference from wild-type animals in the absence of 2-AG (p value of ≤ 0.05). Data are presented as a mean ± SE (n) and analyzed by two-tailed Student t test.

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Figure 3-5. The MAGL inhibitor JZL184 does not inhibit aversive responses in npr-19 null animals or following npr-19 RNAi knockdown.

A screen of the potential cannabinoid receptor null animals, ckr-2 (tm3082), dop-1 (ok398), npr-3 (tm1583), npr-19 (ok2068), octr-1 (ok371) and ser-2 (pk1357) for loss of JZL184-dependent inhibition of aversive responses. Aversive responses to 1-octanol in wild-type (N2) and transgenic animals were examined as described by Harris et al. (Harris et al., 2009). Animals were incubated in vehicle (control) or JZL184 (300μM) for 10 minutes prior to assay. The npr-19p::npr-19 RNAi knockdown construct was generated by PCR fusion as described in Esposito et al. (Esposito et al., 2007). * indicates a significant difference from wild-type animals in the absence of 2-AG (p value of ≤ 0.05). Data are presented as a mean ± SE (n) and analyzed by two-tailed Student t test.

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Figure 3-6. npr-19 or CB1 expression driven by a minimal npr-19 promoter rescues 2-AG sensitivity in npr-19 null animals.

Rescue of 2-AG insensitivity of npr-19 null animals by expression of npr-19 genomic, npr-19 cDNA or the human receptor encoding gene CNR1 cDNA in npr-19 null animals. npr-19 (+) represents the genomic sequence of npr-19 including 1.5 Kb of promoter through 1 Kb 3’ UTR. npr-19::npr-19 and npr-19::CB1 represent a minimal 1.5 Kb npr- 19 promoter fused to npr-19 and CNR1 (CB1) cDNA, respectively. Aversive responses to 1-octanol in wild-type (N2) and transgenic animals were examined as described by Harris et al. (Harris et al., 2009). Animals were incubated in vehicle (control) or 2-AG (3μM) for 10 minutes prior to assay. The npr-19::npr-19 and npr-19::CB1 rescue construct was generated by PCR fusion as described in Esposito et al. (Esposito et al., 2007). * indicates a significant difference from N2 (p value of ≤ 0.05). Data are presented as a mean ± SE (n) and analyzed by two-tailed Student t test.

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Figure 3-7. Comparison of CB1 and NPR-19 amino acid sequences.

CB1/NPR-19 protein alignment. Conserved key amino acid residues involved in AEA binding (F189, L193, L192, F379 and S383) are highlighted in red; identical residues are bolded and indicated with an asterisk.

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3.3. 2-AG and AEA directly activate NPR-19 heterologously expressed in Xenopus laevis oocytes

To demonstrate that 2-AG and AEA activate NPR-19 directly, npr-19 cRNA was co-injected with GIRK1 and GIRK2 cRNAs into Xenopus laevis oocytes. GIRK1 and

GIRK2 encode inwardly rectify potassium channel subunits activated by G-protein βγ subunits and were co-expressed on the assumption that NPR-19 would be Gαo-coupled, based on the observation above that the Gαo-coupled human CB1 rescued aversive phenotypes in npr-19 null animals. As expected, 2-AG and AEA had no effect on oocytes expressing GIRK1 and GIRK2 alone, but initiated a robust inwardly rectifying current in oocytes expressing NPR-19 (Figure 3-8A and B), with EC50s of 395 ± 5.1 nM (Figure 3-

9A) and 14 ± 2.4 nM (Figure 3-9B) respectively (Oakes et al., 2017). These EC50s for 2-

AG and AEA are in the range of EC50s reported for human CB1, 125 nM (Luk et al.,

2004) and 89 nM (McAllister et al., 1999), respectively. Together, these data demonstrate that NPR-19 is a functional CB receptor.

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Figure 3-8. 2-AG and AEA activate NPR-19 after heterologous expression in Xenopus laevis oocytes.

Two-electrode Voltage-Clamp (TEVC) recordings were performed on oocytes expressing NPR-19 and GIRK1/2 subunits and challenged with exogenous 2-AG and AEA 72 hours post-injection, as described previously (Stuhmer, 1998; Bamber et al., 2003; Hansen and + Bräuner-Osborne, 2009). IHK represents the current induced upon a switch from low K to + high K Ringer’s solution. ILigand represents the current induced following ligand application.

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Figure 3-9. 2-AG and AEA dose-response curves for NPR-19 heterologously expressed in Xenopus laevis oocytes.

Dose-response curves for 2-AG (A) and AEA (B) after the heterologous expression in Xenopus laevis oocytes. EC50 values were calculated and curve fitting was performed using GraphPad Prism.

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3.4. DOP-1 is required for the 2-AG-dependent inhibition of 5-HT stimulated aversive responses

Food or exogenous 5-HT stimulates the initiation of aversive responses to 30% 1- octanol to levels reported for 100% 1-octanol through a complex signaling pathway requiring three of the five C. elegans 5-HT receptors, MOD-1, SER-1 and SER-7, operating at different levels within the ASH-mediated aversive circuit (Harris et al.,

2011). As noted above, 2-AG completely blocks this 5-HT stimulation (Figure 3-10).

This 2-AG-dependent inhibition of 5-HT stimulated aversive responses requires NPR-19 and the Gαs-coupled D1-like DA receptor, DOP-1. 2-AG inhibition was absent in npr-19 or dop-1 null animals and could be rescued in dop-1 mutants by the expression of a full- length dop-1 transgene driven by the native promoter, including 1 Kb of 3’UTR (Figure

3-10). Indeed, 2-AG inhibition in the dop-1 rescue animals was more robust than in wild- type animals, presumably as a consequence of dop-1 overexpression. Surprisingly, endogenous DA did not appear to be involved in the 2-AG-dependent inhibition of 5-HT stimulated aversive responses, as 2-AG still inhibited 5-HT stimulated aversive responses in cat-2 null animals (Figure 3-10). Together these data indicate that the 2-AG-dependent inhibition of 5-HT stimulated aversive responses is dependent on the D1-like receptor

DOP-1, but is not dependent on endogenous, suggesting that 2-AG might activate DOP-1 directly.

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Figure 3-10. 2-AG-dependent inhibition of 5-HT stimulated aversive responses requires the D1-like receptor DOP-1.

2-AG-dependent inhibition of 5-HT stimulated aversive responses to 30% 1-octanol are absent in dop-1 null animals and DA receptor quadruple null (DA quad) animals that lack most previously identified DA receptors. 2-AG sensitivity could be restored in dop-1 null animals by the expression of a dop-1 genomic transgene in dop-1 null animals. 5-HT stimulated aversive responses to 30% 1-octanol in wild-type (N2), null and transgenic animals were examined as described by Harris et al. (Harris et al., 2009). Animals were incubated in 5-HT (4mM) or 5-HT (4mM) + 2-AG (30μM) for 30 minutes prior to assay. The dop-1 genomic rescue was generated by PCR and is driven by the native dop-1 promoter and includes 1Kb native dop-1 3’ UTR. * indicates a significant difference from wild-type animals in the presence of 5-HT + 30μM 2-AG (p value of ≤ 0.05). Data are presented as a mean ± SE (n) and analyzed by two-tailed Student t test.

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3.5. Serotonin and octopamine receptors are required for the inhibition of nociception at higher 2-AG concentrations

Surprisingly, at higher exogenous CB concentrations (30 vs 3 µM) the α2A- adrenergic-like receptor, OCTR-1 and the 5-HT1-like receptor, SER-4 were also required for the 2-AG inhibition of nociception, as octr-1 and ser-4 null animals are also resistant to 2-AG inhibition (Figure 3-11) (Oakes et al., 2017). The monoaminergic modulation of aversive responses is complex and involves the synergistic and antagonistic interactions of multiple monoamine receptors interacting at multiple levels in the locomotory decision-making circuit (Wragg et al., 2007; Harris et al., 2011; Mills et al., 2012). An octr-1::gfp transgene is broadly expressed, including both the ASHs and the ventral nerve cord (Wragg et al., 2007), and 2-AG sensitivity could be restored in octr-1 animals by octr-1 expression driven by the predicted 5 kb octr-1 promoter (Figure 3-12) (Oakes et al., 2017). 2-AG activated OCTR-1 directly after heterologous expression in Xenopus laevis oocytes, with an EC50 of 365 ± 24 nM (Figure 3-13A and B) (Oakes et al., 2017).

In contrast, ser-4 is only expressed in a limited number of neurons and 2-AG sensitivity could be restored in ser-4 null animals by ser-4 expression in the two AIB interneurons

(Figure 3-14). In contrast to OCTR-1, 2-AG did not activate SER-4 directly and had no effect on SER-4 affinity for 5-HT (Figure 3-15A and B). These data suggest that both octopaminergic and serotonergic signaling pathways are involved in the modulation of

CB-dependent modulation of aversive responses, OCTR-1 directly and SER-4 indirectly, most probably by increasing endogenous 5-HT release, as examined more fully below.

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Figure 3-11. OCTR-1 and SER-4 are required for inhibition of aversive responses at higher 2-AG concentrations.

The α2-adrenergic-like receptor, octr-1 and the 5-HT1-like receptor, ser-4 null animals are resistant to the 2-AG-dependent inhibition of aversive responses. Aversive responses to 1-octanol in wild-type (N2) and null animals were examined as described by Harris et al. (Harris et al., 2009). Animals were incubated in 2-AG (0, 3, 30 or 300μM) for 10 minutes prior to assay. * indicates a significant difference from N2 at the corresponding 2-AG concentration (p value of ≤ 0.05). Data are presented as a mean ± SE (n) and analyzed by two-tailed Student t test.

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Figure 3-12. octr-1 rescue of 2-AG-dependent inhibition of aversive responses in octr-1 null animals.

Expression of an octr-1 genomic rescue transgene in octr-1 null animals rescues sensitivity of higher 2-AG concentration (3 vs 30 μM) to octr-1 null animals. Aversive responses to 1-octanol in wild-type (N2), null and transgenic animals were examined as described by Harris et al. (Harris et al., 2009). Animals were incubated in 2-AG (0, 3, 30μM) for 10 minutes prior to assay. The octr-1 genomic rescue was generated by PCR and is driven by the native octr-1 promoter and includes 1Kb native octr-1 3’ UTR. * * indicates a significant difference from N2 at the corresponding 2-AG concentration (p value of ≤ 0.05). Data are presented as a mean ± SE (n) and analyzed by two-tailed Student t test.

