THE EFFECTS OF A CANNABINOID INDIRECT AGONIST ON SOCIAL AND NON-SOCIAL

BEHAVIOUR IN MALE MICE

A Thesis

Presented to

The Faculty of Graduate Studies

of

The University of Guelph

by

DARRYL ROBERT BANNON

In partial fulfilment of requirements

for the degree of

Master of Arts

October, 2009

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1+1 Canada ABSTRACT

THE EFFECTS OF A CANNABINOID INDIRECT AGONIST ON SOCIAL AND NON-SOCIAL BEHAVIOUR IN MALE MICE

Darryl Robert Bannon Advisor: University of Guelph, 2009 Professor E. Choleris

Cannabis use and cannabinoid drugs have been shown to affect social activity in both humans and animals. These social effects can be impairing or promoting and are possibly linked to whether the social environment is novel or familiar. The main aim of this research was to reveal some of the social and behavioural consequences resulting from enhancement of the cannabinoid tone in CD-1 mice. The indirect agonist used, URB597, showed a dose-dependent facilitation of a social recognition preference score at the middle dose of 0.1 mg/kg as well as reductions in both social recognition and social approach preferences scores at the high dose of

0.4 mg/kg. The enhancing effect of the 0.1 mg/kg dose appears limited to the 'social' aspect of social recognition since an object recognition test did not reveal any effect at that dose. The impairing effects of the 0.4 mg/kg dose on social preference scores seem to generalize to other forms of learning such as object and place recognition. An olfactory recognition test suggested that the results are not confounded by a URB597 mediated effect on olfaction. These results suggest that URB597 dose-dependently affects social and non-social cognitive behaviours in adult male mice. Acknowledgements

I'd like to acknowledge two undergraduate students, Hyo-Sung Jung and Cristi Orth for

all their hard work on both the social approach test in the home cage and the object

placement test. Without their help the results would not have been attained so rapidly.

Many other students and friends have also contributed in their own small ways to my

success - perhaps less directly, through a few simple words of encouragement for

example - but their help was no less important. A few notables include:

Lorman Ip: for his tireless banter and losing his money at poker

Christian Battista: for showing up to my thesis defense and being a good friend

Doreen Miao: for being there when I was alone

Justin Peterson: for being a good hippie and friend (I still say I should've gotten the

engineering frosh of the year award)

Robin Fraser: for hugs, and for being a lucid and supportive voice in the banshee-

esque maelstrom that is the Psychology Department

My advisor, Dr. Elena Choleris, has been instrumental in helping to see me through this

master's thesis from start to finish. While there are many demands on her time she is still able to manage her graduate students effectively so that they can be successful in their research and in her lab. My committee (Dr. Linda Parker & Dr. Herman Boermans) and chair (Dr. Lana Trick) were also very helpful and I'd like to thank them for their assistance and for reviewing my thesis. Table of Contents

TABLE OF CONTENTS I LIST OF FIGURES II LIST OF ABBREVIATIONS Ill INTRODUCTION 1

CANNABINOID SYSTEM 1 CANNABINOIDS AND SOCIAL BEHAVIOUR 5 CANNABINOIDS AND COGNITION 12 STUDIES RUN IN THE PRESENT THESIS 17 METHODS AND MATERIALS 21

ANIMALS 21 DRUGS 23 MATERIALS 23 PROCEDURES 25 Social Approach Test in Home Cage 25 Social Approach Test in Three Chamber Apparatus 25 Social Recognition Test 26 Object Recognition Test 27 Object Placement Test 28 Chocolate Chip Test 28 Olfactory Recognition Test 29 BEHAVIOURAL SCORING 30 DESCRIPTIONS OF SCORED BEHAVIOURS 31 BEHAVIOURAL SCORES 31 ANALYSIS 33 Social Approach Test in Home Cage & Social Approach Test in Three Chamber Apparatus 34 Social Recognition Test, Object Recognition Test, and Object Placement Test 35 Olfactory Recognition Test 36 RESULTS 37

SUMMARY OF STATISTICAL PARAMETERS 37 SOCIAL APPROACH TEST IN HOME CAGE 38 SOCIAL APPROACH TEST IN THREE CHAMBER APPARATUS 41 SOCIAL RECOGNITION TEST 44 OBJECT RECOGNITION TEST 48 OBJECT PLACEMENT TEST 51 CHOCOLATE CHIP TEST 54 OLFACTORY RECOGNITION TEST 54 DISCUSSION 57

MAIN FINDINGS 57 INJECTION EFFECTS 75 OVERALL CONCLUSIONS 77 OUTLOOK AND IMPLICATIONS 78 REFERENCES 80

1 List of Figures FIGURE 1: 99 FIGURE 2: 100 FIGURE 3: 101 FIGURE 4: 102 FIGURE 5: 103 FIGURE 6: 104 FIGURE 7: 105 FIGURE 8: 106 FIGURE 9: 107 FIGURE 10: 108

ii List of Abbreviations A-9-THC A-9-Tetrahydrocannabinol 2-AG 2-Arachidonoylglycerol 2-HPBCD 2-Hydroxypropyl-beta-cyclodextrin ANOVA Analysis of variance CB1 Cannabinoid-1 CB2 Cannabinoid-2 CD-1 Cluster of differentiation 1 CNS Central nervous system FAAH Fatty acid amide hydrolase FAE Fatty acid ethanolamides fMRI Functional magnetic resonance imaging FRAs Fos-related antigens GABA Y-aminobutyric acid i.p. Intra-peritoneally KO Knock out LTD Long-term depression LTP Long-term potentiation NMDA N-methyl-D-aspartic acid OEA Oleoylethanolamide PEA Palmitoylethanolamide PMSF Phenylmethylsulfonyl fluoride PNS Peripheral nervous system PPAR-alpha Alpha-type peroxisome proliferator-activated nuclear receptors s.c. Subcutaneous TRPV1 Transient receptor potential vanilloid 1 URB597 Cyclohexyl carbamic acid 3'-carbamoyl-biphenyl-3-yl ester

111 Introduction

The cannabis plant, commonly known as marihuana has been around for millennia, being used for medicinal and recreational purposes (reviewed in

Adams & Martin 1996). In recreational usage, marihuana can often be found in social group settings with subjects behaving differently when they use the drug in a group setting (Tart 1970). Indeed cannabis users report social activity as a

main reason for their cannabis use (reviewed in Green et al. 2004). Why there is such a strong linkage between social use and cannabis is not entirely clear given that cannabis use is linked with risk factors for social anxiety disorder (Buckner et al. 2008) but it could be a function of the relaxation effects of the drug (reviewed in Green et al. 2003) enabling social interaction through reduced social stress.

The interaction between the cannabinoid system and social behaviour extends to animal research. In general, it seems that the extent of the activation determines the direction of the effect of cannabinoid manipulation on social behaviour. For example, there can be a promotion or inhibition of social interaction in rat play behaviour dependent upon whether a pharmacological manipulation targeted a single receptor or whether there was a system-wide activation (Trezza &

Vanderschuren 2008a).

