BEHAVIORAL PHARMACOLOGY OF CANNABINOIDS:

TOWARDS AN ANIMAL MODEL FOR STUDYING

CANNABINOID DEPENDENCE/WITHDRAWAL

Thesis Presented

by

Sherrica Tai

to

The Bouvé Graduate School of Health Sciences in Partial Fulfillment of the

Requirements for the Degree of Doctor of Philosophy in Pharmaceutical

Science with Specialization in Behavioral Pharmacology

NORTHEASTERN UNIVERSITY

BOSTON, MASSACHUSETTS

June 2012

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TABLE OF CONTENTS

List of Figures…………………………………………………………………………………….…. ii

List of Abbreviations…………………………………………………………………………….….. iv

Summary…………………………………………………………………………………………...... 1

Acknowledgments……………………………………………………………………….………….. 2

Introduction…………………………………………………………………………………………. 3

Objective and Specific Aims…………………………………………………………….………….. 12

Experimental Design and Methods………………………………………………………………….. 14

Chapter 1: Characterize, in a tetrad assay, a CB1-receptor agonist (AM2389) with a long

duration of effect to facilitate the study of cannabinoid dependence……………….... 18

Chapter 2: Determine if repeated treatment with AM2389 leads to tolerance to CB1-receptor

mediated hypothermia and hypo-locomotion in mice………………..……………….. 29

Chapter 3: Characterize behavioral effects elicited by centrally acting CB1-receptor antagonists

(with either an inverse agonist or a neutral antagonist property) and a neutral

CB1-receptor antagonist with limited acute brain penetration..……………………… 35

Chapter 4: Determine if somatic signs of spontaneous cannabinoid withdrawal can be observed

and quantified after discontinuation of repeated AM2389 treatment............................ 47

Chapter 5: Compare behavioral (locomotion and somatic signs of withdrawal; e.g., front paw

tremors) changes precipitated by a CB1-receptor antagonist/inverse agonist

(rimonabant) and a CB1-receptor neutral antagonist (AM4113) in AM2389-tolerant

mice; determine if somatic signs of withdrawal are primarily attributed to central

and/or peripheral influences; determine if somatic signs of antagonist-precipitated

withdrawal (e.g., hyperactivity) are attenuated by the reinstatement of a cannabinoid

agonist in AM2389-tolerant mice.………………………………………….………… 52

Conclusion………….……………………………………………………………………………….. 70

References……………………………………………………………………………………….….. 73

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LIST OF FIGURES…

Figure 1- Structures and binding data for cannabinoid ligands 15

Figure 2- Open-field: AM2389 measured post single administration 20

Figure 3- Antinociception with AM2389 and ∆9-THC post single administration 22

Figure 4- Inverted screen / catalepsy with AM2389 post single administration 23

Figure 5- Rectal temperatures with AM2389 and ∆9-THC post single administration 24

Figure 6- Rectal temperature time-course with AM2389 and ∆9-THC post single

administration 25

Figure 7- Tolerance to hypothermia developed from repeated AM2389 treatment 31

Figure 8- Tolerance to hypomotility developed from repeated AM2389 treatment 32

Figure 9- Open-field: vehicles grouped from 2008 and 2009 37

Figure 10- Open-field: CB1-receptor antagonists AM251, AM4113 and AM6545

measured post single administration 38

Figure 11- Open-field: AM6545 post single administration 39

Figure 12- Observation chambers: CB1-receptor antagonist AM251, AM4113 and

AM6545 measured post single administration 41

Figure 13- Observation chambers: AM6545 post single administration 42

Figure 14- Observation chambers: spontaneous withdrawal in AM2389-tolerant mice 49

Figure 15.1- Open-field: precipitated withdrawal Phase I 55

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Figure 15.2- Open-field: precipitated withdrawal Phase II 56

Figure 16.1- Open-field: precipitated withdrawal with AM6545 Phase I 58

Figure 16.2- Open-field: precipitated withdrawal with AM6545 Phase II 59

Figure 17.1- Observation chambers: precipitated withdrawal Phase I 60

Figure 17.2- Observation chambers: precipitated withdrawal Phase II 61

Figure 18.1- Observation chambers: precipitated withdrawal with AM6545 Phase I 63

Figure 18.2- Observation chambers: precipitated withdrawal with AM6545 Phase II 64

Figure 19- Open-field: AM2389 attenuation of hyperactivity induced by rimonabant 65

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LIST OF ABBREVIATIONS…

2-AG 2-arachidonoylgycerol

ABHD4 α/β–hydrolase 4

AM1241 1-(methylpiperidin-2-ylmethyl)-3-(2-iodo-5-nitrobenzoyl)indole

AM2389 9-Nor-9β-hydroxy-3-(1-hexyl-cyclobut-1-yl)-hexahydrocannabinol

AM251 N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-

pyrazole-3-carboxamide

AM4113 N-piperidin-1-yl-2,4-dichlorophenyl-1H-pyrazole-3-carboxamide analog

AM630 [6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-

methoxyphenyl)methanone (6-iodopravadoline)

AM6545 [5-(4-[4-cyanobut-1-ynyl]phenyl)]-1-(2,4-dichlorophenyl)- 4-methyl-N-

(1,1-dioxo-thiomorpholino)-1 H-pyrazole-3-carboxamide

anandamide N-arachidonoyl ethanolamine

ANOVA one-way analysis of variance

cAMP cyclic adenosine monophosphate

CB1 type 1 cannabinoid

CB2 type 2 cannabinoid

CB3 type 3 cannabinoid

CNS central nervous system

CP-55,940 2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxypropyl) cyclohexyl]-5-(2-methyloctan-2-

yl)phenol

DAG diacylglycerol

∆9-THC ∆

9-tetrahydrocannabinol

FAAH fatty acid amide hydrolase

G-protein guanine nucleotide binding protein

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GDE1 glycerophosphodiesterase

Gi/o inhibitory G-protein

GP-NAE glycerophospho-N-acyl ethanolamine

GPR55 G protein coupled receptor 55

Gs stimulatory G-protein

ICV intracerebroventricular

lysoNAPE lyso-N-acylphosphatidylethanolamine

MGL monoacylglycerol lipase

NADA N-arachidonoyl-dopamine

NAPE N-arachidonoyl phosphatidylethanolamine

NAPE-PLD NAPE-specific phospholipase D

PGE2-G prostaglandin E2 glycerol ester

PLC phospholipase C

PTPN22 protein tyrosine phosphatase non-receptor type 22

SHIP 1 SH2 domain-containing inositol 5' phosphatase

TRPV1 transient receptor potential vanilloid 1

WIN55,212 (R)-(+)-[2,3-Dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo[1,2,3-de)-

1,4-benzoxazin-6-yl]-1-napthalenylmethanone

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SUMMARY…

The development of improved pharmacotherapeutics for treating cannabinoid dependence has

presented many challenges. This report will discuss these challenges as well as use an animal model to

study cannabinoid dependence by incorporating traditional methodologies (e.g., tetrad assay) and

profiling novel cannabinoid ligands with distinct pharmacological properties/modes of action by

evaluating their pharmacological effects on CB1-receptor related physiological and behavioral endpoints.

Initial steps were made to characterize acutely AM2389, a CB1-receptor agonist, in the tetrad assay

(i.e., locomotor activity, paw thermal stimulation, inverted screen test/catalepsy bar test and rectal

temperature) as a novel approach for inducing dependence with greater potency and a prolonged

functional in vivo duration of action than ∆9-THC. Cannabinoid tolerance was then evaluated with

repeated administration of AM2389. Next, the effects of centrally acting antagonists, rimonabant and

AM4113, and a neutral antagonist with limited acute brain penetration, AM6545, were characterized

behaviorally prior to examining precipitated withdrawal. Antagonist-precipitated withdrawal was

induced in cannabinoid-tolerant mice by either rimonabant or AM4113 but not by AM6545. Finally,

CB1-receptor antagonist induced precipitated withdrawal was reversed by reinstating either AM2389 or

∆9-THC in cannabinoid-tolerant mice. Overall, these findings suggest that cannabinoid precipitated

withdrawal may not be ascribed to the inverse agonist properties of the CB1-receptor antagonist

rimonabant, but rather simply to the rapid displacement of the agonist from the CB1-receptor.

Furthermore, this cannabinoid withdrawal syndrome is likely centrally mediated, since only the centrally

acting CB1-receptor antagonists were able to elicit “withdrawal” responses, i.e., such responses were not

induced by the purported peripherally selective CB1-receptor antagonist AM6545.

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ACKNOWLEDGMENTS…

I would like to express my sincere gratitude to my advisor Dr. Toby Järbe for mentoring me from the

very start of this project. You have taught me many valuable lessons that I will continue to grow from

and put into practice as I establish my career as a researcher. Therefore, I am eternally grateful to you. A

special thanks is extended to my second advisor, Dr. Alexandros Makriyannis, and committee members,

Dr. Jack Bergman, Dr. John Gatley, Dr. David Janero, and Dr. Carol Paronis for their honesty and

tremendous insight throughout the entire process as well as for believing in me. I would also like to thank

the Center for Drug Discovery family past and present for their contributions to pushing this project

forward and for their friendship.

I would like to dedicate this thesis to my amazing mother for her endless love, support and

encouragemeant over the years. Lastly, I am grateful to the rest of my family for allowing me to share all

of my life experiences with you and for allowing me to be a part of yours.