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Figure 3-13. 2-AG dose-response curve for OCTR-1 heterologously expressed in Xenopus laevis oocytes.

A) Two-electrode Voltage-Clamp (TEVC) recordings were performed on oocytes expressing OCTR-1 and GIRK1/2 subunits and challenged with exogenous 2-AG 72 hours post-injection, as described previously (Stuhmer, 1998; Bamber et al., 2003; Hansen and Brauner-Osborne, 2009). IHK represents the current induced upon a switch + + from low K to high K Ringer’s solution. ILigand represents the current induced following ligand application. B) OCTR-1 dose-response curves for 2-AG after heterologous expression in Xenopus laevis oocytes. EC50 values were calculated and curve fitting was performed using GraphPad Prism.

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Figure 3-14. SER-4 expression in the AIB interneurons rescues the 2-

AG-dependent inhibition of aversive responses in ser-4 null animals.

The expression of an AIB::ser-4 transgene, driven by the npr-9 promoter, rescues 2-AG- dependent sensitivity in ser-4 null animals. Aversive responses to 1-octanol in wild-type (N2), null and transgenic animals were examined as described by Harris et al. (Harris et al., 2009). Animals were incubated in 2-AG (0, 3, 30μM) for 10 minutes prior to assay. The AIB-selective ser-4 transgenic rescue was generated by fusion PCR and is driven by the AIB-selective npr-9 promoter fused in-frame to ser-4 cDNA and includes unc-54 3’ UTR. * * indicates a significant difference from N2 at the corresponding 2-AG concentration (p value of ≤ 0.05). Data are presented as a mean ± SE (n) and analyzed by two-tailed Student t test.

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Figure 3-15. 2-AG has no effect on SER-4 heterologously expressed in

Xenopus laevis oocytes.

A) Two-electrode Voltage-Clamp (TEVC) recordings were performed on Xenopus laevis oocytes expressing SER-4 and GIRK1/2 subunits and challenged with exogenous 2-AG 72 hours post-injection, as described previously (Stuhmer, 1998; Bamber et al., 2003; Hansen and Brauner-Osborne, 2009). B) SER-4 dose-response curves for 5-HT and 5-HT + 2-AG (5µM) after heterologous expression in Xenopus laevis oocytes. EC50 values were calculated and curve fitting was performed using GraphPad Prism.

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3.6. Serotonergic signaling is required for the cannabinoid-dependent inhibition of aversive responses to 100% 1-octanol

2-AG inhibition of responses 1to 100% 1-octanol were also dramatically reduced in tph-1 null animals that lack tryptophan hydroxylase, a key enzyme required for 5-HT biosynthesis and, as noted above in ser-4 null animals that lack the Gαo-coupled 5-HT1A- like receptor, SER-4 (Figure 3-16). However, as noted above, 2-AG did not activate

SER-4 directly when heterologously expressed in Xenopus laevis oocytes (Oakes et al.,

2017). Importantly, 2-AG also inhibited aversive responses in mod-5 null animals that lack the key 5-HT reuptake transporter, suggesting that 2-AG stimulated 5-HT release, rather than blocking 5-HT reuptake (Figure 3-16). However, the addition of 5-HT alone had no effect on the initiation of aversive responses to 100% 1-octanol, suggesting that the 2-AG-dependent 5-HT release may be neuron-specific, rather than global or that other

2-AG dependent signaling pathways were required for inhibition. In contrast, dopaminergic signaling did not appear to be involved in this 2-AG-dependent inhibition, as the 2-AG inhibition of aversive responses were wild-type in cat-2 null animals lacking tyrosine hydroxylase, a key DA biosynthetic enzyme, and in a range of DA receptor null animals (Figure 3-17). Together these data suggest that 2-AG stimulates the release of endogenous 5-HT to inhibit aversive responses to 100% 1-octanol.

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Figure 3-16. The 2-AG-dependent inhibition of aversive responses to

100% 1-octanol requires serotonergic, but not dopaminergic signaling.

2-AG-dependent inhibition of aversive responses to 100% 1-octanol at higher 2-AG concentrations (3 vs 30µM) are absent in 5-HT receptor quintuple null (5-HT quint) animals that lack all previously identified 5-HT receptors and tph-1 null animals that lack the 5-HT biosynthetic enzyme tryptophan hydroxylase, are enhanced in mod-5 null animals lacking the 5-HT reuptake transporter, but are present in cat-2 null animals lacking the DA biosynthetic enzyme tyrosine hydroxylase. Aversive responses to 1- octanol in wild-type (N2) and null animals were examined as described by Harris et al. (Harris et al., 2009). Animals were incubated in vehicle (control) or 2-AG (30μM) for 10 minutes prior to assay. * indicates a significant difference from N2 (p value of ≤ 0.05). Data are presented as a mean ± SE (n) and analyzed by two-tailed Student t test.

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Figure 3-17. The 2-AG-dependent inhibition of aversive responses to

100% 1-octanol does not require dopamine receptors.

2-AG-dependent inhibition of aversive responses to 100% 1-octanol are present in a number of dopamine receptor null animals, as well as cat-2 null animals lacking the DA biosynthetic enzyme tyrosine hydroxylase. Aversive responses to 1-octanol in wild-type (N2) and null animals were examined as described by Harris et al. (Harris et al., 2009). Animals were incubated in vehicle (control) or 2-AG (30μM) for 10 minutes prior to assay. * indicates a significant difference from N2 (p value of ≤ 0.05). Data are presented as a mean ± SE (n) and analyzed by two-tailed Student t test.

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3.7. NPR-19 is expressed in a limited number of neurons and expression in the URX sensory neurons is required for the cannabinoid-dependent inhibition of aversive responses

Based on fluorescence from an npr-19::gfp transgene, NPR-19 is expressed in a limited number of neurons, including the two URX sensory neurons (Figure 3-18A and

C) and the two inhibitory, glutamatergic M3 pharyngeal motorneurons (Figure 3-18A and

B) that play key modulatory roles in regulating pharyngeal avoidance behavior and pumping, respectively (Raizen and Avery, 1994; McGrath et al., 2009). As predicted, npr-19 RNAi knockdown in the URXs, using either the URX-selective gpa-8 or flp-8 promoters, mimicked the npr-19 null phenotype and significantly decreased 2-AG- dependent inhibition of aversive responses to 100% 1-octanol (Figure 3-19) (Oakes et al.,

2017). These data demonstrate a key role for NPR-19 and the URXs in the CB-dependent inhibition of aversive responses.

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Figure 3-18. NPR-19 is expressed in a limited number of neurons, including URX and M3.

Expression of an npr-19::gfp transgene in wild-type animals is observed primarily in the URX and M3 neurons. A) DiD-stained wild-type animal expressing npr-19::gfp transgene. Wild-type animals co-expressing npr-19::gfp and either ceh-2::rfp (B) or flp- 8::rfp (C) to identify M3 and URX, respectively.

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Figure 3-19. NPR-19 functions in the URX neurons to inhibit aversive responses to 100% 1-octanol.

Selective npr-19 RNAi knockdown in the URXs, via flp-8 or gpa-8 promoters, dramatically reduces 2-AG-dependent inhibition of aversive responses. egl-36 drives expression in the M3 and ADE neurons. Aversive responses to 1-octanol in wild-type (N2) and transgenic animals were examined as described by Harris et al. (Harris et al., 2009). Animals were incubated in 2-AG (3μM) for 10 minutes prior to assay. The flp- 8::npr-19, gpa-8::npr-19 and egl-36::npr-19 RNAi constructs were generated by PCR fusion as described in Esposito et al. (Esposito et al., 2007). * indicates a significant difference from N2 (p value of ≤ 0.05). Data are presented as a mean ± SE (n) and analyzed by two-tailed Student t test.

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3.8. The cannabinoid-dependent inhibition of pharyngeal pumping and feeding requires NPR-19 expression in the M3 pharyngeal neurons

As noted above, NPR-19 is expressed in the two M3 pharyngeal motorneurons.

The inhibitory M3s repolarize pharyngeal muscle following contraction and ablation of the M3s decreases the rate of pharyngeal pumping and feeding (Raizen and Avery, 1994).

2-AG or AEA also inhibited pharyngeal pumping off food (Figure 3-20), although at higher concentrations than those required for the inhibition of nociception (300 vs 3 µM).

In contrast to nociception, JZL184 or URB597 had no effect of pumping (Figure 3-20), presumably because of the higher CB levels required for inhibition (Oakes et al., 2017).

These higher CB levels also inhibited feeding, as assessed by the uptake of fluorescently-labeled latex beads in wild-type animals (Figure 3-23). The CB-dependent inhibition of both pumping and feeding were npr-19 dependent and, as predicted, could be rescued by the expression of a full-length npr-19 transgene driven by the predicted 1.5 kb promoter, including 1 kb of the npr-19 3’UTR (Figure 3-21 and 3-22). More specifically, npr-19 RNAi knockdown in the M3s, using either the M3-selective glt-1 or egl-36 promoters, mimicked the npr-19 null phenotype and significantly decreased the 2-

AG-dependent inhibition of pharyngeal pumping (Figure 3-22) (Oakes et al., 2017). As predicted these RNAi transgenes had no effect of nociception (Figure 3-19). These data demonstrate a key role for NPR-19 and the M3s in the CB-dependent inhibition of pharyngeal pumping and feeding. Since CB use in humans has been associated with the

“munchies” and increased feeding post use (Sharkey and Pittman, 2005; Kirkham, 2009),

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it is potentially surprising that CBs inhibited feeding in these experiments. It may be useful to examine feeding rates shortly after CB exposure.

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Figure 3-20. Endocannabinoids 2-AG and AEA inhibit pharyngeal pumping in a dose-dependent manner.

2-AG and AEA inhibit pharyngeal pumping in a dose dependent manner, while increasing endogenous levels of either 2-AG or AEA via JZL184 (JZL) or URB597 (URB), respectively, had no effect. Wild-type animals were incubated in 2-AG (0, 3, 30 or 300μM), AEA (0, 3, 30 or 300μM), JZL184 (300µM), or URB597 (300µM) for 30 minutes prior to assay. * indicates a significant difference from 0µM (p value of ≤ 0.05). Data are presented as a mean ± SE (n) and analyzed by two-tailed Student t test.

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Figure 3-21. The 2-AG and AEA-dependent inhibition of pharyngeal pumping requires NPR-19.