Cannabinoid System

l Marihuana through its constituents, such as A-9-THC, acts upon the

endocannabinoid system, in both the central nervous system (CNS) and

peripheral nervous system (PNS) (reviewed in Basavarajappa 2007; and in

Beaulieu 2005). The endocannabinoid system has two identified receptors,

cannabinoid-1 (CB1) and cannabinoid-2 (CB2) (reviewed in Zhu 2006), as well

as a number of other putative receptors, such as the recently described G-protein

coupled orphan receptors, GPR55 and GPR119 (Brown 2007; reviewed in

Pertwee 2007) and the endothelial cannabinoid receptor (Begg et al. 2005). The

CB1 receptor was the first discovered and has received the most study to date.

It is predominantly localized in the central nervous system (CNS) although it has

been shown to be present in a number of peripheral tissues such as the

gastrointestinal tract (reviewed in Svizenska et al. 2008). The CB2 receptor by

contrast was initially believed to be limited to the peripheral nervous system

(PNS) but has recently been detected in microglial cells and neurons (reviewed

in Svizenska et al. 2008).

CB1 receptors are widely distributed within the brain (reviewed in Svizenska et al. 2008). High density regions of CB1 are found in the hippocampus, olfactory

regions, caudate, putamen, nucleus accumbens, the substantia nigra pars

reticulata, globus pallidus, cerebellum and the horizontal limb of the diagonal

band. The high densities of CB1 receptors in the cerebellum can explain the ataxic, immobilizing and cataleptic effects of acute administration of A-9-THC and discrete motor learning in mice with CB1 genetically deleted (Kishimoto & Kano

2 2006). There is also high CB1 receptor expression in areas related to pain

modulation such as the periaqueductal gray and the dorsal horn of the spinal

cord (Lichtman et al. 1996). In the human and rodent cortices, CB1 receptors

are primarily found on axon terminals of cholecystokinin-8 positive GABA

interneurons (Piomelli 2003). Their placement is predominantly presynaptic

(reviewed in Freund et al. 2003; Rodriguez et al. 2001) but can be occasionally

found postsynaptically and on glia (Rodriguez et al. 2001).

There are a number of endogenous compounds that bind to cannabinoid

receptors. Anandamide (N-arachidonoylethanolamine) discovered by Devane et

al. (1992), is the main endocannabinoid targeting the CB1 receptor while 2-AG

(2-arachidonoylglycerol) found not long afterwards (Sugiura et al. 1995) primarily targets the CB2 receptor (reviewed in Svizenska et al. 2008). Other endocannabinoids include virodhamine, noladin ether, and N- arachidonoyldopamine (reviewed in Piomelli 2003). To date, A-9-THC remains the most studied among phytocannabinoids, a family of plant derived compounds that target the cannabinoid system and include cannabidiol, cannabigerol, cannabichromene among others (De Petrocellis et al. 2008).

Endocannabinoids are too lipid soluble to be stored in vesicles as they would pass right through the vesicle membranes. Hence, the brain contains only small amounts of preformed endogenous cannabinoids (reviewed in Piomelli 2003). It is suspected that the majority of endocannabinoids are released postsynaptically

3 and act as retrograde messengers at presynaptic receptors (Alger 2002; reviewed in Kreitzer & Regehr 2002). For example, in the hippocampus endocannabinoids are generated by pyramidal neurons and through retrograde inhibition of GABA release can temporarily cause upswings in pyramidal cell firing (reviewed in Meyer & Quenzer 2005). The inhibition of GABA release in the hippocampus may result in a suppression of one of the mechanisms through which dopamine controls prefrontal cortex excitability, leading to poor integration of transcortical inputs (Pistis et al. 2001). Endocannabinoid regulation has also been described in relation to various other neurotransmitters such as glutamate

(reviewed in Hashimotodani et al. 2007a), acetylcholine (Gessa et al. 1998), dopamine, and noradrenalin (reviewed in Schlicker& Kathmann 2001).

There have been a number of recently developed pharmaceutical agents that enable manipulation of the cannabinoid system. One of these, URB597, indirectly increases endocannabinoid signalling by inhibiting fatty acid amide hydrolase (FAAH), the enzyme that breaks down fatty acid ethanolamides

(FAEs) including the endogenous endocannabinoid anandamide. FAAH is intracellular^ bound and breaks down anandamide after internalization, a process believed to require a carrier-mediated transport system because of anandamide's hydrophobic nature (Fegley et al. 2004; reviewed in Glaser et al.

2005; reviewed in Piomelli 2003). Inhibition of FAAH increases anandamide levels, as well as other FAEs like oleamide, oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) (Cravatt et al. 1995). The breakdown of

4 anandamide by FAAH has been shown to be significant since the deletion of

FAAH results in a 10 to 15 fold increase in brain anandamide levels in mice

(Cravatt et al. 2001). And Fegley et al. (2005) showed that the inhibition of FAAH results in a non-specific increase in activation of the endocannabinoid system in mice that is focused primarily on CB1. This is likely due to the fact that many

FAAH-positive neurons in the CNS are found in close proximity to axon terminals that contain CB1 receptors (reviewed in Freund et al. 2003), in regions of the brain such as the cerebellum, hippocampus and neocortex where the highest densities of FAAH and of CB1 receptors are found (Egertova et al., 1998).

Cannabinoids and Social Behaviour

Social behaviour can be affected by activation or inhibition of the cannabinoid system (Frischknecht 1984; Gorzalka et al. 2008; Malone et al. 2009; Schneider

& Koch 2005; Sieber et al. 1980b; Sundram 2006; Terranova et al. 1996) and thus researchers have hoped that pharmacological manipulation of the system could be used to treat social disorders such as autism. The indirect cannabinoid agonist URB597 has been shown to mediate social behaviour by dose- dependently promoting rat play (Trezza and Vanderschuren 2008a) while a direct

CB1 agonist, WIN 55,212-2, impaired their measure of rat play. The authors explain the differing social effects of the two types of agonists by pointing out that the indirect agonist prolongs endocannabinoid signalling in already active synapses, thus preserving the spatiotemporal specificity of neuronal activations

5 during the performance of specific behaviours, while the direct agonist may non- specifically stimulate CB1 activation in additional brain areas, including those that

are inhibitory towards the social interaction being measured. In Trezza &

Vanderschuren's (2008a) study the indirect agonist would thus activate

predominantly those brain regions that are active when the rat engages in social

play. The additional areas of activation that a direct CB1 agonist could be activating could include the prefrontal cortex and hippocampus, resulting in the cannabinoid mediated impairment of working memory, social discrimination and behavioural flexibility (reviewed in Egerton et al. 2006). Trezza and

Vanderschuren's (2008a) research showing effects of FAAH inhibitors to mediate social play, a behaviour that typically changes over an animal's life span, suggests that areas of social behaviour that are relatively constant throughout an animal's life may also be affected by specific cannabinoid activation. These include social recognition and tests that assess an animal's motivation to approach a social stimulus, both of which have been shown to be affected by cannabinoid manipulation.