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Introduction

The use of cannabis, or marijuana, in medicinal practice was recorded as early as 2737 BC in China

(Jennings, 1968). Currently, cannabis is most frequently used as a recreational drug for its ability to alter

sensory perception, and within limits, producing a state of euphoria (Green et al., 2003). Originating in

south-west states such as New Mexico, increasing concerns for its abuse liability led to the taxation of

cannabis use in 1937 (Musto, 1972); however, this did not prevent cannabis from becoming the most

commonly used illicit drug in the United States to date.

As of 2010, there were an estimated 4.6 million daily cannabis users in the United States, including a

subset of chronic (daily/dependent) users who seek treatment (Substance Abuse and Mental Health

Services Administration, 2011). Approximately 9% of cannabis users develop symptoms of dependence,

such that chronic users who have attempted to abstain from cannabis use have complained about the

severity of withdrawal signs (increased anxiety, restlessness, depression), which can render it difficult for

cannabis users to stop using cannabis (Budney et al., 2004; Hall et al., 2009). Cannabis users have

demonstrated an increased risk of motor vehicle crashes and psychotic symptoms after acute cannabis

intoxication along with an increased risk of impaired cognitive function, lower educational attainment in

adolescents, decreased respiratory function, and exacerbation of psychosis after repeated use (Hall, 2009;

Kalant, 2004).

This thesis will present an animal model for studying cannabinoid dependence to gain insight into

mechanisms implicated in tolerance to cannabinoid receptor stimulation resulting in withdrawal. The

following data may help the development of improved pharmacotherapeutics for cannabinoid

dependence/withdrawal.

Endocannabinoid system

The 1964 identification of the highly lipophilic ∆9-tetrahydrocannabinol (∆

9-THC), the main

psychoactive ingredient of cannabis, led to the 1990 discovery of the first cannabinoid (CB1) receptor

cloned from rat, mouse and human brain with 97-99% amino acid sequence identity across species,

followed three years later by the characterization of the CB2-receptor, cloned from human immune cells

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(Howlett et al., 2002; Montero et al., 2005). An 82% sequence identity exists between mouse and human

CB2 receptors.

CB1 and CB2 receptors are members of the seven transmembrane G protein coupled receptor (GPCR)

superfamily (Begg et al., 2005). The amino acid identity between the two receptor subtypes is 44% for

the full length protein and 68% within the seven transmembrane domains (Szabo, 2008). CB1 receptors

are among the most abundant GPCRs in the mammalian central nervous system (CNS). Outside the

brain, CB1 receptors are expressed at lower levels in a variety of neuronal peripheral tissues, for instance

fat cells, heart, intestine, liver, endocrine pancreas and uterus (Tucci et al., 2006). CB2 receptors are

primarily expressed in the periphery, such as liver, lung and kidney, and are closely associated with the

immune and hematopoietic system (Munro et al., 1993; Teixeira-Clerc et al., 2008). CB1 receptors are

also expressed in immune cells; however, mRNA CB2-receptor expression is significantly greater in the

immune system than mRNA CB1-receptor expression (Pertwee, 2002). Also, increasing evidence

supports the existence of CB2 receptors in the brain, particularly in cerebellum microglia where

inflammatory processes which can further upregulate microglia CB2 receptors (Gong et al., 2006; Onaivi

et al., 2006). Recent in vivo data have strengthened the idea of a functional significance of CB2 receptors

in brain fuction (Garcia-Gutierrez et al., 2012; Xi et al., 2011).

Endocannabinoids

The discovery of the CB1 and CB2 receptors led to the identification of endogenous ligands that bind

these receptors. The most extensively studied endocannabinoids are N-arachidonoyl ethanolamine

(anandamide), discovered in 1992 (Devane et al., 1992), and 2-arachidonoylgycerol (2-AG), isolated

3 years later (Hanus et al., 2001; Sugiura, 2007). Since these lipid messengers are highly lipophilic, they

are not believed to be stored in vesicles; therefore, they are more likely to be synthesized “on demand”.

Anandamide and 2-AG are synthesized by neurons centrally and peripherally by cleavage of their

respective membrane lipid precursors, N-arachidonoyl phosphatidylethanolamine (NAPE) and

diacylglycerol (DAG) (Bisogno et al., 1997; Di Marzo et al., 1994). NAPE was originally thought to

have been hydrolyzed by NAPE-specific phospholipase D (NAPE-PLD) to form anandamide. Currently,

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NAPE-PLD is assumed to be synthesized by a more direct pathway involving a single enzymatic step.

Subsequent reports have found other pathways that liberate anandamide independent of NAPE-PLD (Liu

et al., 2008). NAPE has also been proposed to be hydrolyzed by phospholipase C (PLC) to form

phosphoanandamide which is subsequently dephosphorylated by phosphatases, such as PTPN22 or SHIP

1 (Liu et al., 2006). Another alternative pathway includes the sequential hydrolysis of NAPE and

lysoNAPE by the intermediate serine hydrolase α/β-hydrolase 4 (ABHD4) to form GP-NAE, which is

subsequently hydrolyzed by the metal-dependent phosphodiesterase, GDE1, to form anandamide (Simon

et al., 2010; Simon et al., 2006). 2-AG is synthesized from the precursors phosphatidic acid or

phosphatidylinositol, which are hydrolyzed by PA-phosphohydrolase and PLC, respectively, to yield

DAG (Bisogno et al., 1999; Prescott et al., 1983). 2-AG is primarily generated by the hydrolysis of DAG

by DAG lipases α and β. An alternative, less direct pathway for generating 2-AG involves the hydrolysis

of phosphatidylinositol by phospholipase A1 to generate lysophospholipid, which is subsequently

hydrolyzed by PLC to generate 2-AG (Piomelli, 2003).

Anandamide and 2-AG are rapidly deactivated by uptake into cells where they undergo intracellular

enzymatic hydrolysis. The process by which endocannabinoid uptake occurs prior to its hydrolysis has

received much debate. For instance, it has been suggested that anandamide is transported by a putative

transporter protein (Beltramo et al., 1997). On the other hand, the hypothesized transporter protein has

been refuted by Glaser and colleagues suggesting instead that anandamide uptake occurs through simple

diffusion (Glaser et al., 2003). Anandamide is hydrolyzed by fatty acid amide hydrolase (FAAH) to

generate free arachidonic acid and ethanolamine, and 2-AG is selectively metabolized by monoglyceride

lipase (MGL) in vivo, producing free arachidonic acid and glycerol (Piomelli, 2005). Only under in vitro

conditions is 2-AG also a substrate for FAAH, contributing to ~1-2% of hydrolysis (Blankman et al.,

2007). MGL is the primary hydrolytic enzyme for 2-AG inactivation, responsible for ~85% of 2-AG

hydrolysis in the brain. The remaining 2-AG inactivation occurs through two novel hydrolases, α/β-

hydrolase 6 (ABHD6) and α/β-hydrolase 12 (ABHD12), which are responsible for ~4% and ~9% of 2-

AG hydrolysis, respectively (Simon et al., 2006).

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Anandamide and 2-AG are also substrates for oxygenase enzymes, such as cyclooxygenase (Kingsley

et al., 2009). More precisely, anandamide and 2-AG are oxygenated by cyclooxygenase-2 enzyme to

generate a biologically active, pronociceptive compound, prostaglandin E2 ethanolamide, and

prostaglandin E2 glycerol ester (PGE2-G), respectively (Guindon et al., 2008). These oxygenated

endocannabinoid metabolites have been implicated in cell viability, mobilization of Ca2+

and modulation

of synaptic transmission; for instance, PGE2-G produces hyperalgesia (Fowler, 2007). Rapid synthesis

and deactivation of anandamide and 2-AG suggest that they are synthesized, released, and metabolized

near the site of action. As reviewed, anandamide and 2-AG act at the cannabinoid receptors as a partial

and full agonist, respectively (Szabo, 2008). In addition, basal levels of 2-AG are some 50-500 fold

greater in the brain and periphery in comparison to anandamide (Ho et al., 2007). Recent reports have

suggested the existence of a third cannabinoid receptor, “CB3”, based on several cannabinoid ligands’

ability to alter selectively receptor signaling. For instance, G protein coupled orphan receptor GPR55

was activated by most cannabinoid ligands, except WIN55,212-2 (Johns et al., 2007); however, further

studies are needed to elucidate the physiological and pharmacological functions elicited by this novel

receptor with the use of e.g., GPR55 knock-out animal models.

Endocannabinoid Signaling

In contrast to classical signaling, the endocannabinoid system undergoes retrograde signaling where

information travels from post- to pre-synaptic neurons. The rise in post-synaptic Ca2+

levels leads to the

activation of enzymes that synthesize endocannabinoids from lipid precursors. Endocannabinoids are

released from the post-synaptic site and travel across the synaptic cleft to bind to the cannabinoid

receptors located on the pre-synaptic membrane. This allows the cannabinoid receptor to couple to and

activate the G-protein of the Gi/o class. Even though Gs coupling can occur with CB1 receptors, the

preferred pathway is Gi/o (Glass et al., 1997). Gi/o activation results in inhibition of adenylyl cyclase,

thereby inhibiting the formation of cyclic AMP, as well as certain voltage-sensitive Ca2+

channels, and

activates A-type and inward-rectifying K+ channels and mitogen activated protein kinase (MAPK)

(Guzman, 2003).