The 2-AG and AEA-dependent inhibition of pharyngeal pumping is absent in npr-19 null animals and expression of an npr-19 genomic transgene (npr-19 (+)) in npr-19 null animals restores 2-AG and AEA sensitivity to npr-19 null animals. Animals were incubated in vehicle (control), 2-AG (300μM) or AEA (300μM) for 30 minutes prior to assay. The npr-19 genomic rescue was generated by PCR and is driven by the native npr- 19 promoter and includes 1 kb native npr-19 3’ UTR. * indicates a significant difference from control (p value of ≤ 0.05). Data are presented as a mean ± SE (n) and analyzed by two-tailed Student t test.

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Figure 3-22. NPR-19 functions in the M3 pharyngeal neurons to inhibit pharyngeal pumping.

M3-selective npr-19 RNAi knockdown, driven by the egl-36 or glt-1 promoters, abolishes the 2-AG and AEA-dependent inhibition of pharyngeal pumping. In contrast, URX- selective npr-19 RNAi knockdown had no effect on 2-AG or AEA-dependent inhibition of pharyngeal pumping. Animals were incubated in vehicle (control), 2-AG (300μM) or AEA (300μM) for 30 minutes prior to assay. The flp-8::npr-19, glt-1::npr-19 and egl-36::npr-19 RNAi constructs were generated by PCR fusion as described in Esposito et al. (Esposito et al., 2007). The egl-36 and glt-1 promoters drive expression in the two M3s and a limited number of other neurons and the flp-8 promoter drives expression in the two URXs and a limited number of other neurons (WormBase). * indicates a significant difference from control (p value of ≤ 0.05). Data are presented as a mean ± SE (n) and analyzed by two-tailed Student t test.

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Figure 3-23. The 2-AG and AEA-dependent inhibition of feeding requires NPR-19.

A) The accumulation GFP-tagged latex microspheres of wild-type (N2) animals following incubation in control (vehicle), 2-AG (300µM) or AEA (300µM). B) Quantification of GFP-tagged latex microsphere accumulation in wild-type, npr-19 null and npr-19-genomic rescue animals following treatment in vehicle (control), 2-AG (300µM) or 2-AG (300µM) for 30 minutes. The npr-19 genomic rescue (npr-19 (+)) was driven by 1.5Kb of native npr-19 promoter and 1Kb native npr-19 3’ UTR. * indicates a significant difference from control (p value of ≤ 0.05). Data are presented as a mean ± SE (n) and analyzed by two-tailed Student t test.

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3.9. Cannabinoids modulate locomotory behavior through NPR-19- independent signaling pathways

The addition of exogenous CBs also caused animals to become sluggish and increase turning (Figure 3-24). Interestingly, NPR-19 was not involved in these CB- dependent locomotory phenotypes, CB inhibited locomotion and increased turning similarly in both npr-19 and wild-type animals. In contrast, both SER-4 and OCTR-1 were involved in this CB-dependent locomotory inhibition (Figure 3-25). In fact, this CB- dependent locomotory inhibition mimicked the 5-HT-dependent “locomotory confusion” phenotype mediated by the 5-HT activation of the Gαo-coupled 5-HT1-like receptor,

SER-4, in the two AIB interneurons (Law et al., 2015). The 2-AG-dependent locomotory slowing does not result in a true paralysis, but mimics the locomotory confusion phenotype described for animals on 5-HT, where the SER-4-dependent inhibition of the two AIB interneurons interferes with the processing of antagonistic sensory information, although, as noted above CBs do not activate SER-4 directly (Law et al., 2015). Indeed, ser-4 and 5-HT receptor quintuple null animals were both resistant to CB-dependent locomotory inhibition and, as predicted, could be rescued by ser-4 expression in the AIBs of ser-4 null animals (Figure 3-25). Interestingly, octr-1 null animals were also resistant, potentially through a pathway involving the direct 2-AG activation of the inhibitory OCTR-1 in the motorneurons (Figure 3-25). These results demonstrate that CBs stimulate octopaminergic and serotonergic signaling to modulate both nociception and locomotion.

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Figure 3-24. 2-AG inhibits locomotion in a dose-dependent manner.

Exogenous application of 2-AG dose-dependently inhibits locomotion in wild-type (N2) animals. Locomotion studies were conducted on hypotonic agar-only plates as described in Law et al. (Law et al., 2015) containing control (vehicle) or 2-AG (3, 30 or 300µM). Data are presented as a mean ± SE (n) and analyzed by two-tailed Student t test.

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Figure 3-25. The 2-AG-dependent locomotory inhibition is NPR-19- independent but requires the expression of SER-4 in the AIB interneurons.

Higher concentration of 2-AG (300 vs 30 or 3µM) significantly inhibit locomotion in an NPR-19-independent manner. 2-AG-dependent locomotory inhibition was absent in octr- 1, ser-4 and 5-HT quintuple null (5-HT quint) mutants which lack all identified 5-HT receptors. 2-AG sensitivity could be restored to 5-HT quint animals by expression of ser- 4 selectively in the AIB interneurons. Locomotion studies on wild-type (N2), null and transgenic animals were conducted on hypotonic agar-only plates as described in Law et al. (Law et al., 2015) containing 2-AG (300µM) and were performed as described in Chapter 2. The AIB-selective ser-4 transgenic rescue was generated by fusion PCR and is driven by the AIB-selective npr-9 promoter fused in-frame to ser-4 cDNA and includes unc-54 3’ UTR. * indicates a significant difference from N2 at 15 min (p value of ≤ 0.05). Data are presented as a mean ± SE (n) and analyzed by two-tailed Student t test.

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3.10. The cannabinoid-dependent inhibition of locomotion requires the release of 5-HT

The monoaminergic modulation of locomotory behaviors is complex and involves the differential interaction of dopaminergic, octopaminergic and serotonergic signaling pathways (Horvitz et al., 1982; Chase et al., 2004; Hills et al., 2004; Gray et al., 2005;

Donohoe et al., 2008; Harris et al., 2009; Allen et al., 2011; Vidal-Gadea and Pierce-

Shimomura, 2012). Interestingly, CBs appear to stimulate the release of 5-HT in the

CB-dependent modulation of locomotory behaviors. For example, 2-AG-dependent locomotory inhibition was reduced in tph-1 null animals. In contrast, 2-AG-dependent inhibition was still present and in fact, more robust in mod-5 null animals that lack a key neuronal 5-HT reuptake transporter, suggesting that 2-AG inhibition involved the stimulation of 5-HT release and not the inhibition of 5-HT reuptake. Similarly, as predicted from the results with ser-4 null animals, 2-AG-dependent inhibition was also significantly reduced in ser-5;ser-4;mod-1;ser-7 ser-1 5-HT receptor quintuple (5-HT quints) that lack most if not all 5-HT receptors (Figure 3-26). Similarly, 2-AG-dependent locomotory inhibition was also reduced in cat-1 null animals that lack the vesicular monoamine transporter (VMAT) and in cat-4 null animals that lack the human GCH1 orthologue (GTP cyclohydrolase 1), involved in the synthesis of both 5-HT and DA

(Figure 3-27A). Surprisingly, pre-incubation in either 5-HT or DA rescued the 2-AG- dependent locomotory inhibition in cat-4 null animals, suggesting that the overstimulation of either 5-HT or DA signaling compensated for the absence of the other

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(Figure 3-27B). Together, these data suggest that 2-AG stimulates the release of both 5-

HT to activate a complex monoaminergic signaling network to inhibit locomotion.

The increased 5-HT levels might also explain the requirement for OCTR-1 in the inhibition of nociception at higher CB levels, as 5-HT stimulates aversive responses, in part by activating serotonergic signaling in an array of additional neurons that is antagonized by ASH OCTR-1. Indeed, this complex serotonergic/octopaminergic antagonism in the modulation of ASH-dependent aversive responses has been characterized previously, with at least three different 5-HT receptors, SER-1, SER-5 and

MOD-1, involved in stimulating the initiation of aversive responses (Wragg et al., 2007;

Harris et al., 2009; Mills et al., 2012).

Interestingly, acute exposure (5 min) to higher 2-AG concentrations also significantly increases the frequency of spontaneous reversals off-food in an NPR-19- independent manner and 2-AG stimulation of reversals is absent in tph-1, but not cat-2, null animals lacking 5-HT and DA biosynthesis, respectively (Figure 3-28A). The 2-AG- dependent stimulation of reversal frequency was absent in 5-HT quintuple null animals lacking all known 5-HT receptors, consistent with requirement for 5-HT signaling

(Figure 3-28B). More specifically, mod-1 null animals lacking the 5-HT-gated chloride channel MOD-1 and ser-1 null animals lacking the 5-HT2-like receptor SER-1 were resistant to the 2-AG stimulation of reversals (Figure 3-28B). These results are consistent with previous studies demonstrating a role for MOD-1 and SER-1 in the modulation of reversal frequency (Donohoe et al., 2008; Harris et al., 2009). Together, these data highlight the complex interaction among cannabinoid, serotonergic and octopaminergic signaling and suggest that they may also be relevant to understanding the role of 81

exogenous CBs in the modulation of human behavior, as C. elegans has previously proven to be useful model for understanding monoaminergic modulation in mammals

(Komuniecki et al., 2012; Mills et al., 2012).

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Figure 3-26. 2-AG-dependent locomotory inhibition requires 5-HT signaling.

Locomotory rates at 0 and 15 minutes in wild-type (N2), tph-1 (mg280), mod-5 (n3314) and 5-HT quintuple null animals following 2-AG treatment. Locomotion studies were conducted on hypotonic agar-only plates as described in Law et al. (Law et al., 2015) containing 2-AG (300µM). * indicates a significant difference from N2 (p value of ≤ 0.05). Data are presented as a mean ± SE (n) and analyzed by two-tailed Student t test.

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Figure 3-27. Pre-incubation in 5-HT or DA restores 2-AG-dependent locomotory inhibition in cat-4 null animals.

A) Incubation of wild-type (N2), cat-1 (ok411) and cat-4 (ok342) null animals in 2-AG. B) Pre-incubation of cat-4 (ok342) null animals in 2-AG, 2-AG + 5-HT, 2-AG + DA and 2-AG + 5-HT +DA. . Locomotion was assayed on hypotonic agar-only plates as described in Law et al. (Law et al., 2015) containing 2-AG (300µM), 2-AG (300µM) + 5-HT (2mM), 2-AG (300µM) + DA (5mM) or 2-AG (300µM) + 5-HT (2mM) + DA (5mM). * indicates a significant difference from N2 (A) and N2 + 2-AG (B) (p value of ≤ 0.05). Data are presented as a mean ± SE (n) and analyzed by two-tailed Student t test.