Social recognition is an important component of social memory which has been previously described as an animal's ability to identify and recognize other individual conspecifics (Choleris et al. 2004). Social recognition is often used in rodents to identify and characterize both normal and abnormal social behaviour

(Ricceri et al. 2007). Persistent deficits have been found in social recognition after acute and chronic administration of the CB1 cannabinoid agonist WIN

6 55,212-2 in adolescent rats, and after acute administration in adult rats

(Schneider et al. 2008). Schneider et al.'s (2008) results suggest increased

susceptibility in adolescents as compared to adult rats. Terranova et al. (1996) further showed that recognition memory for an already familiar social stimulus can be restored and memory deficits in aged rats reduced following administration of the CB1 inverse agonist SR 141716A during memory consolidation. Thus these results show that social recognition is impaired by a direct acting cannabinoid agonist and that a cannabinoid inverse agonist can

reverse social recognition memory deficits. And when the results are taken together they suggest that cannabinoid effects on social recognition occur through the CB1 receptor.

Fundamental to all social behaviour is social approach, which in rodents is normally assessed as an animal's preferential approach and investigation of a conspecific when given a choice between a social and a non-social stimulus

(Crawley et al. 2007). This measure is normally considered a measure of social motivation (Crawley et al. 2007). A related measure is sociability, the time that the test animal will spend in proximity of a stimulus conspecific. This measure has been used in rodents as a model of human disorders like autism involving social deficits (Nadler et al. 2004). In humans an earlier study on the effects of smoked marihuana on social behaviour found no increase in time spent in a social area with other subjects, but decreases in verbal interaction and increases in coaction (Foltin & Fischman 1988). Another study by the same group found

7 that smoked marihuana increased total daily social interaction time, but that this

increase was not observed in a group of individuals with very low baseline levels

(Foltin et al. 1987), suggesting an impairing effect of marihuana on sociability.

In rats the chronic effects of exposure to CP 55,940, a CB1 receptor agonist, caused a lasting reduction in social interaction (O'Shea et al. 2006) and also

reduced time in social interaction after acute administration in a familiar

environment (Genn et al. 2004). And low-dose A-9-THC (1 mg/kg) significantly

reduced social interaction between rat pairs in an open field (Malone et al. 2009).

In pairs of CB1 receptor KO mice, decreased social interaction has been found in

a social interaction test in a novel environment (Uriguen et al. 2004) while in pairs of rats injected with oleamide in a bright light version of the same test increased social interaction was seen (Fedorova et al. 2001). Oleamide is a fatty acid which is hypothesized to cause behavioural effects similar to anandamide

(Mechoulam et al. 1997). These studies suggest that social interaction as

measure by the social interaction test is impaired in rodents in both familiar and

novel environments with CB1 receptor KO mice and when cannabinoid agonists are administered. The fatty acid oleamide however, was able to increase social interaction. These studies, while not directly measuring social approach through a choice between a social and a non-social stimulus, suggest an impairing effect of cannabinoid activation on the social motivation to investigate a conspecific.

8 Overall, thus, these studies suggest an impairing effect of non-specific activation of the cannabinoid system by cannabinoid agonists on both social recognition and social motivation. Whether a specific activation of the cannabinoid system of those brain areas that are active during the performance of these social

behaviours (e.g. with URB597) would have similar effects, or have opposite effects as is the case for social play (Trezza & Vanderschuren 2008a), remains to be determined and is one objective of this thesis.

In addition to social recognition and aspects of social motivation, the cannabinoid system has been shown to affect aggression. These cannabinoid effects on aggression appear to be related to whether the study takes place in a familiar or

novel environment. For instance, in an isolation-induced aggression paradigm rats and mice after i.p. injections of A-9-THC (0.5, 2.0 and 10mg/kg) showed decreased aggressive behaviour towards conspecifics (Miczek 1978; Ten Ham &

De Jong 1974; van Ree et al. 1984), while cannabidiol (another active cannabinoid constituent of marijuana) had no effect (van Ree et al. 1984). As well, CB1 receptor knockout (KO) mice have been reported to show increased aggression in the resident-intruder test (Haller et al. 2004; Martin et al. 2002).

These results show that in isolation-induced aggression paradigms in the rodent's home cage (Miczek 1978; Ten Ham & De Jong 1974) cannabinoids reduce aggression while CB1 receptor KO mice have increased aggression in the resident-intruder test which suggests that cannabinoids have an anti- aggressive role. A number of animal studies (Cutler & Mackintosh 1975; Sieber

9 et al. 1980a) have found no association between increased aggressiveness and cannabis use, and this may be a function of the aggression being measured in a novel environment. Aggression was not affected in a novel environment by acute i.p. administration of A-9-THC (5 mg/kg and 20 mg/kg) in male rats (Cutler &

Mackintosh 1975; Sieber et al. 1980a) and mice (Cutler & Mackintosh 1975) in unfamiliar conspecific encounters. Aggression was also not affected after chronic administration (2 months) of A-9-THC (10, 20, 50 mg/kg) in cohabitating pairs of rats (Miczek 1979). CB1 receptor knockout KO mice have been reported to show decreased aggression when placed in pairs in a novel environment

(Haller et al. 2004). These studies show that cannabinoid administration has either no effect on aggression in a novel environment or in the case of CB1 receptor KO mice has the opposite effect to what is found in a familiar environment.

These studies seem to show that aggression is affected by activation of the cannabinoid system in animals and humans and can be decreased when the experimental subject's aggression level is high such as in the resident-intruder test. Stress, particularly environment-mediated stress seems capable of altering cannabinoid effects on aggression. Whether environment-induced anxiety similarly modulates the involvement of the cannabinoid system in other social behaviours, such as social approach or sociability is currently unknown and its investigation, through the use of both familiar and novel testing environments, is one objective of the present thesis.

10 The amygdala is thought to play a role in many social behaviours. Human studies have shown an amygdala involvement in evaluation of familiar versus novel faces with different valences and in the response to threatening faces

(Phan et al. 2008; Somerville et al. 2006). In rodents, the amygdala is critical to the model of social recognition as described by Chdleris et al. (2004). This model involves individual-specific social olfactory cues reaching the medial amygdala where they are processed by oestrogen mediated oxytocin mechanisms which are critical for social recognition in male and female mice

(Choleris et al. 2007; Ferguson et al. 2000; Ferguson et al. 2001). The amygdala outputs in turn facilitate social recognition and a variety of social behaviours.

While the medial amygdala is critical for social recognition, the basolateral amygdala does not appear to be involved (Maaswinkel et al. 1996). Neonatal amygdala lesions in rat pups results in decreased social play behaviour and social interaction (Daenen et al. 2002) and when combined with juvenile isolation severely disrupts social interaction (Diergaarde et al. 2004). The amygdala's is also involved in acute social defeat in mice which caused reductions in brain- derived neurotrophic factor mRNA expression in the amygdala (Pizarro et al.

2004). Furthermore intra-amygdala injections of corticotropin hormone receptor antagonist antalarmin immediately after defeat significantly reduced defensive behaviour in mice (Robison et al. 2004). Taken together, these results suggest that social stress leads to brain plasticity changes in the amygdala which is

11 consistent with social defeat leading to exaggerated fear responses and inhibition of territorial behaviour (Pizarro et al. 2004).

Cannabinoids have been shown to act on social behaviour through the amygdala which contains CB1 intemeurons in the basolateral complex (Piomelli 2003).