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Cannabinoid Ligands

Other endocannabinoids that act as neurotransmitters or neuromodulators include O-arachidonoyl-

ethanolamine (virodhamine), 2-arachidonoylglyceryl ether (noladin ether) and N-arachidonoyl-dopamine

(NADA) (Tanda et al., 2003). Anandamide and NADA both activate TRPV1, although anandamide

binds with lower affinity in comparison to its binding at CB1 receptors. Some synthetic analogs of

anandamide include R-(+)-methanandamide, e.g., AM356 (Abadji et al., 1994; Romero et al., 1996),

arachidonoyl-2’-chloroethylamide (ACEA) and arachidonoylcyclopropylamide (ACPA) (Patel et al.,

2001). Endogenous and exogenous ligands are referred to as eicosanoids. There are four major chemical

classes of exogenous cannabinoid ligands that differ in structure (Palmer et al., 2002). Classical

cannabinoids include ∆9-THC, AM2389, cannabinol, nabilone, HU-210 and other trycyclic terpenoid

derivatives bearing a benzopyran moiety. Non-classical cannabinoids include CP 55,940, HU-308 and

other bicyclic and tricyclic analogs of ∆9-THC lacking the pyran ring of classical cannabinoids.

Aminoalkylindoles including WIN55,212 2 and AM1241, which differ in structure, lipophilicity (less

lipophilic) and binding activity at the cannabinoid receptor(s) in comparison to the above mentioned

classes. Finally, 1,5 biarylpyrazole ligands act as cannabinoid receptor antagonists, such as SR141716A

(rimonabant), AM251, AM4113, AM6545, which are all CB1-receptor selective, and SR144528, which is

CB2-receptor selective.

∆9-THC effects on the Mesolimbic Dopaminergic Pathway

Cannabis, similar to other non-dopaminergic drugs of abuse such as opiates, nicotine and alcohol

activates the reward pathway in the brain termed the mesolimbic dopaminergic pathway, thus elevating

extracellular dopamine levels in the nucleus accumbens (NAc), which is believed important for addiction

(Wise, 2004). This pathway originates in the ventral tegmental area (VTA) of the midbrain and sends

ascending projections to the NAc and other forebrain structures such as the amygdala and prefrontal

cortex (PFC) (Hyman, 2007; Laviolette et al., 2004). These dopamine projections affect the rewarding

properties of food, sex and addictive drugs. Thus, ∆9-THC induced CB1-receptor activation elevates

dopamine levels in the NAc and increases the firing rate of VTA-NAc dopaminergic neurons (Pacher et

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al., 2006). It has been suggested that ∆9-THC lowers the reward threshold for electrical brain stimulation,

although this finding is controversial. In some reports, ∆9-THC decreases the intracranial self-stimulation

threshold at low doses, whereas other data suggest that CB1-receptor agonists including ∆9-THC do not

enhance brain stimulation reward (Panagis et al., 2008).

Tolerance to Receptor Stimulation and Withdrawal

CB1-receptor activation by ∆9-THC and other CB1-receptor agonists produces acute effects such as

antinociception, hypolocomotion, hypothermia, catalepsy, suppression of operant behavior, weight gain at

lower doses and inhibition of gastrointestinal transit at higher doses, all of which have been observed and

quantified in laboratory settings using rodents, as previously reviewed (Maldonado et al., 2002).

Continuous CB1-receptor activation may result in rapid tolerance to the above mentioned effects as well

as cardiovascular actions (e.g., tachycardia), anticonvulsant activity, ataxia, and corticosterone release (a

marker of stress) (Gonzalez et al., 2005). Cannabinoid tolerance is attributable mainly to

pharmacodynamic changes, such as a decrease in CB1-receptor expression/activation and the levels of

CB1-receptor mRNA, with minor contributions resulting from pharmacokinetic effects caused by changes

in cannabinoid metabolism and excretion (Guzman, 2003; Maldonado, 2002; Romero et al., 1997).

Abrupt cessation of chronic ∆9-THC treatment does not typically induce spontaneous signs of

withdrawal, which has contributed to the difficulty in validating the existence of a cannabis withdrawal

syndrome (Jones et al., 1976a). Nevertheless, some human subjects have complained of inner unrest,

irritability, insomnia, hot flashes, loose stools, and anorexia as symptoms of cannabis withdrawal

(Budney et al., 1999). Rimonabant at 20-mg/person is the dose used in launching Acomplia® (the trade

name used by Sanofi-Aventis). Recently, rimonabant (90-mg/person, orally) precipitated cannabis

withdrawal was reported for human chronic marijuana users (Mason et al., 2010). Clinical studies further

suggested that lower doses of rimonabant (20- or 40-mg/person, orally) did not significantly precipitate

cannabis withdrawal according to the pre-specified criteria for antagonist-elicited cannabis withdrawal

(Gorelick et al., 2011). In contrast to ∆9-THC, the synthetic cannabinergic aminoalkylindole

WIN55,212-2 has been reported to induce spontaneous signs of withdrawal, the difference perhaps

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attributable to the different pharmacokinetic properties of these two cannabinergic ligands (Aceto et al.,

2001). The highly lipophilic nature of ∆9-THC promotes its storage in adipose tissue and slows its

elimination such that cessation of cannabis use does not correlate with rapid ∆9-THC whole-body

clearance. It has also been shown that cessation of chronic CP-55,940 administration can elicit

spontaneous withdrawal, which is difficult to reconcile with the above proposed pharmacokinetic

explanation without a direct comparison with ∆9-THC, WIN55,212-2 and CP-55,940 (Oliva et al., 2003;

Oliva et al., 2004). Instances where spontaneous ∆9-THC withdrawal have been detected indicate that the

withdrawal symptoms are mild in comparison to antagonist-precipitated withdrawal, making precipitated

withdrawal a feasible experimental paradigm for studying robust signs of cannabinoid withdrawal, given

the associated phenotypic signs.

∆9-THC has been used as the standard cannabinoid agonist for inducing dependence in animal models

(prior to precipitated withdrawal); however, a CB1-receptor agonist that is capable of producing sustained

CB1 activation, such as AM2389 (discussed in Chapter 1), may be more efficient in this regard than

∆9-THC, a relatively short-acting ligand (Järbe et al., 2012; Nikas et al., 2010). Thus, rats trained to

discriminate between vehicle and the cannabinergic aminoalkylindole AM5983 (0.18 mg/kg) evidenced

an in vivo functional half-life estimate of around 15 hrs when examined at different time-points after

administration of 0.01 mg/kg AM2389 (Järbe et al., 2012). Time course tests with ∆9-THC in rats trained

to discriminate between vehicle and 3 mg/kg ∆9-THC suggested a return to vehicle-like responding at

about 4.5 hrs post-administration (Järbe et al., 1986; Järbe et al., 1981). The ED50 values for the

time-points examined were: 0.85, 1.07 and 4.78 mg/kg for 0.5, 1.5 and 4.5 hrs post-administration (Järbe

et al., 1986). The onset of the pharmacological effects of AM2389 on the other hand was slower. Thus,

dose-response tests in the AM5983 trained animals conducted at 1 and 3 hrs post-administration produced

significantly different ED50 values of 0.0058 and 0.0025 mg/kg for the two time intervals, respectively.

A comparable test with ∆9-THC at 20 min post-administration in the AM5983 trained group produced an

ED50 value of 0.25 mg/kg (Järbe et al., 2012). Thus, these data would suggest that there is

approximately a 100-fold difference in potency between AM2983 and ∆9-THC.

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The CB1-receptor selective antagonist/inverse agonist rimonabant has been a useful experimental tool

for inducing signs of withdrawal from cannabinoid-dependent subjects (Hutcheson et al., 1998).

Rimonabant has proven useful in characterizing experimental withdrawal effects in rodents following

repeated exposure to CB1-receptor agonists such as ∆9-THC. Reported signs of withdrawal in rodents are

wet dog shakes, head shakes, facial rubbing, front paw tremor, ataxia, hunched posture, body tremor,

ptosis, piloerection, hyperlocomotion, mastication, licking, and rubbing (Hutcheson et al., 1998;

Maldonado et al., 2002). The above described behaviors are believed to be CB1-receptor mediated, since

CB1-receptor knock-out mice chronically treated with ∆9-THC and challenged by rimonabant failed to

elicit the above-mentioned withdrawal behaviors (Maldonado et al., 2002). It was also found that

rimonabant administered chronically to ∆9-THC-treated mice reduced the extracellular dopamine levels in

nucleus accumbens, suggesting rimonabant can block at least partially the subjective and rewarding

effects of marijuana (Tucci et al., 2006). Since ∆9-THC and rimonabant bind with reasonably high

affinity to CB1 receptors located both in the CNS and peripheral organs, systemic exposure to these

agents in vivo cannot provide insight as to the contribution of central vs. peripheral compartment(s) to the

withdrawal effects observed. A CB1-receptor antagonist with limited acute brain penetration, such as

AM6545, could help address this issue. AM6545 has been shown to cause a change in neuronal activity

at CB1 receptors located in the periphery with no effect on CB1 receptors in the brain (Tam et al., 2010).

For instances, it has been shown that AM6545, an analog of rimonabant, has limited lipid solubility and

P-glycoprotein mediated extrusion from the brain, which renders it difficult to cross the blood-brain

barrier, while maintaining high affinity and selectivity for the CB1-receptor over the CB2-receptor.

Furthermore, in the behavioral tests, discussed in Chapter 1, AM6545 did not attenuate centrally mediated

behaviors, e.g., hypothermia, catalepsy and immobility, induced by a centrally acting CB1-receptor

agonist. Additionally, in a discriminated drinking aversion procedure using rimonabant as the

discriminative stimulus, AM6545 did not suppress drinking whereas AM4113 did, in a manner similar to

rimonabant (Jarbe et al 2011).