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Figure 3-28. The 2-AG-dependent stimulation of reversal frequency is dependent on serotonergic signaling through MOD-1 and SER-1.

A) 2-AG increases the frequency of spontaneous reversal off food in an NPR-19- independent manner that requires serotonergic, but not dopaminergic, signaling. B) 2- AG-dependent stimulation of reversals is absent in animals lacking the 5-HT-gated chloride channel, MOD-1 and the 5-HT2-like receptor, SER-1. Locomotion studies on wild-type (N2) and null animals were conducted on standard NGM plates as described in WormBook (wormbook.org) containing 2-AG (300µM). * indicates a significant difference from N2 at (p value of ≤ 0.05). Data are presented as a mean ± SE (n) and analyzed by two-tailed Student t test.

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3.11. The cannabinoid-dependent inhibition of locomotion requires DA release and cannabinoids stimulate locomotion in animals deficient in dopaminergic signaling

2-AG-dependent locomotory inhibition was also absent in animals deficient in DA signaling, including cat-2 null animals that lack a key DA biosynthetic enzyme and in dop-2; dop-4 dop-1 dop-3 DA receptor quadruple (DA quads) null animals that lack most if not all DA receptors (Figure 3-29). More specifically, 2-AG-dependent inhibition was absent in the Gαs-coupled DA receptor dop-4 (Figure 3-29). In contrast, dop-3 null animals lacking the Gαo-coupled DA receptor were hypersensitive to 2-AG-dependent inhibition, highlighting the antagonistic relationship between DOP-3 and DOP-4 signaling that has been described previously in the DA modulation of the two ASH sensory neurons (Ezcurra et al., 2011) (Figure 3-29). In fact, 2-AG rapidly (<30 sec) and significantly (>25%) stimulate locomotion in many of these mutant animals with deficient DA signaling. Together, these data strongly suggest that 2-AG stimulates DA release and requires the D1-like receptor DOP-4 to mediate 2-AG-dependent locomotory inhibition.

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Figure 3-29. 2-AG-dependent locomotory inhibition requires DA signaling.

Locomotory rates at 0 and 15 minutes in wild-type (N2), cat-2 (n4547), dop-3 (vs106), dop-4 (ok1321) and DA quadruple null animals following 2-AG treatment. Locomotion studies were conducted on hypotonic agar-only plates as described in Law et al. (Law et al., 2015) containing 2-AG (300µM). * indicates a significant difference from N2 (p value of ≤ 0.05). Data are presented as a mean ± SE (n) and analyzed by two-tailed Student t test.

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3.12. Cannabinoids stimulate the release of both 5-HT and DA in an assay system developed to detect acute 5-HT and DA release

Currently, no assay system is available to measure monoamine release directly in

C. elegans, therefore, changes in monoamine levels have been measured indirectly via reductions in neuronal monoamine immunofluorescence or increases in the transcription of key monoamine biosynthetic enzymes (Zhang et al., 2004). Indeed, changes in GFP fluorescence driven by the tryptophan hydroxylase (tph-1) promoter has been used successfully to approximate 5-HT release (Zhang et al., 2004). However, these methods are time-consuming, indirect and in the case of immunostaining, dependent on the quality of the antisera. Therefore, to measure the release of endogenous 5-HT or DA release directly, we modified a heterologous expression system in remodeled C. elegans used for agonist identification that we had developed previously (Law et al., 2015). In this system, the Gαo-coupled 5-HT1-like 5-HT receptor, SER-4 or the Gαo-coupled D2-like

DA receptor, DOP-3 were expressed in the cholinergic motorneurons of 5-HT quintuple and DA quadruple null animals, respectively. Theoretically, any 2-AG-dependent monoamine release should activate its cognate receptor on the cholinergic motorneurons, inhibit acetylcholine (ACh) release onto the muscle and inhibit locomotion. As predicted,

5-HT or DA had no effect on the locomotory rate of 5-HT quintuple and DA quadruple null animals, but rapidly inhibited locomotion after the selective expression of the appropriate Gαo-coupled monoamine receptor in the motorneurons (Figure 3-30A).

Similarly, as noted above, the 5-HT quintuple and DA quadruple null animals were resistant to 2-AG-dependent inhibition, but following expression of SER-4 in the

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motorneurons of the 5-HT quints and DOP-3 in the motorneurons of the DA quads, 2-AG rapidly inhibited locomotion in both heterologously expressed animals (Figure 3-30B).

Together, these data confirm our initial hypothesis that 2-AG stimulates the release of both 5-HT and DA and validates the use of transgenic 5-HT quint and DA quad animals to detect CB-dependent 5-HT and DA release.

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Figure 3-30. 2-AG inhibits locomotion in remodeled C. elegans designed to detect acute 5-HT or DA release.

A-B) Gαo-coupled 5-HT (SER-4) and DA (DOP-3) receptors were expressed in the cholinergic motorneurons of ser-5;ser-4;mod-1;ser-7 ser-1 5-HT receptor quintuple null animals (5-HT quint) or dop-1;dop-2;dop-3;dop-4 DA receptor quadruple null animals (DA quad) null animals that lack most, if not all, 5-HT and DA receptors, respectively, on the assumption that any CB-dependent monoamine release would activate its cognate receptor on the motorneurons, inhibit ACh release onto the muscle and inhibit locomotion. A) Expression of unc-17β::ser-4 in the 5-HT quintuple null and unc- 17β::dop-3 in DA quadruple null animals restores sensitivity to 5-HT and DA, respectively. B) Expression of unc-17β::ser-4 in the 5-HT quintuple null and unc- 17β::dop-3 in DA quadruple null animals restores sensitivity to 2-AG. Locomotion studies were conducted on hypotonic agar-only plates as described in Law et al. (Law et al., 2015) containing 5-HT (2mM), DA (5mM) or 2-AG (300µM) and were performed as described in Chapter 2. The unc-17β::ser-4 and unc-17β::dop-3 transgenic rescues were generated by PCR fusion as described in Esposito et al. (Esposito et al., 2007) of a short (250bp) unc-17 promoter fused to ser-4 or dop-3 cDNA and includes the unc-54 3’ UTR. * indicates a significant difference from 0 min (p value of ≤ 0.05). Data are presented as a mean ± SE (n) and analyzed by two-tailed Student t test.

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3.13. Identification of the neurons involved in the cannabinoid- dependent 5-HT and DA release

To identify the monoaminergic neurons involved in CB-dependent locomotory inhibition, we used a combination of selective RNAi knockdown/rescue in the serotonergic neurons, where selective promoters were available. Indeed, expression of tph-1 selectively in the ADFs rescued 2-AG-dependent locomotory inhibition in tph-1 null animals (Figure 3-31A). In contrast but in agreement with the ADF-selective rescue,

ADF-selective RNAi knockdown of tph-1 in wild-type animals abolished 2-AG- dependent inhibition (Figure 3-31A). Together these data suggest that CBs stimulate endogenous 5-HT release selectively in the serotonergic ADF neurons to mediate CB- dependent locomotory inhibition.

To identify the dopaminergic neurons involved in the 2-AG-dependent locomotory inhibition, we measured CB-dependent increases in cat-2::gfp fluorescence, on the assumption that following CB-dependent DA release, DA biosynthesis will be upregulated to replenish DA stores. We expressed mCherry driven by the DA biosynthetic enzyme tyrosine hydroxylase cat-2 promoter in wild-type animals and assessed changes in cat-2p::mCherry fluorescence following 2-AG exposure. In the anterior of the worm we observed cat-2 expression in the 2 ADE and 4 CEP neurons, confirming correct expression of cat-2 (Figure 3-31B). Our preliminary results suggest that 2-AG robustly increases cat-2::mCherry fluorescence in the ADEs, but not the CEPs, after 1 hour 2-AG exposure (Figure 3-31B and C). While these results need to be supported with direct detection of neuronal DA changes via immunofluorescence or

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induced fluorescence, these data suggest that 2-AG may stimulate DA release selectively from the ADEs and the 2-AG effects on DA biosynthesis are most likely indirect in order to replenish DA stores.

Figure 3-31. 2-AG-dependent inhibition of locomotion requires 5-HT and DA from the sensory ADFs and ADEs neurons, respectively.

A cat-2p::mCherry transgene was expressed in 6 dopaminergic neuron pairs in the amphid, 4 CEPs and 2 ADEs, and fluorescence was analyzed after 2-AG incubation. B) 2-AG significantly increases cat-2p::mCherry fluorescence in the ADE, but not the CEP, sensory neurons. C) Quantification of selective 2-AG-dependent increase in cat- 2p::mCherry fluorescence in the ADE neurons. Images were captured using an Olympus IX81 confocal microscope and analyzed using ImageJ. * indicates a significant difference from control (p value of ≤ 0.05). Data are presented as a mean ± SE (n) and analyzed by two-tailed Student t test.

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3.14. TRP channels are involved in the cannabinoid-dependent inhibition of locomotion

Interestingly, 2-AG rapidly (< 30 seconds) and significantly (> 25 %) stimulate locomotion in cat-2 and DA receptor quadruple null animals (Figure 3-32). Therefore, due to this rapid response, we hypothesized that to mediate such a rapid response, 2-AG may activate surface receptors in the amphids or phasmids. Therefore, to further define the 2-AG-dependent signaling pathways leading to monoamine release and locomotory inhibition, we used a reverse genetics approach and screened a number of sensory signaling mutants for loss of or reduced 2-AG-dependent inhibition of locomotion. Using this approach, we identified a number of TRP channel subunits that were resistant to 2-

AG-dependent inhibition, including ocr-1, ocr-4 and osm-9, that encode TRPV1-like subunits, and trp-4 that encodes the pore-forming subunit of a mechanosensitive TRPN

(transient receptor potential nompC (no mechanoreceptor potential C)) channel expressed in the dopaminergic neurons (Figure 3-32). Importantly, locomotion in these TRPV channel mutant animals was largely wild-type in the absence of 2-AG. These results were of particular interest since pCBs and eCBs activate mammalian TRP channels in the low nM range, although little is known about the role of these channels in CB-dependent behavioral modulation (Di Marzo et al., 1998; De Petrocellis et al., 2001; Di Marzo and

Maccarrone, 2008; Maccarrone et al., 2008). In C. elegans, many of these TRP channel subunits form heteromeric channels, that are involved in the sensory-mediated modulation of a host of behaviors, including nociception, locomotion and egg-laying

(Colbert et al., 1997; Colbert and Bargmann, 1997; Tobin et al., 2002; Ezak et al., 2010).