Older cannabinoid research indicated changes in patterns of electrical activity in the amygdala, hippocampus and septal region in monkeys under the influence of marihuana (Harper et al. 1977; Heath et al. 1980). These older studies have some methodological concerns such as very small sample sizes but also point to the amygdala as a possible site of action of cannabinoid effects on social behaviour. Together these studies strongly implicate the amygdala in various social behaviours including social recognition, avoidance, and defeat. It further appears that the cannabinoid system is involved social behaviours through the amygdala. While none of the tests conducted in this thesis assess amygdala involvement, because of the social nature of a number of the tests, amygdala activation via the cannabinoid system is a possibility.

Cannabinoids and Cognition

The long term cognitive effects of marihuana use in humans remain largely inconclusive, in part due to methodological biases such as subject inclusion criteria (reviewed in Karila & Reynaud 2003). The acute effects of marihuana in humans include impaired short-term episodic and working memory, decrements

12 in attentional performance, and psychomotor disturbances (reviewed in

Ranganathan & D'Souza 2006; Indlekofer et al. 2008; Roser et al. 2009;

reviewed in Kano et al. 2009), with heavy cannabis users being likely to suffer

less than occasional users on some tasks such as perceptual motor control and

dual task processing (Ramaekers et al. 2008), suggesting the development of

tolerance to some acute cognitive effects of THC. In general it appears that

short-term memory or working memory is vulnerable to cannabinoid agonists while retrieval of previously learned information is resistant (Kano et al. 2009).

In summary, while studies suggest decrements in learning and remembering new

information during cannabis use, some researchers argue that cannabis compounds would have an acceptable margin of safety for use as therapeutic

agents (Grant et al. 2003) in conditions such as drug dependence (reviewed in

Le Foil & Goldberg 2005; Lallemand et al. 2004) or nausea (Cross-Mellor et al.

2007; Parker et al. 2006).

In animal models the cannabinoid system has been shown to have both

improving and impairing functions in memory and learning (Arenos et al. 2006;

Mishima et al. 2001; Nakamura et al. 1991; Terranova et al. 1996) with spatial

memory being frequently found to be impaired (e.g. Boucher et al. 2009). CB1 agonists such as A-9-THC, when administered into the medial pre-frontal cortex of rats, pre-acquisition, disrupt spatial working memory in the radial arm maze

(Silva de Melo et al. 2005). Similarly, in the water maze, acquisition of spatial learning is impaired in rats with i.p. injections of the cannabinoid agonist HU-210

13 (Robinson et al. 2007) and with daily injections of A-9-THC and HU-210 (Cha et al. 2007; Ferrari et al. 1999) before task acquisition. In the object placement test of spatial memory in rats, the cannabinoid agonist WIN 55,212-2 has been found to impair performance when given intra-hippocampally pre-acquisition (Suenaga

& Ichitani 2008). Similar to what is found with pre-acquisition injections, post- acquisition injections impair spatial memory in a variety of tasks. In water maze tasks, post-acquisition injections have been found to have no spatial effect in rats with HU-210 (Robinson et al. 2007) and to impair spatial memory in mice with A-

9-THC (Varvel et al. 2001). In the radial arm maze in rats, post-acquisition intra- hippocampal injections of cannabinoid agonists CP-55,940 (Wise et al. 2009) and A-9-THC (Lichtman & Martin 1996) disrupted spatial memory and this effect was blocked by inverse agonist, SR 141716A (Wise et al. 2009), suggesting that the effect was mediated by the CB1 receptor. Accordingly, treatment with SR

141716A reversed the improved acquisition in a spatial water maze task observed in FAAH (-/-) KO mice as well as the increased acquisition and extinction of wild type mice after daily treatment with FAAH inhibitor OL-135

(Varvel et al. 2007).

In summary it appears that both pre- and post-acquisition activation of the cannabinoid system impairs spatial memory. The general impairing effects of cannabinoids on spatial memory appear to be linked to both CB1 receptors and

FAAH and the use of either FAAH (-/-) KO mice or pharmacological FAAH inhibition can improve acquisition.

14 The neural structure where cannabinoid effects on spatial learning likely occur is the hippocampus where lesions result in spatial memory impairments (Winters et al. 2004) and which has a high density of CB1 receptors in the rat brain

(Egertova et al., 1998). And indeed, the radial arm maze A-9-THC post- acquisition impairment in rats has been identified as being in the dorsal and ventral hippocampus, and possibly the dorsomedial thalamus nucleus (Egashira et al. 2002). The specific mechanism could be through the modulation of GABA release in addition to the inhibition of glutamate, acetylcholine, and dopamine, altering neuronal firing (reviewed in Ranganathan & D'Souza 2006). This modulation may take the form of changes in long-term potentiation (LTP) and long-term depression (LTD) since cannabinoid receptor activation inhibits both

LTP and LTD induction in the hippocampus (Terranova et al. 1996). One consequence of these changes is likely cannabinoid interference with old memory traces due to cannabinoid inhibition of hippocampal LTD of CA1 field potentials.

Object recognition learning is an important component of memory needed to identify familiar versus unfamiliar surroundings. It has been examined in relation to cannabinoid effects with researchers alternately finding either impairments or no effects. The impairment finding appears to relate to the timing of cannabinoid manipulation (pre-acquisition or consolidation), and modality (i.p. or s.c. versus intra-hippocampally).

15 The impairing effects of acute systemic administration of cannabinoid agonists on acute object recognition memory extend to both acquisition and consolidation phases of memory. Pre-acquisition acute exposure i.p. injection in rats of an anandamide analogue and CP 55,940 impaired short-term object recognition memory (Kosiorek et al. 2003) as did s.c. injections of WIN 55,212-2 during consolidation (Baek et al. 2009). In chronic exposure to the cannabinoid agonist

CP 55,940, object recognition memory has also been reported to be either impaired (O'Shea et al. 2006) or had no effect (Higuera-Matas et al. 2009). The explanation for these differences may lay in the degree of difficulty of the object recognition tasks with more difficult tests being more likely to show subtle impairments (Higuera-Matas et al. 2009).

The hippocampus has been shown to be involved in the long-term effects of cannabinoid activation on object recognition memory with an intra-hippocampal injection of WIN 55,212-2 during consolidation causing object recognition impairment (Clarke et al. 2008). However, hippocampal involvement does not appear to extend to short-term object recognition memory as intra-hippocampal injections of WIN 55,212-2 in rats during consolidation (Clarke et al. 2008) and pre-acquisition (Suenaga & Ichitani 2008) reveal no impairments. It has been suggested that the perirhinal-entorhinal cortex is involved in short-term recognition memory (Petrulis & Eichenbaum 2003), and lesions to the perirhinal cortex result in object recognition deficits (Winters et al. 2004), thus it is possible

16 that the effects of activation of the cannabinoid system in a short-term object recognition test would be mediated by cannabinoid receptors in this brain area.

In summary it appears that both object recognition and spatial memory is impaired with cannabinoid injections both pre- and post-acquisition. Some research has implicated FAAH inhibition in spatial memory and increased acquisition, while its effects on short-term object recognition are unknown. Given that FAAH inhibition has already been implicated in enhanced social play (Trezza

& Vanderschuren 2008a) and that no study has systematically compared its effects across social recognition, social approach, object recognition and spatial memory, its involvement in these tests has been studied in this thesis.

Studies Run in the Present Thesis

The aim of the present thesis was that of assessing the involvement of the cannabinoid system on social behaviour, recognition memory, and olfaction, thus furthering our understanding of the cannabinoid system's behavioural correlates.