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In Europe, rimonabant was once marketed for the treatment of obesity and related metabolic risk

factors. As reviewed, 20 mg rimonabant produced significant weight loss, reduced triglycerides and

increased high-density lipoprotein (HDL) cholesterol (McLaughlin, 2012). Its removal from the

European market and denial for FDA approval was related to clinical reports of suicidal tendencies,

depression, anxiety and seizures (Janero et al., 2009; Jones, 2008; McLaughlin, 2012). Subsequent

studies suggested the adverse effects induced by rimonabant were attributed to its inverse agonist

properties (Bergman et al., 2008). By virtue of its inverse agonist property, rimonabant engagement of

the CB1-receptor can not only prevent the receptor from binding an agonist, but it can also alter

constitutive CB1-dependent cellular signaling/activity in vitro. For instance, rimonabant increased cAMP

levels in the forkolin stimulated cAMP assay (Mato et al., 2002; Sink et al., 2008). To address the above

mentioned adverse effects induced by rimonabant it was suggested that a neutral antagonist would be an

alternative approach for treating obesity and related metabolic risk factors (Chambers et al., 2007). A

neutral antagonist, such as AM4113, would behave similarly to rimonabant, with its ability to prevent a

CB1-receptor agonist from engaging the receptor but is devoid of negative efficacy such that it does not

affect constitutive CB1-receptor activity. For instance, AM4113 had no effect on forskolin stimulated

cAMP production (Sink et al., 2008). Preclinical studies revealed behavioral differences between an

inverse agonist and a neutral antagonist, discussed further in Chapter 3. Briefly, AM4113 failed to induce

nausea (i.e., conditioned gaping) and vomiting (i.e., emesis) in rodents as reported with inverse agonists,

such as rimonabant and AM251 (Chambers et al., 2007; McLaughlin et al., 2005; Salamone et al., 2007).

It was our belief that in vivo signs of cannabinoid withdrawal induced by rimonabant may potentially

result not only from CB1-receptor blockade, but also as a negative efficacy effect of rimonabant.

Therefore, we sought to investigate if AM4113 would be an alternative approach to optimize the benefits

of CB1-receptor antagonists while minimizing the severity of cannabinoid withdrawal signs. Overall, this

thesis will present an animal strategy for studying cannabinoid dependence to gain insight into

mechanisms implicated in tolerance to cannabinoid receptor stimulation resulting in withdrawal.

~ 12 ~

Objective and Specific Aims

The proceeding studies examined the ability of the CB1-receptor agonist AM2389 for inducing

dependence in an animal model, with greater potency than ∆9-THC and having a prolonged functional

in vivo duration of action (Järbe et al., 2012; Nikas et al., 2010). In addition, the effects of CB1-receptor

antagonists, with either neutral or negative-efficacy property (AM4113 and rimonabant, respectively),

were studied in an attempt to elucidate the mechanisms by which cannabinoid withdrawal transpires.

Finally, use of a neutral antagonist with limited acute brain penetration, i.e., AM6545, will address

whether or not there are peripheral contributions to the current withdrawal phenomena indices.

Therefore, the overall objective of this thesis was to profile CB1-receptor ligands with distinct

pharmacological properties/modes of action as potential pharmacological tools for studying dependence

by evaluating their pharmacological effects on CB1-receptor related physiological and behavioral

endpoints.

The specific aims of this dissertation research are to:

1) Characterize, in a tetrad assay, a CB1-receptor agonist (AM2389) with a long duration of effect to

facilitate the study of cannabinoid dependence.

Hypothesis: AM2389 exerts pronounced central effects that can be measured in the tetrad assay with

greater potency than ∆9-THC.

2) Determine if repeated treatment with AM2389 leads to tolerance to CB1-receptor mediated

hypothermia and hypo-locomotion in mice.

Hypothesis: Repeated treatment of AM2389 leads to tolerance by attenuating hypothermia and

hypo-locomotion that was induced by an acute administration of AM2389.

3) Characterize behavioral effects elicited by centrally acting CB1-receptor antagonists (with either an

inverse agonist or a neutral antagonist property) and a neutral CB1-receptor antagonist with limited acute

brain penetration.

~ 13 ~

Hypothesis: There are behavioral differences between a centrally active CB1-receptor antagonist/inverse

antagonist, neutral antagonist and a neutral antagonist with limited acute brain penetration.

4) Determine if somatic signs of spontaneous cannabinoid withdrawal are observed and quantifiable after

discontinuation of repeated AM2389 treatment.

Hypothesis: Repeated treatment of AM2389 does not lead to spontaneous physical signs of withdrawal,

possibly because of a slow, protracted elimination.

5) Compare behavioral (locomotion and somatic signs of withdrawal; e.g., front paw tremors) changes

precipitated by a CB1-receptor antagonist/inverse agonist (rimonabant) and a CB1-receptor neutral

antagonist (AM4113) in AM2389-tolerant mice; determine if somatic signs of withdrawal are primarily

attributed to central and/or peripheral influences; determine if somatic signs of antagonist-precipitated

withdrawal (e.g., hyperactivity) are attenuated by the reinstatement of a cannabinoid agonist in

AM2389-tolerant mice.

Hypotheses: A neutral antagonist, such as AM4113, elicit cannabinoid withdrawal symptoms in vivo that

differs quantitatively and/or qualitatively from those elicited by an antagonist/inverse agonist such as

rimonabant; somatic signs of withdrawal are primarily attributed to central influences; somatic signs of

antagonist-precipitated withdrawal are attenuated by the reinstatement of a cannabinoid agonist in

AM2389-tolerant mice.

~ 14 ~

Experimental Design and Methods

Animals

Male CD-1 and C57BL/6J mice (Charles River Breeding Laboratories, Wilmington, MA, USA)

weighing 30-35 g were group housed, 5 to a cage, in a temperature controlled (~20OC) animal facility.

Mice were habituated to the animal facility for at least 1 week prior to experiments with a light/dark cycle

of 12:12 h (lights on at 7 a.m.). Mice were given access to food and water ad libitum. Experimentally

naïve mice were used for all procedures and the subjects were tested during the light phase. CD-1 mice

were used for all behavioral assays, except where otherwise indicated. The Animal Care and Use

Committee of Northeastern University, Boston, MA, USA approved all procedures. The principles

outlined in the “Guidelines of Principles of Animal Laboratory Care” (National Institute of Health, 1996)

were followed.

Drugs

(-)-∆9-THC [(-)-delta-9-tetrahydrocannabinol (Figure 1) was delivered (National Institute of Drug

Abuse) as an ethanol (200 mg/ml) solution and stored at -20OC until used. Upon use, appropriate amounts

were withdrawn followed by the evaporation of ethanol under a stream of nitrogen. The residue was

dissolved (w/v) in an aqueous solution of dimethyl sulfoxide (DMSO, 2%), propylene glycol (PG, 4%)

and Tween-80 (T-80, 4%) prior to a final suspension with saline slowly added before animal dosing, the

exception being 100 mg/kg ∆9-THC where the vehicle used was DMSO 2%, PG 10% and T-80 8%.

Suspensions were prepared daily shortly before injection. AM2389 was stored and prepared in the same

manner as ∆9-THC. The ligands AM2389, AM251, rimonabant, AM4113 and AM6545 were synthesized

at the Center for Drug Discovery (CDD), Northeastern University, Boston, MA. Doses were

administered intraperitoneally (i.p.) in a volume of 10 ml/kg, except for 100 mg/kg ∆9-THC where the

volume was 15 ml/kg.

~ 15 ~

Agonist

(-)-Δ9-THC AM2389

CB1 Ki = 39.5 nM CB1 Ki = 0.16 nM

CB2 Ki = 40.0 nM CB2 Ki = 4.21 nM

Selectivity CB1/CB2 = 1/1 Selectivity CB1/CB2 = 26/1

Antagonist

N-piperidin-1-yl-2,4-dichlorophenyl-

1H-pyrazole-3-carboxamide analog

AM251 Rimonabant AM4113 AM6545 CB1 Ki = 3.43 nM CB1 Ki = 10.0 nM CB1 Ki = 0.89 nM CB1 Ki = 1.73 nM

CB2 Ki = 124 nM CB2 Ki = 931 nM CB2 Ki = 92 nM CB2 Ki = 523 nM

Selectivity CB1/CB2 = 36/1 Selectivity CB1/CB2 = 93/1 Selectivity CB1/CB2 = 103/1 Selectivity CB1/CB2 = 308/1

Figure 1. Structures and binding data for cannabinoid ligands (Thakur et al., 2005).

~ 16 ~

Open Field

Locomotor activity (horizontal exploration) was examined using an open-field photobeam activity

monitoring system (San Diego Instruments, San Diego, CA, USA). Activity was recorded by infrared

photobeam sensors in a 16- x 16-in acrylic chamber, which was placed inside a sound attenuating

ventilated box to produce a controlled environment. The photobeam sensors consisted of 4 infrared beam

bars placed at 90O angles enclosing the bottom of the chamber, lined with a second row (only for 2 of the

bars located parallel to each other) that recorded rearing (vertical exploration). All chambers were wiped

with a soap/water solution between tests.

Paw Withdrawal

Paw withdrawal was tested using a method previously described (Hargreaves et al., 1988) and

subsequently modified for mice. Mice were placed inside a Plexiglas chamber above a glass plate heated

to 30OC. An acclimation period of ~1 hr was allowed prior to testing. A focused projection bulb serving

as the thermal nociceptive stimulus, located underneath the glass plate, was aimed at the plantar surface of

the hind limb. The heat current was set to 6.0 amperes. Measurements were carried out by an automated

motion sensor, which terminated the heat stimulus and recorded the latency to withdrawal. The glass

plate was cleaned to remove any waste such as urine and feces prior to taking measurements.