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For example, OSM-9 and OCR-2 form a functional heteromeric channel that is expressed in number of sensory neurons and involved in variety of sensory-mediated behaviors.

Unfortunately, we were not able to examine ocr-2 null animals in these 2-AG dependent locomotory assays, as the ocr-2 null animals rapidly slowed when placed on a plate in the absence of food and 2-AG. Importantly, osm-9 is more broadly expressed than ocr-2

(Colbert et al., 1997; Tobin et al., 2002). Interestingly, CBs initially stimulated locomotion in the trp-4 null animals, a phenotype also observed in animals deficient in dopaminergic signaling. In contrast, the temporal pattern of slowing in the TRPV1-like mutants mimicked that observed in animals with compromised serotonergic signaling, as outlined above (Figure 3-33).

To confirm a more direct role for TRP channels in 2-AG-dependent locomotory inhibition, uncompromised by potential compensation in the mutant animals, we examined the effects of 2-aminoethoxydiphenyl borate (2-APB), a non-selective TRP channel antagonist that at low concentrations inhibits TRP channels (Xu et al., 2005;

Togashi et al., 2008). 100μM 2-APB alone inhibited locomotion in wild-type C. elegans, but had no effect at 10μM (Figure 3-34). Interestingly, higher 2-APB concentrations have been reported to function as agonists at some TRP channels (Xu et al., 2005; Togashi et al., 2008). In fact, although 100μM 2-APB dramatically inhibited locomotion in wild- type animals, it had no effect on locomotion in cat-2 null animals with deficient dopaminergic signaling, suggesting that 2-APB at these higher concentrations might be activating TRP-4 on the dopaminergic neurons to stimulate DA release. Importantly, although 2-APB at 10μM had no effect on locomotion, it abolished aversive responses to dilute 1-octanol that requires the activation of a heteromeric OSM-9/OCR-2 TRPV1-like 94

channel in the two ASH sensory neurons, validating the use of 2-APB as a TRPV-1-ilke antagonist in these studies (Figure 3-35A). As predicted, 2-APB at 10μM also had no effect on 5-HT-dependent locomotory inhibition in 5-HT quint mutants expressing SER-4 in the motorneurons, but dramatically reduced 2-AG-dependent inhibition in these same animals (Figure 3-35B). In contrast, 2-APB at 10μM had no effect on either the DA or 2-

AG-dependent inhibition in DA quad mutants expressing DOP-3 in the motorneurons

(not shown). The reason for inability of 2-APB to prevent 2-AG dependent locomotory inhibition in the DA quads expressing DOP-3 in the motorneurons is unclear. TRP-4 may be insensitive to 2-APB; indeed, 2-APB does not appear to inhibit similar TRPN channels in mammals (Xu et al., 2005; Togashi et al., 2008). Alternatively, 2-APB at 10μM may not have access to TRP-4 during the time course of these experiments. Indeed, cilia of the mechanosensory dopaminergic neurons, where TPR-4 is expressed, end embedded directly in the cuticle, presumably making these neurons less responsive to chemical perturbations in the external environment, in contrast to dendrites from most sensory neurons found in the amphids that open to the outside and are directly exposed to the external environment (Inglis et al., 2005). Together, observations support that hypothesis that 2-AG activates TRPV-1-like channels that, in turn, either directly or indirectly simulate 5-HT release to inhibit locomotion.

To determine if OSM-9 and TRP-4 function in the monoaminergic neurons to mediate 2-AG-dependent locomotory inhibition, we expressed either a tph-l::osm-9 or cat-2::trp-4 RNAi transgene in wild-type animals to drive knockdown in the serotonergic and dopaminergic neurons, respectively. As predicted, osm-9 RNAi knockdown in the serotonergic neurons and trp-4 RNAi knockdown in the dopaminergic neurons 95

significantly reduced 2-AG sensitivity in wild-type animals (Figure 3-36). These data strongly suggest that 2-AG requires OSM-9 on the serotonergic, and TRP-4 on the dopaminergic, neurons to stimulate the release of 5-HT and DA, respectively. Together, these data demonstrate that the expression of specific TRP channels is required in monoaminergic neurons for the CB-dependent release of 5-HT and DA. Whether these

TRP channels are activated directly by CBs and whether they are functioning in a specific subset of dopaminergic or serotonergic neurons remains to be determined.

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Figure 3-32. Several TRP channel subunits are required for 2-AG- dependent locomotory inhibition.

N2, ocr-1 (ok132), ocr-2 (ak47), ocr-4 (vs137), osm-9 (ky10) and trp-4 (ok1605) animals were screened for loss of 2-AG dependent locomotory inhibition and rapid (<30sec) 2- AG-dependent initial stimulation of locomotion in trp-4 (ok1605) null animals. Locomotion studies were conducted on hypotonic agar-only plates as described in Law et al. (Law et al., 2015) containing 2-AG (300µM) and were performed as described in Chapter 2. * indicates a significant difference from N2 (p value of ≤ 0.05). Data are presented as a mean ± SE (n) and analyzed by two-tailed Student t test.

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Figure 3-33. The temporal pattern of 2-AG-dependent locomotory inhibition in osm-9 and trp-4 null animals is similar to that of 5-HT and

DA deficient animals, respectively.

The temporal patterns of 2-AG-dependent locomotory inhibition of osm-9 and trp-4 null animals are identical to the temporal patterns of 2-AG inhibition of tph-1 and cat-2 null animals, respectively. Locomotion studies were conducted on hypotonic agar-only plates as described in Law et al. (Law et al., 2015) containing 2-AG (300µM). Data are presented as a mean ± SE (n) and analyzed by two-tailed Student t test.

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Figure 3-34. At high concentrations, the TRP channel inhibitor, 2-APB, inhibits basal locomotion in wild-type animals.

The non-selective TRP channel inhibitor 2-aminoethoxydiphenyl borate (2-APB) has no effect on basal locomotion in wild-type (N2) animals at low concentrations (10μM) but mimics 2-AG addition at higher concentrations (100μM). Locomotion studies were conducted on hypotonic agar-only plates as described in Law et al. (Law et al., 2015) containing 2-AG (300µM), 2-APB (0, 10, 20, 50 and 100μM) and were performed as described in Chapter 2. * indicates a significant difference from N2 at 0μM 2-APB (p value of ≤ 0.05). Data are presented as a mean ± SE (n) and analyzed by two-tailed Student t test.

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Figure 3-35. The TRP channel inhibitor, 2-APB, abolishes aversive responses to 100% 1-octanol in wild-type animals and the 2-AG locomotory inhibition in remodeled C. elegans designed to detect acute

5-HT release.

A) Treatment of wild-type (N2), 5-HT quintuple null animals expressing unc-17β::ser-4 in the cholinergic motor neurons and DA quadruple null animals expressing unc- 17β::dop-3 with 2-aminoethoxydiphenyl borate (2-APB), a non-specific TRP channel blocker inhibits 2-AG-dependent 5-HT release and locomotory inhibition. B) 2-APB inhibits basal aversive responses to 100% 1-octanol and mimics the octanol-resistant phenotype of osm-9 null animals. Locomotion studies were conducted on hypotonic agar- only plates as described in Law et al. (Law et al., 2015) containing vehicle (control), 2- AG (300µM), 2-APB (10µM) or 2-AG (300µM) + 2-APB (10µM) and were performed as described in Chapter 2. Aversive responses to 1-octanol in wild-type and null animals were examined as described by Harris et al. (Harris et al., 2009) and in Chapter 2. For aversive assays using 100% 1-octanol, worms were incubated in vehicle (control), 2-APB (10μM) for 10 minutes prior assay. * indicates a significant difference from 2-AG treatment (p value of ≤ 0.05). Data are presented as a mean ± SE (n) and analyzed by two-tailed Student t test. 100

Figure 3-36. OSM-9 and TRP-4 function in the serotonergic and dopaminergic neurons, respectively, to mediate 2-AG inhibition

Expression of an tph-1::osm-9 or cat-2::trp-4 RNAi transgene in wild-type (N2) animals abolishes 2-AG-dependent locomotory inhibition. The tph-1 and cat-2 promoters drive expression in the serotonergic and dopaminergic neurons, respectively. Locomotion studies were conducted on hypotonic agar-only plates as described in Law et al. (Law et al., 2015) containing vehicle (control) or 2-AG (300µM) and were performed as described in Chapter 2.The tph-1::osm-9 and cat-2::trp-4 transgenic rescues were generated by PCR fusion as described in Esposito et al. (Esposito et al., 2007). * indicates a significant difference from N2 at 15 min (p value of ≤ 0.05). Data are presented as a mean ± SE (n) and analyzed by two-tailed Student t test.

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Chapter 4

Discussion

4.1. Overview

Cannabis or marijuana alters sensory perception and has been purported to be useful for the treatment of a range of conditions, including anxiety, epilepsy, nausea, loss of appetite, and pain management. In addition, anecdotal evidence suggests that cannabis is useful for nematode control in agricultural settings and that may help to control parasitic gut nematodes in humans, although the mechanism underlying these purported medicinal effects is unclear (Walker and Huang, 2002). Cannabis sativa contains more than 100 bioactive compounds, or phytocannabinoids (pCBs), including cannabidiol (CBD) and the , Δ9-tetrahydrocannabinol (THC) that interact with an endogenous cannabinoid (eCB) signaling system initiated by the eCBs, arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine (anandamide or AEA).

Both eCBs and pCBs differentially activate a canonical eCB signaling pathway initiated by two Gαo-coupled CB receptors, CB1 and CB2. CB1 and CB2 are differentially expressed and, in part, mediate a retrograde signal from postsynaptic to presynaptic neurons to inhibit neurotransmitter release and CB1-mediated signaling has been

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associated with short and long-term depression in both excitatory and inhibitory synapses

(Diana and Marty, 2004; Kheirbek, 2007; Adermark et al., 2009; Ohno-Shosaku and

Kano, 2014). Although structurally distinct, 2-AG and AEA are considered the eCB mimics of CBD and THC, respectively. For example, 2-AG plays a key role in neuromodulation and pain suppression. Conversely, AEA activates reward centers in the brain, and is the major neurotransmitter responsible for exercise-induced euphoria, or

“runner’s high” (Fuss et al., 2015). Two membrane-bound pre-synaptic enzymes, monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH) degrade 2-AG and AEA, respectively, to terminate CB signaling.