To this aim the effects of URB597, an indirect cannabinoid agonist that selectively activates already active synapses were assessed in a number of social and non-social behavioural paradigms.

In order for social behaviour to occur, it is necessary for the initiating conspecific to have the appropriate motivation, without which social behaviour will either not

17 occur or be deeply affected. Most of the results of the studies in the cannabinoid literature involving social behaviour (e.g. aggression, social play, social interaction, etc.) may well be explained by effects on motivation to approach social stimuli. Yet no studies have specifically investigated the involvement of the cannabinoid system in social approach by giving the animals a choice between social and non-social stimuli. Thus, to assess preference for a social stimulus a social approach test in the mouse's home cage in which the experimental mouse was given a binary choice between social and non-social stimuli was performed. The social approach paradigm used has been confirmed to be social in nature by research comparing sniffs directed towards the novel mouse to sniffs directed towards the non-social stimulus (Crawley 2007).

Levels of anxiety in animals are known to be mediated by novel vs. familiar environments, which in turn can influence how manipulation of the cannabinoid system affects social and non-social behaviour (Haller et al. 2004; Haller et al.

2009). Therefore to elucidate environmental effects in a social context, the social approach test was also conducted in a three-chamber apparatus novel to the experimental mouse. The three chamber apparatus is similar to one previously described by Nadler et al. (2004). The two social approach paradigms taken together assessed URB597's effects on the desire of the experimental mouse to be in close proximity to the social versus the non-social (object) stimulus and how environmental anxiety modulated this relationship.

18 One other behaviour that underpins most social behaviour and interaction is social recognition. This is the ability to recognize individual conspecifics, and is necessary for discriminating friendly conspecifics from foes, identifying potential mates and diseased conspecifics, and recognizing rank within a social hierarchy

(reviewed in Choleris et al. 2009). At present two cannabinoid studies in rats directly assess the effects of cannabinoids on social recognition. Social recognition impairments were shown with the CB1 agonist WIN 55,212-2

(Schneider et al. 2008) while facilitation was seen with the CB1 inverse agonist

SR 141716A (Terranova et al. 1996). While these two studies use different pharmacological agents to assess how social recognition is affected by cannabinoids, both utilize drugs that when administered i.p. result in system-wide activation of the cannabinoid system. Targeting only a subset of cannabinoid receptors through the use of a more selective pharmacological agent might reveal different results and allow a better understanding of how social recognition facilitation or impairment occurs. Thus to investigate the results from these two studies further, a social recognition test was performed using the indirect cannabinoid agonist URB597. The procedure that was followed has been outlined by Choleris et al. (2006) using pre-acquisition injections. It was expected that a facilitatory dose-dependent relationship between URB597 and social recognition would be found, similar to what Trezza and Vanderschuren

(2008a) found with social play.

19 Social recognition memory is one specific subset of general memory and is likely to have some common neural pathways with other forms of memory. Our results for the social recognition test led to the examination of other aspects of memory to assess whether the results seen were specific or whether they would be found in other behavioural tests of memory. Two additional memory paradigms were run, a novel object recognition test and an object placement test. Our procedure involved habituation to two object stimuli followed by either a new object or a new location at test. Rats or mice are known to usually explore the novel object or novel location more than the other (Dere et al. 2007). Our intention was to parse out exactly whether object recognition or placement memory was being affected by cannabinoid system manipulation to help further interpret the social recognition test results. The expectation was that performance in the object placement test would be impaired as has been seen with direct acting CB1 agonists (Cha et al. 2007; Robinson et al. 2007; Suenaga & Ichitani 2008) as well as in the novel object recognition test as has been seen dose-dependently in short-term recall paradigms with direct acting CB1 agonists (Baek et al. 2009;

Kosiorek et al. 2003).

Social behaviour in mice, particularly recognition and social approach, is highly dependent on olfaction (Matochik 1988; Ryan et al. 2008). And regions of the olfactory bulb, olfactory nuclei and the olfactory portion of the anterior commissure are known to have dense localisation of CB1 receptors (Svizenska et al. 2008) and activation of the cannabinoid system has been shown to affect

20 odour processing (Czesnik et al. 2007). Given the importance of olfaction to

social behaviour in mice it was possible that a cannabinoid-mediated effect on

olfaction would be capable of explaining or confounding any results found in our

paradigms on social behaviour. Hence, two tests for olfaction were run, one to

assess general olfactory capabilities, and one to assess discrimination between

different olfactory stimuli.

Methods and Materials

Animals

All mice used were experimentally naive CD-1 IGS, 2-3 month old male mice

from Charles River, Quebec. There were a total of 368 experimental mice used

in the seven tests included in this thesis. The stimulus mice were castrated

males and the experimental mice were gonadally intact. The castrations were

performed by the provider. The body weight of the male experimental mice

ranged from approximately 30 to 40g. Mice were drug and test naive and were

used only once in all experiments. Food (Harlan Teklad Rodent Chow 14%) and water were available ad libitum except in the chocolate chip olfactory test as

described later. Room temperature was maintained at 23 +/- 3°C. Humidity was

maintained at approximately 50%. A reversed 12 hour day:night cycle was

maintained in the colony room with lights off at 0800. Testing was conducted during the dark phase, which since mice are nocturnal, is their active phase.

Experiments were conducted in red light since the sensitivity of mouse

21 photoreceptors at wavelengths above 600nm has been shown to be incredibly small (Hattar et al. 2003; Yoshimura & Ebihara 1996).

After arrival mice were given a one-week rest period to acclimatise to the new environment. Intact male experimental mice were individually housed and castrated stimulus mice were group housed three per cage. Dimensions of the mouse home cage are shown in figure 2. The bedding present in the home cage was corn cob bedding from Harlan Teklad with a depth of approximately 2cm.

The home cage also contained one toy, either a yogurt cup or a small plastic object under which the mouse could hide and a piece of brown paper towel which the mouse would typically make into a nest. In experiments where plexiglass cylinders were used, stimulus mice were habituated to being inside the cylinder prior to starting the experiment. This research was approved by the University of

Guelph Animal Care and Use Committee and conducted in accordance with the regulations of the Canadian Council on Animal Care (CCAC).

Stimulus mice were castrated to reduce hormone-mediated activity differences and to make the stimuli appear more uniform since as observed in social play research, the social activity of one partner determines the related activity of the other (Pellis & McKenna 1995). Intact adult male mice will normally fight when paired but when castrated before puberty do not fight (Beeman 1947) which suggests that the stressful and aggressive response of the experimental mouse will be minimized by using castrated stimuli.