Inverted Screen

The inverted screen was used to measure muscle strength and motor coordination (Coughenour et al.,

1977; Lichtman et al., 2004). An invertible wire mesh grid (14 x 14 cm2) was suspended horizontally

30 cm over a cushioned surface, fixed between two metal supports.

Catalepsy

Catalepsy was assessed with a bar test, i.e., both forelimbs were positioned on a stainless steel rod

(0.40 cm in diameter) that was elevated 4.5 cm above the surface (Lichtman et al., 2002).

~ 17 ~

Rectal Temperature

Rectal temperature was measured in mice using a rectal probe of a digital laboratory thermometer,

RET-3-ISO, type T thermocouple (Physitemp Instruments Inc, Clifton, NJ). The lubricated probe was

inserted ~2.0 cm into the rectum for ~30 sec prior to each recording.

Observation Chambers

Observation chambers were used to score individual mouse behaviors. Chambers consisted of a

circular clear glass jar (radius 4.25 cm, height 16 cm) sealed with a ventilated cover, providing clear

visibility of movement and ample air circulation.

Data Analysis

Results are presented as means ± SEM. Significant differences between group means were calculated

with ANOVA [based on the Fisher (F) distribution], followed by Holm-Sidak multiple comparison

post-hoc statistical test procedure. Differences between group means at the P < 0.05 level were

considered significant.

~ 18 ~

Chapter 1. Characterize, in a tetrad assay, a CB1-receptor agonist (AM2389) with a long duration

of effect to facilitate the study of cannabinoid dependence

AM2389, a potent and long-acting CB1-receptor agonist (Järbe et al., 2012; Nikas et al., 2010), was

evaluated as an improved pharmacological tool for studying cannabinoid dependence. Initial experiments

aimed at establishing a dependence model that could produce sustained activation of the CB1-receptor by

utilizing a CB1-receptor agonist with a long duration of effect, AM2389. AM2389 was characterized

using the tetrad assay, a family of behavioral end-points commonly used to study ligands that activate the

CB1 receptors. We hypothesized that AM2389 exerts pronounced central effects that can be measured in

the tetrad assay with greater potency than ∆9-THC. Thus, we studied locomotor activity, paw thermal

stimulation, inverted screen test/catalepsy bar test and rectal temperature. To increase generality, two

different mice types (the outbred stock CD-1 and the inbred strain C57BL/6J) were used for the rectal

temperature assay and comparisons made with ∆9-THC.

Procedures

Drugs were administered 20 min (∆9-THC) or 90 min (AM2389) prior to testing, unless otherwise

indicated. Time-points listed above were chosen based on preliminary data examining onset and duration

of effect (Tai et al., 2008). Each assay was carried out in naïve mice that had not prior exposure to drug

treatment or apparatus.

Locomotor activity was measured as the total number of beam breaks within a 60 min period

following a 90 min post-injection with AM2389. For analysis, measurements were sampled in 15 min

bins at the 15, 30, 45 and 60 min time-points. Rearing was recorded in the same manner.

Paw withdrawal latencies were measured from the time of initial heat stimulus contact up to the time

of the paw withdrawal. The maximum time allowed for heat exposure was 40 sec to prevent tissue

damage.

~ 19 ~

The inverted screen assay consisted of placing mice in the center of the wire mesh grid that was

inverted 180O so that the mice were oriented upside down on the bottom of the screen. The latency to

climb onto the reverse (top) side within 60 sec was recorded.

Catalepsy was measured as the length of time each mouse maintained both forelimbs in an elevated

position on the bar. Mice that remained immobile (with the exception of respiratory movements) were

considered cataleptic.

In CD-1 mice, rectal temperature recordings with ∆9-THC or AM2389 were taken at a single

time-point either 20 or 90 min post-administration. For rectal temperature recordings over time in

C57BL/6J mice, antagonism was tested by administering the CB1-receptor antagonist/inverse agonist,

AM251 immediately preceding AM2389-administration. For a more detailed description of the methods,

refer to the Experimental Design and Methods section and the corresponding figure legends.

Results

Open-field (Figure 2): Vehicle-treated and AM2389-treated groups evidenced reduced activity over

time (Figure 2A,B). In Figure 2A, two-way repeated measure ANOVA indicated significance for Dose

[F(3,32) = 9.87; P < 0.001], Time [F(3,96) = 30.89; P < 0.001] and the Dose x Time interaction [F(9,96) =

5.83; P < 0.001]. At the 15 min time-point, all doses of AM2389 (0.03, 0.1 and 0.3 mg/kg) produced a

significant decrease in activity in comparison to the vehicle group at the corresponding time-point, which

persisted to the 30 min time-point. Habituation to locomotor activity in the vehicle group occurred, as the

activity at the 30, 45 and 60 min time-points was significantly lower compared to the 15 min time-point.

In Figure 2B, two-way repeated measure ANOVA indicated significance for Dose [F(3,32) = 10.06;

P < 0.001], Time [F(3,96) = 11.68; P < 0.001] and the Dose x Time interaction [F(9,96) = 4.29;

P < 0.001]. Similar to locomotor activity, rearing bouts were significantly suppressed in mice treated

with all doses of AM2389 at the 15 and 30 min time-points, as well as 0.3 mg/kg AM2389 at the 45 min

time-point, compared to the vehicle group at the corresponding time-points. The vehicle group at the 45

and 60 min time-points showed a significant decrease in rearing bouts compared to vehicle at the 15 min

time-point, indicating habituation to the open-field arena.

~ 20 ~

AM2389

15 30 45 60

Activity C

ounts

0

500

1000

1500

2000

2500

Vehicle

0.03 mg/kg

0.1 mg/kg

0.3 mg/kg

#

#

*

*

#

Time (min)

15 30 45 60

Num

ber

of R

earing B

outs

0

20

40

60

80

100

120

140

160

Vehicle

0.03 mg/kg

0.1 mg/kg

0.3 mg/kg

*

#

#

*

AM2389

#

#

A. Locomotor Activity

B. Rearing Activity

#

#

#

#

#

#

*

Figure 2. Locomotor activity was measured as the total number of beam breaks within a 60 min period following 90 min

post-injection with AM2389 (n = 6 per dose) or vehicle (N = 18; n = 6): “N” refers to the total number of mice used in statistical

analysis, while “n” refers to the number of mice run in parallel with each AM2389 dose. (A) For statistical analysis,

measurements were sampled in 15 min bins at the 15, 30, 45, 60 min time-points. (B) Rearing was recorded in the same manner

as locomotor activity. Each data point represents the mean ± SEM for total beam breaks or rearing bouts for 15 min at the

corresponding time-points. *, indicate significant difference from vehicle at the 15 min time-point at P < 0.05. #, indicate

significant difference from vehicle at the same time-point at P < 0.05. Holm-Sidak all pair-wise multiple comparisons following

two-way repeated measure ANOVA.

~ 21 ~

Paw withdrawal latencies (Figure 3): All doses of AM2389 (0.03, 0.1 and 0.3 mg/kg) produced a

significantly greater paw withdrawal latency in comparison to the corresponding vehicle group

[F(3,32) = 27.70; P < 0.001]. ∆9-THC did not induce any significant change in latency compared to the

corresponding vehicle group [F(3,32) = 0.88; P > 0.05].

Inverted screen and catalepsy bar tests (Figure 4): In the inverted screen test, AM2389 at 0.03, 0.1 and

0.3 mg/kg produced no significant difference [F(3,32) = 2.61; P > 0.05] from the vehicle group, since

there was only a trend towards the vehicle group reaching the reverse side of the inverted screen more

quickly in comparison to the highest doses of AM2389 (0.1 and 0.3 mg/kg) (Figure 4A). In the catalepsy

bar test, 0.1 mg/kg AM2389 resulted in immobility significantly greater than the vehicle group [F(3,32) =

4.06; P < 0.02] (Figure 4B). The dose of 0.3 mg/kg AM2389 yielded a large variation in response; hence,

no significant difference in comparison to vehicle.

Rectal temperature in CD-1 mice (Figure 5): Vehicle-treated mice had a stable body temperature. All

doses of AM2389 (0.03, 0.1 and 0.3 mg/kg) induced a dose-dependent decrease in temperature in

comparison to the corresponding vehicle groups [F(5,30) = 76.37; P < 0.001]. The highest doses of

AM2389 (0.1 and 0.3 mg/kg) resulted in a significant decrease in temperature compared to 0.03 mg/kg

AM2389. The highest doses of ∆9-THC (10 and 30 mg/kg) induced a greater decrease in temperature

than the corresponding vehicle groups, along with a decrease in temperature significantly greater than that

induced by 3 mg/kg ∆9-THC [F(5,30) = 11.34; P < 0.001].

Rectal temperature in C57BL/6J mice (Figure 6): Two-way repeated measure ANOVA indicated

significance for Dose [F(5,35) = 50.28; P < 0.001], Time [F(4,140) = 163.12; P < 0.001] and the Dose x

Time interaction [F(20,140) = 40.83; P < 0.001]. Vehicle treated mice had stable body temperatures over

the whole time period with a slight decrease at the 360 min but not the 1440 min time-point post-injection

(i.e., 20 min to 24 hrs post-injection; Figure 6) Significant initial decreases in body temperature were

exhibited at the 20 min time-point in mice treated with 0.3 mg/kg AM2389 and 30 mg/kg ∆9-THC alone

compared to vehicle at the same time-point; this hypothermia persisted at the 60, 180, 360 and 1440 min

~ 22 ~

-THC

Vehicle 3 10 30P

aw

Withdra

wal Late

ncy (

sec) Vehicle

-THC

AM2389

Vehicle 0.03 0.1 0.3

Paw

Withdra

wal Late

ncy (

sec)

0

10

20

30

40

50

Vehicle

AM2389

*

*

*

Figure 3. Paw withdrawal was measured as the amount of time utilized to remove the hind paw from a thermal stimulus after injection with AM2389 (n = 6 per dose),

∆9-THC (n = 6 per dose), or vehicle (N = 18; n = 6): “N” refers to the total number of mice used in statistical analysis, while “n” refers to the number of mice run in

parallel with each drug dose. Cut off time was 40 sec. *, indicates significant difference from corresponding vehicle groups at P < 0.05. Holm-Sidak multiple

comparisons versus vehicle following one-way ANOVA.