Although initial reports suggested that nematodes lacked canonical CB receptors, the present study demonstrates that C. elegans contains an endogenous CB signaling system mediated by a canonical CB receptor that modulates an array of key behaviors. 2-

AG and AEA have been identified previously in C. elegans extracts by GC/MS

(Lehtonen et al., 2011) and in the present study, both eCBs caused a “dazed and confused” phenotype, inhibiting aversive behavior, feeding, locomotion and increasing turning when applied exogenously. Indeed, in contrast to previous reports that suggested

C. elegans does not contain a canonical CB receptor, based on failed attempts to identify

CB receptors by sequence analysis (McPartland et al., 2001; Pastuhov et al., 2016), we have identified a predicted neuropeptide receptor, NPR-19, that is essential for CB- dependent behaviors and that responds directly to eCBs with high affinity with affinities similar to human CB1. As predicted, the CB-dependent inhibition of feeding, as measured by pharyngeal pumping and fluorescent microsphere uptake, and nociception, as measured by the initiation of aversive responses to the noxious repellant, 1-octanol, are 103

absent in npr-19 null mutants and can be rescued by the expression of the human CB1, driven by the npr-19 promoter, confirming the orthology of the two receptors.

Conversely, inhibiting the enzymes responsible for the breakdown of endogenous 2-AG or AEA, monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH), respectively, mimics eCB addition and inhibits nociception in an NPR-19 dependent manner (Oakes et al., 2017). Interestingly, NPR-19 and CB1 exhibit only limited sequence identity (23%), key amino acids involved in AEA binding appear to be conserved and, importantly, both 2-AG and AEA activate NPR-19 directly after heterologous expression in Xenopus laevis oocytes, with nM affinities similar to those of human CB1.

eCBs also activate octopaminergic, serotonergic and dopaminergic signaling in C. elegans to modulate aversive responses and locomotory behaviors by acting as agonists for the α2A-adrenergic-like OA receptor, OCTR-1, and increasing the release of endogenous 5-HT and DA through pathways requiring the expression of TRPV-1 and mechanosensitive TRPN (NOMPC) channels on serotonergic and dopaminergic neurons, respectively. Together, these results highlight the advantages of studying CB signaling in a genetically-tractable, whole-animal model and might also explain the proposed anthelmintic properties of Cannabis, given that the CB-dependent locomotory inhibition mimics the “locomotory confusion” phenotype previously proposed as a potential anthelmintic target (Law et al., 2015). These studies also provide insights into the potential effects of Cannabis on monoaminergic signaling in humans and suggest a role for CBs in activating the dopaminergic and serotonergic systems involved in reward and anxiety/depression, respectively. 104

4.2. Cannabinoid signaling through NPR-19-independent pathways stimulates 5-HT and DA release

CBs also signal through NPR-19 independent pathways to inhibit locomotion and increase turning behavior. These NPR-19 independent signaling pathways involve the endogenous release of both 5-HT and DA from subsets of monoaminergic neurons. For example, CB-dependent locomotory inhibition is dramatically reduced in tph-1 and cat-2 null animals that encode key enzymes in 5-HT and DA biosynthesis, respectively, and ser-4 and dop-4 null animals that encode 5-HT and DA receptors, respectively. In contrast, locomotion in dop-3 null animals that lack a Gαo-coupled DA receptor is hypersensitive to CB inhibition, highlighting the complex effects of CBs on dopaminergic signaling. CB-stimulated 5-HT release activates SER-4 in two key interneurons, leading to “locomotory confusion” and transient paralysis, as described previously (Law et al., 2015). Importantly, CBs inhibit both aversive behavior and locomotion in mod-5 or dat-1 null animals that lack reuptake transporters for either 5-HT or DA, respectively, suggesting that CBs exert their effects by stimulating monoamine release, not exclusively by inhibiting monoamine reuptake. CB-dependent locomotory inhibition is also absent in cat-4 null animals. cat-4 encodes an orthologue of human GTP cyclohydrolase 1 and contains reduced levels of both 5-HT and DA. CB-dependent locomotory inhibition can be rescued in the cat-4 null animals by incubation in either 5-

HT or DA, suggesting that the overstimulation of either 5-HT or DA signaling compensates for the absence of the other. CBs also increase reversals and omega turns

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through a pathway requiring 5-HT, but not DA or NPR-19 signaling, suggesting that CBs might be mimicking the transition to starvation conditions.

Importantly, CBs also inhibit locomotion in remodeled chimeric mutant animals designed to detect the acute release of either 5-HT or DA. To construct this model, Gαo- coupled 5-HT (SER-4) and DA (DOP-3) receptors were expressed in the cholinergic motorneurons of ser-5;ser-4;mod-1;ser-7 ser-1 5-HT receptor quintuple null animals (5-

HT quint) or dop-2; dop-4 dop-1 dop-3 DA receptor quadruple null animals (DA quad) null animals that lack most, if not all, 5-HT and DA receptors, respectively, on the assumption that any CB-dependent monoamine release would activate its cognate receptor on the motorneurons, inhibit ACh release onto the muscle and inhibit locomotion. As predicted, 5-HT or DA have no effect on locomotion in the 5-HT quint or

DA quad mutants, respectively, but rapidly inhibit locomotion after the selective expression of the appropriate inhibitory Gαo-coupled monoamine receptor in the motorneurons, supporting the hypothesis that CBs stimulate monoamine release.

Importantly, these results validate our use of this chimeric C. elegans model as a tool in future studies to identify the neurons and receptors responsible for mediating CB- dependent monoamine release, as well as the neuronal pathways responsible for the behavioral consequences.

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4.3. TRP channels are essential for Cannabinoid-dependent 5-HT and

DA release

CB-dependent locomotory inhibition is also absent in ocr-1, ocr-4, osm-9 and trp-

4 null animals that lack functional TRP channel subunits. ocr-1, ocr-4, osm-9 encode

TRPV-1 like channel subunits expressed in a number of sensory neurons and trp-4 encodes the pore-forming subunit of a mechanosensitive TRPN (NOMPC) channel expressed in the dopaminergic neurons. Similarly, 2-aminoethoxydiphenol (2-APB), a non-selective TRP channel blocker also inhibits CB-dependent locomotory inhibition.

More importantly, 2-APB also differentially inhibits CB-dependent locomotory inhibition in remodeled mutant animals used to measure acute 5-HT or DA release, suggesting that

CB-dependent TRP channel activation is required for monoamine release and locomotory inhibition. Interestingly, the temporal pattern of 2-AG inhibition differs significantly in the 5-HT/OSM-9 and DA/TRP-4 deficient signaling mutants. For example, in mutants with disrupted serotonergic or TRPV-1 signaling, 2-AG-dependent inhibition is initially delayed, but eventually these mutant animals begin to slow (25% at 30 min). In contrast, in mutants with disrupted DA or TRP-4 signaling, 2-AG never inhibits locomotion and, in fact, 2-AG rapidly (<30 sec) and significantly (>25%) stimulates locomotion in these mutants, suggesting that TRP-4 might be modulating DA release. Indeed, previous studies have demonstrated functional correlations between TRP-4 function in the dopaminergic neurons and DA-dependent locomotory behaviors. For example, trp-4 null animals are deficient in the basal slowing behavior, which is dependent on DA release

(Kindt et al., 2007).

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4.4. Cannabinoids modulate additional behaviors in nematodes.

CBs appear to mediate entry into dauer, a quiescent stage designed to survive harsh environments, based on studies involving the application of exogenous N- acylethanolamines (NAEs), including AEA, the decrease of endogenous AEA levels by the overexpression of FAAH responsible for AEA degradation or the addition of mammalian CB receptor agonist/antagonists in both wild-type and dauer-constitutive mutant backgrounds (Lucanic et al., 2011). Dauer entry is a complex process mediated by the interaction of both the TGFβ and insulin/IGF signaling pathways (Shaw et al., 2007).

For example, animals with mutations in either daf-2 that encodes an insulin/IGF receptor or daf-7 that encodes a TGFβ ligand constitutively form dauer larvae in the absence of nutrient depletion (Shaw et al., 2007; Lucanic et al., 2011; Reis-Rodrigues et al., 2016).

NPR-19 is most highly expressed during the dauer stage and the levels of NAEs, including AEA, peak during the second larval stage (L2), during which the animal commits to reproductive growth rather than entry into dauer (Lucanic et al., 2011). In fact, NAE levels are highest at the early pre-dauer L2 (L2d) and rapidly decline in the late L2d and subsequent dauer stages (Lucanic et al., 2011). These data suggest that

NAEs, including AEA, signal nutrient availability to modulate dauer entry. However, although all of these studies demonstrate an impact of CBs on dauer formation and life- span, the results are sometimes conflicting. For example, exogenous NAEs, including eicosapentaenoyl ethanolamide (EPEA) and AEA, prevent dauer entry and stimulate reproductive growth in dauer-constitutive animals, including daf-2 mutants with compromised insulin/IGF signaling (Lucanic et al., 2011). In addition, eCBs appear to regulate the transport/mobilization of cholesterol required for the production of 108

dafachronic acids that govern dauer formation (Galles et al., 2018). For example, C. elegans is unable to synthesize cholesterol and cholesterol depletion leads to dauer formation and 2-AG or AEA rescue dauer formation induced by 1) cholesterol depletion by increasing cholesterol mobilization in wild-type animals or 2) impaired TGFβ signaling in daf-7 animals (Galles et al., 2018). In contrast, AM251, a CB1 receptor antagonist, suppresses constitutive dauer entry and promotes reproductive growth in daf-

2 mutants, while one, O-2545, but not all CB1 agonists, interfere with AM251 dauer suppression and promote dauer formation in daf-2 animals at semi permissive temperatures (Reis-Rodrigues et al., 2016). However, it is important to note that the selectivity or specificity of these mammalian CB1 effectors have never been examined on nematode GPCRs. For example, AM251 is also a potent agonist for GPR55 and potentially its putative nematode homologue, NPR-9, based on sequence analysis (Kapur et al., 2009).

Endogenous NAE levels are regulated by food availability. For example, NAE levels are significantly reduced in starved vs well fed first-stage larvae and are dramatically increased following feeding (Lucanic et al., 2011). Dietary restriction (DR) via starvation or deficiencies in pharyngeal pumping significantly increases life-span in adult C. elegans (Klass, 1977; Hosono et al., 1989; Avery, 1993a; Lakowski and Hekimi,

1998; Vanfleteren and Braeckman, 1999; Finch and Ruvkun, 2001; McKay et al., 2004).