22 Drugs

On the basis of a literature review (Bortolato et al. 2007; Fegley et al. 2005;

Gobbi et al. 2005; Haller et al. 2009; Holt et al. 2005; Moise et al. 2008; Naidu et

al. 2007; Patel et al. 2005; Patel & Hillard 2006; Rademacher & Hillard 2007;

Schlosburg et al. 2009; Trezza & Vanderschuren 2008a) 3 doses of the FAAH

inhibitor cyclohexyl carbamic acid 3'-carbamoyl-biphenyl-3-yl ester (URB597)

were selected 0.05 mg/kg, 0.1 mg/kg and 0.4 mg/kg. URB597 was purchased

from Sigma-Aldrich Co., Canada and dissolved in a 45% 2-hydroxypropyl-beta-

cyclodextrin (2-HPBCD) vehicle purchased from ONBIO, Richmond Hill, ON,

Canada. The three URB597 doses were run along with the vehicle (2-HPBCD)

and non-injected groups for all tests except the chocolate chip test and the olfactory recognition test, both of which are explained later. Injections were

administered intra-peritoneally (i.p.) at a volume of 10 ml/kg, one hour prior to

experimentation since it takes one hour for maximal anandamide levels to be

reached (Fegley et al. 2005). Each mouse was only injected once. Preliminary observations on a 0.05-1.5 mg/kg dose range were used to ensure the levels of the drug used in this study would not cause obvious behavioural impairments.

Materials

The dimensions for the three chamber apparatus, used in the social approach test, are shown in figure 1. The apparatus as shown in the schematic is a large

23 plexiglass rectangular enclosure subdivided into 3 distinct chambers which are interconnected by a hole in the walls dividing the chambers. The holes in the walls leading to the other two chambers could be blocked with small, thin, square, plexiglass pieces in order to confine the experimental mouse to the central chamber. These thin plexiglass pieces slotted into grooves in the middle of the plexiglass walls dividing the chambers. The dimensions for the cylinders used in both of the social approach tests, the social recognition test and the olfactory recognition test were 12.1 x 6.9 cm (outer diameter) with an inner diameter of 6.25 cm. There were three rows of twenty holes (of diameter 0.5 cm) situated in the bottom 3.2 cm of the cylinder. The holes in the base of the cylinders allow the transfer of olfactory information but not tactile contact. This method of interaction has been shown to elicit high social interest by the experimental mouse (Kudryavtseva et al. 2002). Behaviours were recorded using a Sony Handycam video camera recorder in nightshot mode, model CCD-

TRV138, placed 70cm above the apparatuses. The dimensions for the object used in the object placement test (a metal drain catch) were 1.1 by 6.3 cm (outer diameter) with an inner diameter of 3.7 cm. The objects used in the object recognition test were the a metal drain catch of the same dimensions as just described and a near-cubic translucent glass ornament of dimensions 3.8 x 4.0 x

3.9 with a triangular equilateral chip missing from one corner of dimensions 1.1 x

1.1 x 1.1 cm. The dimensions for the transparent plexiglass lid used for experiments involving the home cage were 14.2 x 35 x 1cm.

24 Procedures

Social Approach Test in Home Cage

A total of 13 experimental mice were used in each group except the 0.05 mg/kg

URB597 group in which 12 were used. All forms of environmental enrichment were removed from the experimental mouse's home cage and the wire lid was replaced with a clear lid so that the mouse could be videotaped from above. The experimental mouse was given 5 minutes to habituate to the modified home cage environment. Two empty cylinders were then placed in two of the cage corners for the experimental mouse to habituate. After 5 minutes the two empty cylinders were replaced with two new cylinders, one containing a stimulus mouse and the other a few drips of vanilla extract (diluted 1:100). The vanilla extract drips were placed on a small piece of pipe cleaner which was placed in the cylinder in a 'V shape in such a way so that the point where the extract was placed on the pipe cleaner was at the level of the holes in the base of the cylinder. This test trial lasted 15 minutes. Both the second 5 minute habituation and the 15 min test session were taped for subsequent behavioural scoring.

Social Approach Test in Three Chamber Apparatus

A total of 13 experimental mice were used in each injected group, while 15 were used in the non-injected group. A thin layer of bedding was placed throughout the apparatus prior to experimentation. The experimental mouse was first placed and confined in the middle chamber for 5 minutes to habituate. Empty cylinders

25 were then placed in the middle of the two end chambers and the plexiglass dividers removed allowing the experimental mouse to roam freely within the apparatus. The experimental mouse was given 5 minutes for this second habituation. This was followed by a 15 minute test session in which both cylinders were replaced with two new cylinders, one of them containing a stimulus mouse and the other empty. There was a brief (30 seconds to 1 minute) interlude between the second habituation and the test session during which the experimental mouse was confined to the middle chamber while the cylinders were replaced. Both the second 5 minute habituation and the test session were taped for subsequent behavioural scoring.

Social Recognition Test

A total of 12 experimental mice were used in each injected group, while 15 were used in the non-injected group. All forms of environmental enrichment were removed from the experimental mouse's home cage and the wire lid was replaced with a clear lid so that the experimental mouse could be videotaped.

Two empty cylinders were placed in two of the cage corners for 10 minutes for the experimental mouse to habituate. Two new cylinders containing stimulus mice were then placed in the same locations in the cage for a 5 minute habituation session, then removed for 15 minutes (during which clean empty cylinders were placed in the same positions). This was repeated four times. The fifth 5 minute session was the test session in which one of the stimulus mice was

26 replaced with a novel stimulus mouse. Each of the 5 minute sessions were

taped for subsequent behavioural scoring.

Object Recognition Test

A total of 12 experimental mice were used in each group. All forms of

environmental enrichment were removed from the experimental mouse's home

cage and the wire lid was replaced with a clear lid so that the mouse could be

videotaped. The objects used in this test were the glass cubes described

previously. The experimental mouse was given 10 min to habituate to the altered

environment. An object was then placed in each of two corners on the same side

of the cage for a 5 minute habituation session after which they were removed for

15 minutes. The objects were secured to the cage wall with Velcro. This was

repeated four times with the objects being washed with baking soda and Alconox

(New York) between each trial to remove olfactory cues. The fifth 5 minute session was the test session in which one of the glass cubes was replaced with a

novel object. The novel object was a metal drain catcher. It has previously been shown in our lab that when a mouse was presented with these two objects in their home cage they showed a roughly equal preference for both. Each of the 5

minute sessions were taped for subsequent behavioural scoring.

27 Object Placement Test

A total of 12 experimental mice were used in each group. All forms of environmental enrichment were removed from the experimental mouse's home cage and the wire lid was replaced with a clear lid so that the mouse could be videotaped. The objects used in this test were the metal drain catchers described previously. The experimental mouse was given 10 min to habituate to the altered environment. An object was then placed in each of two corners on the same side of the cage for a 5 minute habituation session after which they were removed for 15 minutes. The objects were secured to the cage wall with

Velcro. This was repeated four times with the objects being washed with baking soda and Alconox (New York) between each trial to remove olfactory cues. The fifth 5 minute session was the test session in which one of the metal drain catchers was moved to a new location diagonally opposite to the unmoved drain catcher. Each of the 5 minute sessions were taped for subsequent behavioural scoring.

Chocolate Chip Test

Two groups were run in this test; one was a URB597 dose at which previous effects had been seen and a vehicle (0.1 mg/kg URB597 and 2-HPBCD). A total of 12 experimental mice were used in each group. Mice were food deprived starting the night prior to experimentation and left with a tiny sample of chocolate chip (Hershey Chipits, pure semi-sweet chocolate chips) of approximate weight

0.09-0.18g, to familiarize with its flavour. Shortly before experimentation (15-30

28 minutes) mice were again given a tiny sample of chocolate chip. For the test, another tiny piece of chocolate chip was placed just beneath the bedding away from the nest area and the experimental mouse in the home cage. A timer was started as soon as the experimenter's hand was removed. The mouse was given up to 5 minutes to find the chocolate chip piece. Latency times to find the chocolate chip piece were recorded. There was both a 0.1 mg/kg URB597 group and a vehicle group. The 0.1 mg/kg URB597 group was chosen since a significant result was noted at this treatment level for the social recognition test.