~ 23 ~

Catalepsy Bar Test

Dose (mg/kg)

Vehicle 0.03 0.1 0.3

Imm

ob

ility

Tim

e (

se

c)

0

5

10

15

20

25*

B. Catalepsy Bar Test

Dose (mg/kg)

Vehicle 0.03 0.1 0.3

Clim

bin

g T

ime

(se

c)

0

10

20

30

40

50

60A. Inverted Screen

AM2389

Vehicle

Figure 4. Each data point represents the mean ± SEM in sec after increasing doses of AM2389 (n = 6 per dose) or

vehicle (N = 18; n = 6): “N” refers to the total number of mice used in statistical analysis, while “n” refers to the

number of mice run in parallel with each AM2389 dose. Cut off time was 60 sec. (A) Inverted Screen: mice that

failed to climb to the reverse side or fell off were given a score of 60 sec. (B) Catalepsy: the duration of immobility

was recorded until the mouse removed both forepaws from the bar or climbed onto the bar with one or both hind

paws. *, indicates significant difference from the vehicle group at P < 0.05. Holm-Sidak multiple comparisons

versus vehicle following one-way ANOVA.

~ 24 ~

Dose (mg/kg)

0.03 0.1 0.3

Change in T

em

pera

ture

(0C

)

-8

-6

-4

-2

0

Vehicle

AM2389

3 10 30

Vehicle

THC

*

*

*

*

#

#

#

#

*

Figure 5. Changes in temperature compared between vehicle and AM2389 or ∆9-THC 90 min or 20 min post-injection, respectively. AM2389 (n = 6 per dose), ∆9-THC (n = 6

per dose), vehicle (N = 18; n = 6): “N” refers to the total number of mice used in statistical analysis, while “n” refers to the number of mice run in parallel with each drug dose.

Temperature readings prior to dosing averaged 37.48oC ± 0.51, 37.74oC ± 0.60 and 37.58 ± 0.10 for AM2389, ∆9-THC and vehicle groups, respectively. *, indicates significant

difference from corresponding vehicle group at P < 0.05. #, indicate significant difference from the lowest dose of its corresponding drug group at P < 0.05. Holm-Sidak all

pair-wise multiple comparisons following one-way ANOVA.

~ 25 ~

Time (min)

20 60 180 360

Ch

an

ge

in

Te

mp

era

ture

(0C

)

-12

-10

-8

-6

-4

-2

0

2

mg/kg

*

*

*

*

*

*

*

*

*

*

*

**

*

1440

Vehicle

AM2389 (0.1)

AM2389 (0.3)

THC (30)

AM2389 (0.3) + AM251 (3)

AM2389 (0.3) + AM251 (10)

#

Figure 6. Changes in temperature compared between vehicle (N = 11; n = 2 to 3), AM2389 (n = 5 – 6 per dose), Δ9-THC alone

(n = 6) and AM2389 (0.3 mg/kg) plus AM251 (3 and 10 mg/kg; n = 6 to 7 per dose) in C57BL/6J mice: “N” refers to the total

number of mice used in statistical analysis, while “n” refers to the number of mice run in parallel with each drug dose. Changes

in temperature were recorded over time at the 20, 60, 180, 360 and 1440 min time-points. Rectal temperatures prior to dosing

averaged 37.40oC ± 0.18, 37.33oC ± 0.28, 37.30oC ± 0.18, 37.07oC ± 0.18, 37.61oC ± 0.18 and 37.20oC ± 0.30 for vehicle,

AM2389 (0.1 mg/kg), AM2389 (0.3 mg/kg), AM2389/AM251 (3 mg/kg), AM2389/AM251 (10 mg/kg), and Δ9-THC groups,

respectively. *, indicate significant difference from the corresponding vehicle group at the same time-point at P < 0.05.

#, indicate significant difference from vehicle group at the 20 min time-point at P < 0.05. Holm-Sidak all pair-wise multiple

comparisons following two-way repeated measure ANOVA.

~ 26 ~

time-points for 0.3 mg/kg AM2389 and at the 60 and 180 min time-points for ∆9-THC. These

temperature changes were significantly different from vehicle at the same time-points. Significant

decreases in body temperature for 0.1 mg/kg AM2389 alone and 0.3 mg/kg AM2389 plus 3 mg/kg

AM251 were initially exhibited at the 60 min time-point in comparison to vehicle at the same time-point;

this decrease was even greater at the 180 min and 360 min time-points. The maximum hypothermic

effect was observed for all groups treated with AM2389 alone or in combination with 3 mg/kg AM251 at

the 360 min time-point, whereas hypothermia elicited by 30 mg/kg ∆9-THC was maximal at the 60 min

time-point. AM251dose-dependently antagonized the hypothermic response induced by 0.3 mg/kg

AM2389 alone and the dose of 10 mg/kg AM251 completely blocked the hypothermic response. In

addition, all groups (except 0.3 mg/kg AM2389 alone) reached temperatures not statistically different

from the vehicle group at 1440 min post-injection. ∆9-THC (30 mg/kg) treated mice had body

temperatures not statistically different from vehicle controls at the 360 and 1440 min time-points.

Discussion

In this study, we characterized physiological and behavioral responses in mice administered AM2389,

a high-affinity and potent agonist with ~26-fold selectivity for the CB1- over the CB2-receptor (Järbe et

al., 2012; Nikas et al., 2010). The influence cannabinergics have on the basal ganglia and cerebellum has

been implicated in the regulation of motor function, while the hypothalamus appears more involved in

thermal regulation (Neychev et al., 2008; Romanovsky, 2007). This would suggest that activation of

central CB1 receptors induces hypoactivity, hypoalgesia, catalepsy and hypothermia. Thus, the

psychomotor and temperature alterations observed in the current study support previous findings that

suggest these responses were likely due to activation of CB1 receptors in the brain (Kishimoto et al.,

2006; Rawls et al., 2002). Furthermore, the behavioral effects of AM2389 measured here share

similarities with other CB1-receptor agonists, including Δ9-THC and WIN55,212-2 (Fan et al., 1994;

Lichtman et al., 2001a).

AM2389 suppressed locomotor and rearing activity to a greater degree than vehicle. In addition, the

vehicle group(s) exhibited a decrease in activity over time. This probably reflects an increased familiarity

~ 27 ~

with the chamber, reducing novelty exposure (habituation). Furthermore, the results from the inverted

screen and catalepsy bar test showed a trend towards increased immobility time in a dose-dependent

manner. The inverted screen test outcome may imply that AM2389 induces biphasic effects, i.e.,

hyperactivity at low doses and hypoactivity at higher doses. This would follow suit with previous work

suggesting that CB1-receptor agonists may induce biphasic effects (Chaperon et al., 1999). On the other

hand, if this was truly a biphasic effect one might expect a statistically significant increase in activity at

the lowest AM2389 dose (0.03 mg/kg) compared to vehicle and the opposite response for the highest dose

of AM2389 (0.3 mg/kg) but this was not the case. In the catalepsy bar test, mice given 0.3 mg/kg

AM2389 appeared unable to maintain their posture on the bar and they fell off; this explains the large

error bars and thus the lack of significance with respect to the controls. This suggests that the inverted

screen and the catalepsy bar test may not be measuring the same aspects of motor control.

Hypoalgesic effects of CB1-receptor agonists are mediated through the spinal and supraspinal levels of

the CNS, including the periaqueductal gray area, the rostral ventromedial medulla and the dorsal horn of

the spinal cord (Hohmann et al., 2006; Lichtman et al., 1991). AM2389 increased the paw withdrawal

latency in the presence of a thermal noxious stimulus at lower doses than Δ9-THC. A concern for using

high doses of cannabinoids is the potential for binding to other targets, i.e., off-target binding. As

mentioned previously AM2389 is ~26-fold selective for the CB1- over the CB2-receptor, in addition to

having an affinity ~250-fold greater for the CB1-receptor and ~10-fold greater for the CB2-receptor in

comparison to Δ9-THC. Taking everything into consideration, the efficacious and potent agonist,

AM2389, has the potential to induce maximal effects while minimizing the likelihood of acting on off-

target sites that may promote non-specific responses. It was unclear why there was no significant

increase in the paw withdrawal latency noted after Δ9-THC treatment, even at the highest dose. It is

likely that the heat stimulus intensity used to induce a response for the lowest dose of AM2389 surpassed

the heat sensitivity threshold for inducing a dose-dependent latency in Δ9-THC-treated mice. Also, it is

possible that other analgesia instruments (e.g., hot-plate and tail-flick) may be more sensitive at detecting

Δ9-THC hypoalgesia than the thermal stimulator used here. However, the sensitivity of this paw

~ 28 ~

stimulator to cannabinoids might be increased during hyperalgesia (e.g., carrageenan induced

inflammation), as shown previously (Richardson et al., 1998).