Decreasing endogenous NAE levels via faah-1 over-expression, especially in the pharynx, mimics DR, delays development and increases adult lifespan. Similarly, fat-4 null animals that lack a delta-5 fatty acid desaturase required for NAE synthesis also have reduced NAE levels and an extended lifespan (Lucanic et al., 2011). Conversely, 109

exogenous NEAs, including AEA significantly reduce DR-mediated increase in lifespan in wild-type animals (Lucanic et al., 2011). Taken together, these observations demonstrate that NAEs and potentially CBs play a role in nutrient sensing and act as a

“food availability” signal that controls dauer entry and determines lifespan.

CBs also inhibit axon regeneration following neuronal injury (Pastuhov et al.,

2012). For example, laser severed axons begin regeneration within 24 hours and make a full recovery in wild-type animals (Pastuhov et al., 2012). In contrast, axon regeneration is reduced and recovery delayed in faah-1 null animals with increased EPEA and AEA, suggesting that EPEA and AEA inhibit axon regeneration (Pastuhov et al., 2012).

Similarly, the over-expression of NAPE-1, a specific phospholipase D responsible for

AEA synthesis, mimics the loss of faah-1 and also significantly inhibits axon regeneration (Pastuhov et al., 2016). As predicted, exogenous EPEA or AEA also significantly reduce axon regeneration in wild-type animals (Pastuhov et al., 2012).

Importantly, AEA had a more pronounced inhibitory effect on axon regeneration than

EPEA (Pastuhov et al., 2012). The AEA-dependent inhibition of axon regeneration is absent in animals lacking the G-protein Gαo subunit, GOA-1, suggesting AEA inhibits axon regeneration via a Gαo-coupled receptor and two predicted neuropeptide receptors,

NPR-19 and NPR-32, were identified as potential mediators, based on the ability of npr-

19;faah-1 and npr-32;faah-1 double mutants to rescue the axon regeneration deficiency of faah-1 single mutants (Pastuhov et al., 2016). Since npr-19 and npr-32 null animals do not have axon regeneration deficiencies similar to faah-1, these two receptors were proposed to function redundantly to inhibit axon regeneration. Unfortunately, these studies did not rescue axon regeneration phenotypes in the npr-19 or npr-32 null animals 110

and axon regeneration was not measured in npr-19;npr-32 double mutants (Pasuhov et al., 2016). Together with the results outlined in chapter 3, these observations suggest that

CB signaling in C. elegans is extensive, with much still remaining to be uncovered

(Figure 4-1).

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Figure 4-1. Comprehensive diagram of CB-dependent signaling in C. elegans.

The eCBs 2-AG and AEA are synthesized by diacylglycerol lipase (DAGL) and N- arachidonylphosphatidylethanolamine (NAPE)-phospholipase D (NAPE-PLD), respectively. AEA modulates lifespan and inhibits dauer formation by stimulating cholesterol mobilization and subsequent activation of DAF-9 (Galles et al., 2018; Rodrigues et al., 2016; Lucanic et al., 2016). 2-AG and AEA activate the canonical cannabinoid receptor NPR-19 in the URX and M3 neurons to inhibit nociception and feeding, respectively (Oakes et al., 2017). NPR-19 is also involved in the AEA- dependent modulation of axon regeneration (Pastuhov et al., 2016). 2-AG also activates the α2-adrenergic-like receptor OCTR-1 directly to inhibit locomotion and nociception (Oakes et al., 2017). 2-AG and AEA also activate cannabinoid receptor independent signaling pathways to inhibit locomotion and stimulate reversal and omega turns. 2-AG and AEA activate TRPV1-like channels which lead to serotonergic neuron activation and 5-HT release to increase reversal and omega turn frequency, as well as inhibiting locomotion via the 5-HT1-like receptor SER-4 in the AIB interneurons. 2-AG also activates TRP-4 directly on the dopaminergic neurons to stimulate the release of DA to activate the D1-like DA receptor DOP-4 to inhibit locomotion. eCB signaling is terminated when 2-AG and AEA are hydrolyzed by monoacylglycerol lipase (MAGL) and fatty acid amide hydroxylase (FAAH), respectively.

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4.5. Similarities between cannabinoid signaling in nematodes and mammals

Given the complexity of the mammalian nervous it has been difficult, if not impossible, to relate studies at the neuronal level with changes in individual behaviors and most correlations of CB signaling with behavior have been inferred from observations at the molecular/cellular level. In contrast, our recent work with the genetically-tractable, nematode model, Caenorhabditis elegans, has examined the molecular/neuronal consequences of CB signaling with whole-animal behavior and dissected the role of CB signaling on sensory integration and decision-making, using the wide range of molecular tools available in this model system (forward and reverse screens, calcium imaging, neuron-specific activation, knockdown and rescue) (Riddle et al., 1997; Sengupta and Samuel, 2009; Boulin and Hobert, 2012; Chung et al., 2013).

Importantly, the utility of C. elegans as a translational tool for understanding basic processes and drug action in the mammalian nervous system is well documented

(Engleman et al., 2016). For example, C. elegans contains a compact nervous system

(only 302 neurons and about 7000 synapses), but still exhibits complex behaviors modulated by serotonergic, dopaminergic, adrenergic (octopaminergic) and opiate signaling that are mediated by receptors with clear orthology to their mammalian counterparts (Mills et al., 2016; Oakes et al., 2017). Similarly, the noradrenergic/octopaminergic inhibition of pain/aversive responses is also similar in mammals and nematodes, with α2-ARs inhibiting primary nociceptors and α1-like adrenergic receptors stimulating the release of an array of inhibitory . C.

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elegans has also been useful for deciphering roles for mammalian TRPs (Colbert et al.,

1997; Tobin et al., 2002; Zhang et al., 2004; Ezak et al., 2010; Kang et al., 2010;

Upadhyay et al., 2016).

C. elegans has provided useful insights into mammalian biology and many aspects of serotonergic, adrenergic and opiate signaling in C. elegans mimic similar processes in mammals, often involving orthologous receptors. Similarly, many aspects of CB signaling are also conserved between mammals and nematodes and both systems contain an endogenous cannabinoid signaling system mediated by canonical cannabinoid receptors. For example:

1) Mammalian and nematode systems contain canonical CB signaling systems with identical eCBs, 2-AG and AEA, and orthologous receptors, CB1/CB2 in mammals and

NPR-19 in nematodes (Lehtonen et al., 2011; Oakes et al., 2017). The function of these canonical CB receptors between the two systems is also conserved, as CB-mediated antinociceptive behaviors in mammals and nematodes require CB1 and NPR-19, respectively. Most importantly, expression of human CB1 robustly restores the CB- dependent inhibition of nociception in C. elegans mutants lacking NPR-19, demonstrating that the function In addition, the eCBs 2-AG and AEA activate NPR-19 directly with affinities in the nM range and are similar to mammalian CB1 affinities

(McAllister et al., 1999; Luk et al., 2004; Oakes et al., 2017). Together these studies

2) Mammalian and nematode systems degrade eCBs through similar pathways mediated by monoacylglycerol lipase (MAGL) for 2-AG hydrolysis and FAAH for AEA hydrolysis and both nematode degradative enzymes, y97e10al.2 and faah-1, respectively, are inhibited by antagonists specific for their mammalian counterparts and most 114

importantly, produce antinociceptive effects in both systems. For example, JZL184, a selective and irreversible inhibitor of MAGL, and URB597, a selective inhibitor of

FAAH, suppresses peripheral nociception in mammalian (Guindon et al., 2010; Kinsey et al., 2013; Kwilasz et al., 2014) and nematode models (Oakes et al., 2017). These studies highlight the conservation and orthology of the endocannabinoid signaling system between mammals and nematodes and demonstrate the usefulness of the C. elegans model system to study the complex interactions of endocannabinoid signaling that can be translated to mammalian systems.

3) Mammalian and nematode systems exhibit significant CB1/CB2 or NPR-19- independent CB signaling, mediated by additional GPCRs and TRP channels. For example, CBs also differentially activate the recently deorphanized mammalian GPCRs,

GPR18, GPR35, GPR55 and GPR119 (Sagan et al., 1999; Begg et al., 2005; Kohno et al., 2006; Overton et al., 2006; Brown, 2007; Ryberg et al., 2007; Lauckner et al., 2008;

Waldeck-Weiermair et al., 2008; McHugh et al., 2010; McHugh et al., 2012; Martinez-

Pinilla et al., 2014). However, the interactions of CBs with GPRs are complex and often antagonistic to one another. For example, 2-AG, AEA and THC function as agonists for

GPR55, while CBD functions as an antagonist (Ryberg et al., 2007). CBs also activate at least five different mammalian TRP channels with affinities in the nM range, including the capsaicin-sensitive, TRPV1 channel (De Petrocellis et al., 2010). For example, AEA inhibits nociception by activating CB1 (Iskedjian et al., 2007; Clapper et al., 2010), but at higher AEA concentrations appears to inhibit nociception by activating the TRPV1 channels (Zygmunt et al., 1999, 2013; Di Marzo et al., 2001, 2002; Morisset et al., 2001;

Horvath et al., 2008). Recent studies have also recorded 2-AG-dependent activation of 115

heterologously expressed TRPV1 in mammalian cells (Zygmunt et al., 2013).

Interestingly, N-arachidonoyl-serotonin (AA-5-HT), an eCB that functions as a dual blocker of FAAH and the TRPV1 channel, induces anxiolytic, or anti-anxiety effects and may inhibit contextual fear memory by stimulating CB1 highlighting the potentially complex interactions between these different signaling pathways (Gobira et al., 2017). In addition, another eCB, N-arachidonoyldopamine (NADA) functions as a CB1 receptor and TRPV1 channel agonist (Huang et al., 2002; Grabiec and Dehghani, 2017), as well as a MAGL and FAAH inhibitor (Bisongo et al., 2000; Bjorklund et al., 2010). In C. elegans, 2-AG appears to activate TRP channels directly to stimulate the release of 5-HT and DA to mediate 2-AG-dependent inhibition of locomotion (Oakes et al., 2017). In summary, the interaction between CBs and TRP channels are complex and because many of these interactions were discovered within the recent years, little is known about the behavioral consequences of such interactions. These studies highlight the usefulness of the C. elegans model system to study the interactions between CBs and TRP-like channels and most importantly, characterize the receptors and neuronal pathways mediating 2-AG-dependent behavioral changes.