Olfactory Recognition Test

Three groups were run in this test; one was a URB597 dose at which previous effects had been seen, a higher URB597 dose and a vehicle (0.1 mg/kg, 1.0 mg/kg and 2-HPBCD). A total of 11 experimental mice were used in both the vehicle and 0.1 mg/kg URB597 groups while 8 were used in the 1.0 mg/kg

URB597 group. All forms of environmental enrichment were removed from the experimental mouse's home cage and the wire lid was replaced with a clear lid so that the mouse could be videotaped. An empty plexiglass cylinder was placed in the mouse's home cage for 5 minutes prior to experimentation so that the mouse could habituate. The empty cylinder was then removed and a cylinder containing either a few drips of pure vanilla extract or pure almond extract (both

Club House brand) diluted 1:100 was inserted into the same position in the cage.

The experimental mouse was allowed to habituate to the odour for 5 minutes after which it was removed for 15 minutes and an empty cylinder inserted. This

29 was repeated for four habituation trials. This was followed by a 5 minute dishabituation test session in which the odour in the cylinder was replaced with the as yet unused odour stimulus. Each of the 5 minute sessions were taped for subsequent behavioural scoring. Half of the mice received vanilla as the habituation odour and the other half received almond.

Behavioural Scoring

Scoring was performed using Observer 3.0 & 5.0 software from Noldus

Information Technology Inc. (Virginia, U.S.). Behaviours to be scored were decided in advance based on previous observations and findings within our lab

(eg: Clipperton et al. 2008) and the mouse ethogram (Grant & Mackintosh 1963).

A period of tape watching training occurred prior to actual scoring for each test to allow the watcher time to develop a familiarity with experimental mouse behaviours in the test environment. Observers were unaware of the animal's treatment. Tapes were then visually reviewed using media software on a PC connected to a Sony Handycam (with AV cables) while the scorer simultaneously indicated which behaviours were being observed by pressing the keyboard key defined for that behaviour in Observer 3.0. The first 10 trials that were scored were rescored at the end of scoring. This was done because a certain scoring routine develops only after having scored a few trials and uniform scoring throughout all sessions was desired.

30 Descriptions of Scored Behaviours

Target social Investigation directed at the cylinder that at testing contained stimulus the novel social stimulus. Target object Active investigation of the object stimulus that at testing is stimulus novel including sniffing and biting. Vertical Non-directed movement by the experimental mouse in which Movement the front two paws are elevated off the bedding. The fore paws may be resting against the wall of the chamber or simply elevated into the air. Horizontal Activity by the experimental mouse about the experimental Movement confines with all four paws on the bedding that was not directed towards any of the social or non-social stimuli used in the experiments. Horizontal and vertical movement when taken together aid in assessing patterns of mouse activity. Grooming Scratching, pawing, licking or similar bodily maintenance by the experimental mouse. Intended to assess self- maintenance behaviour. Sitting The mouse remains in a stationary position. The mouse may be completely immobile or exhibit some sniffing while in an idle sitting position. Assesses a combination of mouse inactivity and lack of exploratory motivation. Digging Any pawing or head movements that noticeably displaced bedding. Intended to assess the repetitiveness and intensity of digging behaviour.

Behavioural Scores

The following variable definitions apply to all behavioural scores defined below.

Tc/o = time spent exploring the target cylinder or object

31 nT = time spent exploring the non-target cylinder or object hM = time spent in horizontal movement

VM = time spent in vertical movement

D = time spent digging

Social recognition preference score is the active investigation time directed at the target cylinder containing the mouse that at testing is novel divided by the total active investigation time of either cylinder. It is calculated as:

Social recognition preference score = Tc/0 / (Tc/0 + nT) * 100

Social approach preference score is the combined active investigation time directed at the target cylinder containing the social stimulus mouse divided by the total active investigation time for behaviours directed at either empty or social cylinders. It is calculated as:

Social approach preference score = Tc/0 / (Tc/0 + nT) * 100

Object recognition/placement preference score is the active investigation time directed at the target object that at testing is either novel or moved to a different location divided by the total active investigation time for behaviours directed at either object. It is calculated as:

Object recognition/placement preference score = Tc/0 / (Tc/0 + nT) * 100

32 Total object exploration time is the total time spent exploring both objects. It is

calculated as:

Total object exploration time = Tc/0 + nT

Total social behaviour time is the total time spent exploring the cylinder(s)

containing the social stimulus/stimuli. It is calculated as:

Total social behaviour time = Tc/0 + nT

Total cylinder exploration time is the total time spent exploring both cylinders.

This is equal to the total social behaviour time in the case of the social

recognition test. It is calculated as:

Total cylinder exploration time = Tc/0 + nT

Total activity time is the combined duration of all behaviours excluding the non-

locomotor behaviours of sitting and grooming. It is calculated as:

Total activity time = Tc/0 + nT + hM + VM + D

Analysis

Percent change was calculated for the tests with preference scores (all tests

except the olfactory recognition test) from habituation to test. This was calculated as:

(T-H)/(T+H)*100

33 where:

H = habituation preference score

T = test preference score

A one-way analysis of variance (ANOVA) was run on the percent change values for each test.

Social Approach Test in Home Cage & Social Approach Test in Three Chamber Apparatus

Durations and frequencies of behaviours and behavioural categories during habituation ('home cage habituation' for the Social Approach Test in Home Cage and 'three chamber habituation' for the Social Approach Test in the Three

Chamber Apparatus) and test were analyzed using one-way ANOVA. This was followed by Tukey's post hoc tests and where appropriate paired tests using t- tests. For all tests, normality and equality of variance were assessed to determine if the data distribution was parametric. When data failed the requirements for parametric statistics the non-parametric Kruskal-Wallis one-way

ANOVA on ranks was used. This was followed by post hoc comparisons using the non-parametric Dunn's test and where appropriate paired tests using the

Wilcoxon signed rank test. Separate analyses were run on habituation and test because of differing trial lengths.

In the two social approach tests the analysis of the preference score was done using a two-way repeated measures ANOVA from habituation to the test trial. A two-way repeated measures ANOVA could not be done with the durations and

34 frequencies of behaviours because of different trial lengths, however preference scores are measured as a percent of total stimuli exploration time and thus can be used as an independent variable in a repeated measure. The other independent variable was drug treatment group. This was followed by post hoc comparisons using Tukey's test. Planned apriori comparisons using paired t- tests were performed examining within group increases from social approach habituation to test and independent samples t-tests examining differences from vehicle on the test trial. When the conditions for parametric tests were violated, a

Kruskal-Wallis one-way ANOVA on ranks was used for each trial followed by the

Mann-Whitney Rank Sum Test for mean comparisons and the Wilcoxon signed rank test for paired comparisions.

Social Recognition Test, Object Recognition Test, and Object Placement Test

A two-way repeated measures ANOVA (repeated over the 4 habituations and 1 test) was used to analyze duration and frequencies of the behavioural results. In addition to the trial number, the other independent variable was drug treatment group. The five trials are referred to as social/object hab-1, -2, -3, -4 and test.