Core body temperature in vehicle-treated mice is maintained with great stability, but is reduced after

administration of AM2389 or Δ9-THC alone. In CD-1 mice, the highest dose of AM2389 (0.3 mg/kg)

induced a significant decrease in temperature (-6.63 ± 0.55OC), which was some 2-fold greater than the

hypothermia elicited by a 100-fold higher dose (30 mg/kg) of Δ9-THC (-3.36 ± 0.56 OC). The rectal

temperature changes observed in CD-1 mice generalize across mice strains and species, including

C57BL/6J mice (current study) and Sprague-Dawley rats (Järbe, 1978). Järbe (1978) also found that

hypothermia induced by 8 mg/kg Δ9-THC peaked ~1 hr post-injection, similar to our current study. It

could be argued that the Δ9-THC doses examined here were not sufficient to induce a greater degree in

hypothermia. However, maximal effects on hypothermia with doses 32 and 100 mg/kg Δ9-THC have

been reported in C57BL/6J mice (McMahon et al., 2007). Temperature recordings over time showed that

0.3 mg/kg AM2389 induced hypothermia was blocked or attenuated, respectively, by 10 and 3 mg/kg

AM251, a CB1-receptor selective antagonist. This dose-related attenuation indicates that AM2389

induced hypothermia is a CB1-receptor mediated phenomenon. AM2389 produced maximum

hypothermic effects at the 360 min time-point whether it was administered alone or when combined with

the lower dose of the antagonist AM251 (3 mg/kg). It would be presumptuous to consider that all ligands

peak at the same time when determining the maximum response of cannabinoid ligands to physiological

and behavioral changes. Different cannabinoid ligands, like other drugs, produce maximum effects at

different time-points (Järbe et al., 1989; Järbe et al., 1981); undoubtedly, compared to Δ9-THC’s effect on

hypothermia, AM2389 had a slower onset, peaked later and had a considerably longer duration of effect.

Overall, we have presented evidence that AM2389 exerts pronounced central effects that can be measured

in classical behavioral assays associated with CB1 agonist activity, and does so with greater potency than

Δ9-THC.

~ 29 ~

Chapter 2. Determine if repeated treatment with AM2389 leads to tolerance to CB1-receptor

mediated hypothermia and hypo-locomotion in mice

In the previous chapter, it was demonstrated that AM2389 in vivo behaved similarly to Δ9-THC in the

tetrad assay as would be expected of CB1-receptor cannabinergics, with greater potency and having a

prolonged functional in vivo duration of action. More importantly, these effects were more pronounced

than those of Δ9-THC. For instance, AM2389 produced a greater hypothermic effect and had a protracted

time course greater than Δ9-THC. However, these findings alone do not suggest that repeated treatment

with AM2389 leads to tolerance. Δ9-THC administered twice daily at 10 mg/kg or increasing daily doses

of Δ9-THC for 6.5 days produced tolerance to the behavioral tetrad, e.g., antinociception, catalepsy,

hypothermia and increased immobility (McKinney et al., 2008). However, mice that were administered

vehicle twice daily for 6.5 days with a single treatment of Δ9-THC showed profound cannabinoid

mediated effects. Before attempting to study AM2389 associated withdrawal, e.g., either by abrupt

discontinuation of the agonist or with the aid of an antagonist, it seemed prudent to determine if repeated

treatments of AM2389 indeed led to tolerance. To test this hypothesis, two cannabinoid mediated effects

were measured: hypothermia and hypo-locomotion.

Procedures

To measure tolerance, rectal temperature recording were taken from mice administered AM2389 for

5 consecutive days. Initial recordings were obtained prior to dosing followed by another recording

90 min post-injection of 0.1 mg/kg AM2389, day 1 and on days 2-5, 90 min post-injection of 0.3 mg/kg

AM2389. The dosing regimen used was based on preliminary data that determined an appropriate

regimen that did not compromise mice health. Acutely administered 0.3 mg/kg AM2389 produced

marked effects such that mice did not eat or drink for at least 24 hrs. Therefore, 0.1 mg/kg AM2389 was

used for the initial tolerance induction.

Tolerance was also measured in the open-field by recording locomotor and rearing activity in mice that

were treated similarly to the above mentioned dosing regimen. In brief, AM2389 was administered for

~ 30 ~

5 days (repeated treatment) followed by placing these experimentally naïve mice (i.e., mice not

previously exposed to the open-field apparatus) into the open-field arena to measure locomotor and

rearing activity. Another group of experimentally naïve mice were dosed 0.1 mg/kg AM2389 (acute

treatment) and placed into the open-field 90 min post-injection. Locomotor activity was measured as the

total number of beam breaks within a 60 min period following an acute or repeated treatment of AM2389.

For analysis, measurements were sampled in 15 min bins at the 15, 30, 45 and 60 min time-points.

Rearing was recorded in the same manner.

Results

Rectal Temperature (Figure 7): On day 1, AM2389 induced maximum hypothermia, a 5.7oC decrease

in temperature from baseline. This hypothermic effect was attenuated to -3.9, -1.1, 0.9 and -1.4 with

repeated AM2389 administration at days 2, 3, 4 and 5, respectively [F(4,12) = 36.04; P < 0.001]. Thus,

hypothermia was attenuated after repeated dosing resulting in temperatures approaching baseline.

Open-field (Figure 8): Mice that were repeatedly treated with AM2389 exhibited greater locomotor

activity at the 15 min time-point in comparison to mice that received an acute treatment of AM2389

[F(1,12) = 6.81; P = 0.02] (Figure 8A). At the subsequent time-points (e.g., 30, 45 and 60 min),

repeatedly treated mice showed an attenuation of locomotor activity approaching that of acutely treated

mice. Similarly, mice that were repeatedly treated with AM2389 reared significantly more than acutely

treated mice at the 15 min time-point [F(1,12) = 4.91; P < 0.05] (Figure 8B). Since acutely treated mice

exhibited minimal rearing bouts initially (e.g., 15 min time-point), there was no further change present;

however, repeatedly treated mice exhibited habituation to rearing, which were observed as a downward

trend in rearing bouts at the 30, 45 and 60 min time-points.

Discussion

It has been well established that Δ9-THC produces measureable pharmacological signs of tolerance

upon repeated administrations in both humans and experimental animals (Carlini, 1968; Hart et al., 2002;

Jones et al., 1976a; Jones et al., 1981). For instance, humans treated with oral Δ9-THC (70-210 mg/day)

~ 31 ~

AM2389, 5 day treatment

Day 1 2 3 4 5

Cha

ng

e in T

em

pe

ratu

re (

0C

)

-8

-6

-4

-2

0

Dose 0.1 0.3 0.30.3 0.3 mg/kg

*

* * *

Figure 7. Changes in temperature measured once daily for 5 days in mice administered AM2389 daily (n = 4). Rectal

temperatures prior to dosing averaged 38.9oC ± 0.1. *, indicate significant difference from day 1 at P < 0.05. Holm Sidak

multiple comparisons versus day 1 following one-way repeated measure ANOVA.

~ 32 ~

A. Locomotor Activity

15 30 45 60

Activity C

oun

ts

0

500

1000

1500

2000

2500

B. Rearing Activity

Time (min)

15 30 45 60

Nu

mbe

r of R

eari

ng

Bou

ts

0

20

40

60

80

100

120

140

160

*

*

Acute

Chronic

Vehicle

Figure 8. Locomotor activity was measured as the total number of beam breaks within a 60 min period following acute or

repeated exposure to AM2389 (n = 6 - 7). Acute refers to mice administered 0.1 mg/kg AM2389 and repeated refers to mice

administrated 0.1 mg/kg AM2389 on day 1 followed by 0.3 mg/kg AM2389 on days 2 - 5. For comparative purposes,

performances after vehicle administration were replotted from Figure 2. (A) For statistical analysis, measurements were

sampled in 15 min bins at the 15, 30, 45, 60 min time-points. (B) Rearing was recorded in the same manner as locomotor

activity. Each data point represents the mean ± SEM for total beam breaks or rearing bouts for 15 min at the corresponding

time-points. *, indicate significant difference between acute ( ) and repeated ( ) administration at the corresponding time-point

at P < 0.05. Holm-Sidak multiple comparisons versus acute following one-way ANOVA.

~ 33 ~

for 30 days developed a loss of the subjective high that was experienced with initial treatment (Carlini,

1968; Hart et al., 2002; Jones et al., 1976a; Jones et al., 1981). The current study sought to determine if

AM2389 would lead to tolerance upon repeated administration by evaluating psychomotor and

temperature alterations. Changes in rectal temperatures indicated that by days 3-5 maximum attenuation

of hypothermia was reached after repeated AM2389 dosing, which suggested that maximum tolerance

was also produced. Further evidence of tolerance was revealed by measuring psychomotor changes that

occurred in the open-field arena. Immediately after being placed in the chambers, there was a profound

difference in locomotor as well as rearing activity in mice repeatedly treated with AM2389 in comparison

to mice that were acutely treated. The change in activity measured several days post initial treatment

would suggest that the mice showed tolerance to the psychomotor effects of AM2389. It was also found

that there was a decrease in overall activity at the 15 min time-point, suggesting that the mice became

habituated to the chambers over time. This habituation is consistent with early findings, which showed

mice that are exposed to the chamber for an extended period of time increase their familiarity with the

chambers, regardless of whether treated with a cannabinergic or not, Chapter 1.

Tolerance to cannabinoid-mediated effects is mainly attributable to pharmacodynamic changes at the

cellular level, with the down regulation and desensitization of cannabinoid receptor mediated G protein

activation (Oviedo et al., 1993; Sim-Selley, 2003); pharmacokinetic changes, e.g., cannabinoid

metabolism, plays a minor role (Maldonado, 2002; Romero et al., 1998). For that reason, investigating

pharmacodynamic tolerance has become the center of attention for elucidating molecular changes leading

to cannabinoid induced tolerance.