In mammals, CBs modulate the firing of monoaminergic neurons and the release of serotonin (5-HT), dopamine (DA) and norepinephrine (NE) and some of the behavioral effects of CBs appear to be mediated either, directly or indirectly, through changes in serotonergic, adrenergic and dopaminergic signaling. For example:

4) Mammalian and nematode systems exhibit CB-dependent modulation of endogenous 5-HT signaling. In mammals, pCBs and eCBs have antagonistic effects on endogenous 5-HT levels, as pCBs stimulate 5-HT release (Sagredo et al., 2006) and eCBs 116

generally inhibit 5-HT release depending on the localization and eCBs involved (Nakazi et al., 2000; Egashira et al., 2002; Darmani et al., 2003; Tzavara et al., 2003; Aso et al.,

2009). For example, chronic THC exposure increases endogenous 5-HT levels in the rat prefrontal cortex (Sagredo et al., 2006). In contrast, pharmacological inhibition or genetic knockdown of CB1 increased endogenous 5-HT levels, suggesting that ECS inhibits 5-HT release (Darmani et al., 2003; Tzavara et al., 2003; Mato et al., 2007; Aso et al., 2009), however, eCBs can also inhibit 5-HT reuptake to increase endogenous 5-HT levels in a dose-dependent manner (Steffens and Feuerstein, 2004). CBs also modulate the firing of serotonergic neurons and modulate the efficacy of 5-HT receptors in mammals. For example, genetic knockdown of CB1 in mice resulted in an increase in 5-HT1A and 5-

HT2A receptor efficacy, but a decrease in serotonergic neuronal activation (Mato et al.,

2007; Aso et al., 2009). Indeed, electrophysiological studies demonstrate an eCB- dependent increase in serotonergic neuron excitability (Gobbi et al., 2005; Bambico et al., 2007; Haj-Dahmane and Shen, 2005, 2009). In C. elegans, CBs do not activate 5-HT receptors directly or increase their efficacy for 5-HT (Oakes et al., 2017), but stimulate the release of endogenous 5-HT via a mechanism requiring TRPV1-like channel activation. In conclusion, the CB-dependent modulation of serotonergic signaling is complex and CBs extensively modulate the serotonergic signaling system in mammals and nematodes. Importantly, the intricate CB-dependent modulation of 5-HT signaling may explain the anti-anxiety and anti-depression effects of cannabinoids. However, the behavioral consequences of CB-dependent modulation of serotonergic signaling have yet to be fully characterized and C. elegans is a useful model to study the correlation between

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the cellular/neuronal CB effects and the behavioral outcomes in an intact whole-animal system.

5) Mammalian and nematode systems exhibit CB-dependent modulation of endogenous DA signaling. For example, in mammals, CBs regulate the firing of dopaminergic neurons and release of DA indirectly via depolarization induced suppression of excitation (DSE) and inhibition (DSI) of glutamatergic and GABAergic neurons, respectively, upstream to dopaminergic neurons (Uchigashima et al., 2007;

Wang et al., 2015). Indeed, the eCBs 2-AG and AEA increase the firing rate of dopaminergic neurons and stimulate DA release both directly by activating TRP channels expressed on dopaminergic neurons (Mezey et al., 2000; Marinelli et al., 2003; Starowicz et al., 2007; Zygmunt et al., 2013) and indirectly by inhibiting GABA release upstream of dopaminergic neurons via CB1 activation on GABAergic neurons (Cheer et al., 2004;

Cheer et al., 2005; De Luca et al., 2014). In C. elegans, CBs stimulate DA release directly via a mechanism that requires the TRPN-like channel TRP-4 expressed in dopaminergic neurons to mediate CB-dependent inhibition of locomotory behavior

(Oakes, unpublished data). However, CB-stimulated DA release is not required for the

CB-dependent inhibition of nociception, as CB-dependent inhibition is present in animals lacking the DA biosynthetic enzyme CAT-2. Additionally, preliminary studies suggest that 2-AG activates the D1-like DA receptor DOP-1 directly, as the 2-AG-dependent inhibition of 5-HT stimulated nociceptive responses is present in animals lacking DA biosynthesis but absent dop-1 null animals. Although these results are preliminary and need to be confirmed with heterologous expression, the present study demonstrates that

CB interaction with dopaminergic signaling is extensive and the conservation of this 118

interaction between mammals and nematodes justifies the use of C. elegans as a model to study the effects of CBs on dopaminergic signaling and the behavioral outputs of such interactions.

6) Mammalian and nematode systems exhibit CB-dependent modulation of endogenous adrenergic signaling. For example, eCBs and pCBs activate the hypothalamic-pituitary-adrenal (HPA) axis and increase corticosterone and norepinephrine release in mammals (Kurihara et al., 2001; McLaughlin et al., 2009), suggesting that the analgesic of antinociceptive effects of CBs may be mediated, at least in part, by adrenergic signaling. Indeed, CB1 and α2A-AR co-localize in certain regions of the brain (Cathel et al., 2014) and the α2-adrenoceptor antagonist yohimbine and the CB1 receptor antagonist AM25120 abolish the peripheral antinociceptive effects of AEA, suggesting that the antinociceptive effects of AEA may be mediated by a combination of

CB and adrenergic signaling, at least in regions of the brain where CB1 and α2A- adrenoceptors co-localize. The suppression of chronic pain in mammals involves the modulation of signaling to pain receptors via the Gαq-coupled α1 and Gαo-coupled α2 AR- dependent regulation of neurotransmitter and neuropeptide release (Pertovaara, 2006). In

C. elegans, a similar system functions to mediate nociceptive behaviors, with the Gαq and

Gαo-coupled α-adrenergic like receptors TYRA-3 and OCTR-1, respectively, modulating and fine-tuning the release of monoamines and neurotransmitters from the ASH and ASI sensory neurons to elicit escape behaviors to noxious stimulus (Harris et al., 2009;

Hapiak et al., 2013; Mills et al., 2016; Oakes et al., 2017; Clark and Hapiak et al., 2018).

CB-dependent inhibition of nociception requires the α2-like adrenergic octopamine receptor OCTR-1 at higher CB concentrations (Oakes et al., 2017), but does not require 119

octopaminergic signaling (data not shown). Indeed, CBs activate OCTR-1 directly in the nM range when heterologously expressed in Xenopus laevis oocytes (Oakes et al., 2017).

While there are very few studies examining the direct activation of adrenoceptors by eCBs in mammalian systems, (CBG), a less commonly known pCB, is a potent α2A-AR agonist with an affinity in the low nM range (Cascio et al., 2010). These studies demonstrate the complexity of the interaction between CB and adrenergic signaling and suggests that CBs modulate adrenergic signaling directly and indirectly to mediate antinociceptive behaviors.

4.6. Cannabinoids as anthelmintics

The global impact of parasitic nematode infections in medical, veterinary, and agricultural settings has often been underestimated, given the focus on mortality and not morbidity, but new anthelmintics are needed in all settings, especially with the rapid and increasing onset of resistance to our current pharmacopeia. Interestingly, the literature contains a wealth of anecdotal evidence; mostly from primitive communities where the use of folk medicine is still common, suggesting that cannabis might be used to control worm infestations. For example, hemp (Cannabis sativa L.) is rarely infested with nematodes and mixing dried C. sativa leaves and flowers with soil appears to protect the surrounding plants and repels pathogenic nematodes, bacteria, fungus and insects.

Similarly, C. sativa has been planted as a companion crop to control plant parasitic nematodes (McPartland, 1997; McPartland and Glass, 2001, Kayani et al., 2012). In addition, crude cannabis extracts paralyze the human liver fluke, Fasciola buski and more 120

recently, a study of the Aka foragers in the Congo basin demonstrated that cannabis ingestion is strongly associated with reduced worm burdens and reinfection, suggesting an anthelmintic action (Roulette et al. 2016; Roy and Tandon, 1996). Despite this wealth of anecdotal evidence, the potential molecular targets of eCBs in nematodes have never been identified. Our recent work suggests that CBs inhibit feeding directly and cause locomotory confusion through the activation of 5-HT, DA and OA signaling, two phenotypes previously associated with anthelmintic modes of action, although we are not aware of any controlled studies supporting the utility of cannabis as an anthelmintic.

4.7. Conclusion

In conclusion, CB signaling in mammals and nematodes is extremely complex and although CBs interact with a variety of CB1/CB2 and non-CB1/CB2-dependent signaling pathways, the relative significance of CB1/CB2-dependent and CB1/CB2- independent signaling in the modulation of behavior is not fully understood. In addition, it is important to recognize that given the complexity of the mammalian nervous system and the wide range of potential CB receptor ligands, generalizations about specific signaling pathways can be complicated. CBs stimulate DA release in both C. elegans and mammals and the direct CB-dependent activation of the reward system could have substantial implications into the physiological addiction associated with CB use. In C. elegans, CB-dependent DA release is NPR-19-independent and instead requires a TRP-4 channel directly on the dopaminergic neurons, although a potential role for CBs in modulating inputs into the dopaminergic neurons remains to be examined. Similarly, CBs

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increase 5-HT release in both C. elegans and mammals, however, interactions between

CB and serotonergic signaling are complex. In addition, CBs also activate excitatory 5-

HT2A receptors directly and serotonergic neurons release CBs, even though they do not appear to express canonical CB receptors (Haj-Dahmane and Shen, 2005; 2009). In C. elegans, CBs do not appear to activate 5-HT1-like receptor directly but instead stimulate serotonergic signaling and increase 5-HT release through a pathway requiring TRPV1- like channel activation. Finally, CBs also stimulate adrenergic signaling in both C. elegans and mammals. For example, CB1 agonists increase neuronal activity in rat locus coeruleus and activate α2A-adrenergic receptors in both systems (Cascio et al., 2010;

Oakes et al., 2017).

Together, these studies highlight the similarities between mammalian and nematode systems and the potential benefit for additional study of CB signaling in C. elegans. For example, CBD is non-hallucinogenic, but appears to be involved in many of the proposed medicinal benefits of CB signaling, suggesting that the study of CBD signaling in C. elegans is warranted. Similarly, the effects of CBs are quite rapid in C. elegans and the direct CB activation of TRP channels is essential for the release of both

5-HT and DA, suggesting that CBs might begin to alter sensory processing almost immediately upon exposure. Indeed, CBs gate at least 5 distinct TRP channels in mammals, some of which are expressed on monoaminergic neurons, suggesting that the relationship between CB-dependent TRP channel activation and monoaminergic signaling in mammals might benefit from additional examination (Di Marzo et al., 1998;

De Petrocellis et al., 2001; Bandell et al., 2004; McIntosh et al., 2007). Finally, much remains to be learned about how eCB and pCB signaling interact and how ligands with 122

similar affinity for canonical CB receptors initiate such dramatic differences in downstream signaling and ultimately behavior. C. elegans may prove to be a useful and simple model to decipher these subtle interactions operating in more complex nervous systems.

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