Tolerance that develops as a result of repeated cannabinoid administration has been reported to display

regional differences in terms of onset of reduction and magnitude in the brain (Romero et al., 1998). For

instance, repeated administration of Δ9-THC resulted in a decrease in CB1-receptor binding and

cannabinoid receptor mediated G protein activation in the hippocampus and cerebellum more rapidly and

to a greater magnitude compared to the striatum/globus pallidus (Breivogel et al., 1999). It should be no

surprise that there are also differences in the rate of recovery after cessation of cannabinoid use, with the

~ 34 ~

hippocampus recovering at a slower rate in comparison to the striatum/globus pallidus (Sim-Selley et al.,

2006). Subsequent experiments demonstrated that even in humans, higher doses of Δ9-THC and longer

treatment durations would lead to greater tolerance (Hart et al., 2002; Jones et al., 1981). Similarly, the

cellular changes that occur as a result of repeated cannabinoid administration are manifested at the

behavioral and physiological level. For instance, repeated administration of Δ9-THC induced tolerance to

hypothermia more rapidly in comparison to tolerance to hypo-locomotion (Whitlow et al., 2003). The

recovery rate of tolerance has infrequently been studied at the behavioral level; however, it has been

shown that cessation of cannabinoid administration in mice resulted in tolerance to hypo-locomotion

recovering more quickly in comparison to tolerance to antinociception (Bass et al., 2000).

This study has provided further evidence that AM2389 exhibits functional activity in vivo that is

parallel to other CB1-receptor cannabinergic ligands. For instance, repeated administration of AM2389

leads to tolerance by attenuating the hypothermia and hypo-locomotion that was induced by an acute

administration of AM2389. Therefore, the findings reported from Chapters 1 and 2 have for the first time

provided an in vivo pharmacological characterization of AM2389 in the tetrad assay, in addition to

presenting further evidence for using AM2389 as a pharmacological tool for studying cannabinoid

dependence/withdrawal.

~ 35 ~

Chapter 3. Characterize behavioral effects elicited by centrally acting CB1-receptor antagonists

(with either an inverse agonist or a neutral antagonist property) and a neutral CB1-receptor

antagonist with limited acute brain penetration

Prior to conducting CB1-receptor antagonist precipitated withdrawal studies, it was important to

characterize behaviorally the CB1-receptor antagonists alone in the behavioral paradigm to be used in the

precipitated withdrawal studies (Chapter 5). It was hypothesized that there were behavioral differences,

in the behaviors describe below, between a centrally active CB1-receptor antagonist/inverse antagonist

(e.g., AM251), a centrally active neutral antagonist (e.g., AM4113) and a neutral antagonist with limited

acute brain penetration (e.g., AM6545) relative to typical CB1-receptor antagonists/inverse agonists

(Cluny et al., 2010; Rinaldi-Carmona et al., 1994; Sink et al., 2008). AM251, AM4113 and AM6545 are

diarylpyrazole analogs structurally related to rimonabant. Characterization of all three CB1-receptor

antagonists was carried out in the open-field and the observation chamber test.

Procedures

Drugs were administered immediately prior to the mice being placed into the open-field arena or

observation chambers. Dose curves were determined for each ligand (AM251, AM4113, and AM6545)

in both behavioral assays.

Locomotor activity was measured as the total number of beam breaks within a 120 min period

immediately following a single injection of either a CB1-receptor antagonist or vehicle. For analysis,

measurements were sampled in 5 min bins at the 5-10, 15-20, 25-30, 35-40, 45-50, 55-60 and

115-120 min time intervals. Rearing was recorded in the same manner.

Observation chambers were used to score key behaviors (e.g., duration of rearing, grooming and

scratching) within a 120 min period immediately following a single injection of either a CB1-receptor

antagonist or vehicle. Definitions for each behavior were: rearing, upward movements of the mouse with

their entire weight being supported by the hind paw(s); grooming, licking/brushing of the fur; scratching,

using the hind paw to scrape the skin. These behaviors were chosen based on previous findings that

~ 36 ~

reported a change in activity levels of rearing, grooming and scratching after a single administration of a

CB1-receptor antagonist or when used to induce CB1-receptor antagonist precipitated withdrawal (Cook

et al., 1998; Hutcheson et al., 1998). For statistical analysis, measurements were sampled in 5 min bins at

the 5-10, 15-20, 25-30, 35-40, 45-50, 55-60 and 115-120 min time intervals.

Results

Open-field (Figure 9): Characterization of CB1-receptor antagonists were initially carried out in year

2008 and completed in year 2009. Vehicle groups from 2008 and 2009 were compared to determine if

the data were comparable. Both the 2008 and 2009 vehicle groups evidenced reduced activity over time.

However, no significant differences were found between the 2008 and 2009 vehicle groups at any time

interval (P > 0.05).

Open-field (Figure 10): All groups evidenced reduced activity over time. AM251, AM4113 and

AM6545 at 3 mg/kg produced no distinguishable effects on locomotion or rearing bouts in comparison to

vehicle at all time intervals (P > 0.05) (Figure 10A, C). AM6545 at 10 mg/kg displayed the greatest

suppression in locomotion and rearing bouts at all time intervals in comparison to the other treatment

groups at the corresponding time intervals (Figure 10B, D), whereas AM4113 produced significant

sporadic suppressions in activity at select time intervals.

Open-field (Figure 11): The effects of AM6545 were further characterized by expanding the

dose-response curves to include higher and lower doses. All groups treated with either AM6545 or

vehicle had reduced activity over time. An initial significant suppression in locomotor activity from

vehicle was observed at the 5-10 min [F(6,43) = 5.86; P < 0.001] time interval for the higher doses of

AM6545 (5.6 to 18 mg/kg), which persisted through the 15-20, 25-30, 35-40 and 45-50 min time intervals

(Figure 11A), with 5.6 mg/kg AM6545 not significantly different from vehicle at the 45-50 min time

interval. Similarly, rearing bouts were initially significantly suppressed at the 5-10 min [F(6,43) = 6.19;

P < 0.001] time interval in mice treated with the higher doses of AM6545 (5.6 to 18 mg/kg) in

comparison to vehicle, which persisted through the 15-20, 25-30 and 35-40 min time intervals

(Figure 11B).

~ 37 ~

Time (min)

5-10 15-20 25-30 35-40 45-50 55-60

Nu

mb

er

of R

ea

rin

g B

ou

ts

0

20

40

60

80 B. Rearing Activity

115-120

115-1205-10 15-20 25-30 35-40 45-50 55-60

Activity C

ou

nts

0

200

400

600

800

2008 Vehicle

2009 Vehicle

A. Locomotor Activity

Figure 9. Locomotor activity was measured as the total number of beam breaks within a 120 min period immediately following vehicle

treatment (2008 vehicle group, n = 6 and 2009 vehicle n = 14). (A) For statistical analysis, measurements were sampled in 5 min bins at

the 5-10, 15-20, 25-30, 35-40, 45-50, 55-60 and 115-120 min time intervals. (B) Rearing was recorded in the same manner as locomotor

activity. Each data point represents the mean ± SEM for total beam breaks or rearing bouts for 5 min at the corresponding time intervals.

No significant differences were found between groups at any time interval at P > 0.05. Holm-Sidak multiple comparisons versus 2008

following one-way ANOVA.

~ 38 ~

*

* * * **

*

3 mg/kg

Time (min)

5-10 15-20 25-30 35-40 45-50 55-60

Activity C

ou

nts

0

200

400

600

800

Vehicle

AM251

AM4113

AM6545

10 mg/kg

5-10 15-20 25-30 35-40 45-50 55-60

*

*

*

A. B.

3 mg/kg

Time (min)

5-10 15-20 25-30 35-40 45-50 55-60

Nu

mb

er

of

Re

arin

g B

ou

ts

0

20

40

60

80 10 mg/kg

5-10 15-20 25-30 35-40 45-50 55-60

* *

* * ***

C. D.

115-120115-120

115-120115-120

Time (min)

Figure 10. Locomotor activity was measured as the total number of beam breaks within a 120 min period immediately following drug administration (n = 6 per dose) or

vehicle (N = 14): “N” refers to the total number of mice used in statistical analysis. (A, B) For statistical analysis, measurements were sampled in 5 min bins at the

5-10, 15-20, 25-30, 35-40, 45-50, 55-60 and 115-120 min time intervals. (C, D) Rearing was recorded in the same manner as locomotor activity. Each data point

represents the mean ± SEM for total beam breaks or rearing bouts for 5 min at the corresponding time intervals. *, indicate significant difference from the corresponding

vehicle group at the same time interval at P < 0.05. Holm-Sidak multiple comparisons versus vehicle following one-way ANOVA.

~ 39 ~

Locomotor Activity

AM6545

Time (min)

5-10 15-20 25-30 35-40 45-50 55-60

Activity C

ounts

0

200

400

600

800Vehicle

0.3 mg/kg

1.0 mg/kg

3.0 mg/kg

5.6 mg/kg

10.0 mg/kg

18.0 mg/kg

A. Locomotor Activity

****

*

**

*

***

**

*

Time (min)

5-10 15-20 25-30 35-40 45-50 55-60

Num

ber

of R

earing

Bouts

0

20

40

60

80 B. Rearing Activity

****

*

**

***

**

115-120

115-120

Figure 11. Locomotor activity was measured as the total number of beam breaks within a 120 min period immediately following

AM6545 (n = 6 per dose) or vehicle (N = 14): “N” refers to the total