This was followed by post hoc comparisons using Tukey's test.

For the purposes of analysis of the preference scores the four habituation trials were averaged into a single value, social HAB for the social tests and object HAB for the object tests.

35 In the social and object recognition tests and the object placement test the analysis of the preference score was done using a two-way repeated measures

ANOVA from social/object HAB to the test trial. This was followed by post hoc comparisons using Tukey's test. Planned apriori comparisons using paired t- tests were performed examining within group increases from social/object HAB to test and independent samples Wests examining differences from vehicle on the test trial. When the conditions for parametric tests were violated, a Kruskal-

Wallis one-way ANOVA on ranks was used for each trial followed by post hoc comparisons using the non-parametric Dunn's test and the Mann-Whitney Rank

Sum Test for mean comparisons.

Olfactory Recognition Test

A two-way repeated measures ANOVA (repeated over the 4 habituations and 1 test) was used to analyze duration and frequency of the behavioural results. In addition to the trial number, the other independent variable was drug treatment group. The five trials are referred to as olfactory hab-1, -2, -3, -4 and test. This was followed by post hoc comparisons using Tukey's test.

The order of scent presentation for both vehicle and 0.1 mg/kg URB597 was examined across trials using a two-way repeated measures ANOVA, comparing

36 vanilla to almond scent order with almond to vanilla scent order for the same treatment group.

Results

Whenever the results of the analyses run for duration and frequency were in general agreement with each other, only the results of duration are reported.

Non-significant results are not reported unless meaningful. Drug effects are reported in comparison to vehicle while effects of the injection of vehicle per se are reported in comparison to the non-injected mice.

For brevity the names of the various statistical tests are not repeated below. The reported statistical parameters indicate from which specific test the various results were obtained.

Summary of Statistical Parameters

Statistical test Reported as:

ANOVA F

Tukey's post hoc test Q ttest t

Kruskal-Wallis test H

Dunn's test Q

Mann-Whitney Rank Sum test T

37 Wilcoxon signed rank test W

Eta Squared* rf

Cohen's d d

*Since parametric effect sizes are affected by non-normality and heterogeneity of scores (Leech & Onwuegbuzie 2002), rf values for the non-parametric Kruskal-Wallis test were calculated by transforming scores into ranks, performing a one-way ANOVA on the transformed data, then calculating n.2 values as shown by Gardner (2001).

Figures are not shown for some behaviours and behavioural scores including

digging, percent change, time spent investigating the stimulus, and sit on object.

Social Approach Test in Home Cage

Overall, when given a choice between a social and a non-social stimulus all

groups showed a preference for the social stimulus, with the 0.4 mg/kg URB597

group showing a reduced preference as indicated by a lower preference score

then all other groups (figure 3A).

The social approach preference score (figure 3A) gave no significant effects

between treatment groups but did show a significant within group increase

across trials, (F(1,59) = 157.70, p < .001, n,2 = -73). Significant increases from

home cage habituation to test were found for non-injected, (q(25) = 9.27, p <

.001), vehicle, (g(25) = 9.56, p< .001), 0.05 mg/kg URB597, (q(23) = 5.43, p<

.001), 0.1 mg/kg URB597, (g(25) = 10.01, p< .001), and 0.4 mg/kg URB597,

(g(25) = 5.56, p < .001). On the test trial the 0.4 mg/kg URB597 group had a significantly lower preference score than vehicle, (f(25) = -2.25, p < .05, d= .92),

38 than 0.05 mg/kg URB597 group, (7(24) = 197.00, p< .05), and than 0.1 mg/kg

URB597 group, (7(25) = 237.00, p< .01).

The percent change from home cage habituation to test based on preference

scores was nearly significant, (H(4) = 8.97, p = .062, q2 = .14) due to the 0.05

mg/kg URB597 having significantly less percent change than 0.1 mg/kg URB597,

(7(24) = 104.00, p < .01) and then vehicle, (1(23) = -2.23, p< .05, d= .95). This

appears to be due to a higher baseline at habituation for the 0.05 mg/kg on the

social approach preference score (figure 3A).

The main factor of treatment was significant for social behaviour time (figure 4A) for habituation, (F(4,59) = 2.77, p < .05, rv2 = -16) and for test, (F(4,59) = 6.57, p <

.001, n2 = -31). The effect on the test trial was explained by the non-injected

mice engaging in significantly more social time than vehicle, (g(25) = 4.29, p <

.05).

The treatment factor for total cylinder (social & non-social) home cage investigation was significant for habituation, (F(4,59) = 4.90, p < .01, n2 = -25) and test, (F(4,59) =7.43, p < .001, rf = .33). The test trial difference was explained by the non-injected group showing significantly more investigation than vehicle, (g(25) = 4.78, p < .05) and reflects the results of the higher investigation of the social cylinder since the vehicle injected group was not different from the non-injected group in investigation of the non-social cylinder.

39 Total activity time (figure 4B) had a significant effect for the factor of treatment on

both home cage habituation, (H(4) = 16.01, p < 0.01, n2 = -25) and test, (H(4) =

15.68, p < .01, n2 = -25). No significant differences were found when binary

comparisons were performed.

There was a significant effect of treatment on horizontal movement time (figure

4D) for home cage habituation, (H(4) = 21.20, p < .001, q2 = .34), and treatment

at test, (F(4,59) = 7.86, p < .001, rf = .35) which is explained by the non-injected

group having significantly less horizontal movement time than vehicle, (q(25) =

4.78, p < .05). A significant effect of treatment on vertical movement time (figure

4C) for home cage habituation, (F(4,59) = 10.89, p < .001, n2 = -42) and test,

(F(4,59) = 5.09, p < .001, q2 = -26) were found and was attributable to higher

levels of vertical movement time in the non-injected group than in the vehicle group (g(25) = 5.79, p< .01).

Total digging time was not significantly affected by treatment for home cage

habituation, but was at test, (F(4,59) = 3.32, p < .05, n2 = -18) as non-injected

mice performed significantly more digging than vehicle, (q{25) = 4.19, p < .05).

Sitting (figure 4F) duration levels showed a significant effect of treatment for

home cage habituation, (H(4) = 13.95, p < .01, n2 = -22), and test, (H(4) = 13.22, p < .05, n2 = -21) while no effects for grooming (figure 4E) were found.

40 Social Approach Test in Three Chamber Apparatus

In the social approach test in the three chamber apparatus all groups showed a significant preference (figure 3B) for the target social stimulus but there was no significant differences between any of the groups.

No statistically significant effects were found between treatment groups for the social approach preference score (figure 3B). However, a significant within group increase in preference score from three chamber habituation to test was found (F(1,62) = 79.10, p < .001, n2 = -56) for all groups, for non-injected, (q(29)

= 4.44, p < .01), vehicle, (q(25) = 5.61, p < .001), 0.05 mg/kg URB597, (g(25) =

5.89, p< .001), 0.1 mg/kg URB597, (g(25) 5.68, p< .001), and 0.4 mg/kg

URB597, (q/(25) = 6.42, p < .001). This indicates that all groups preferred to investigate a social stimulus over an empty cylinder.

Total cylinder exploration time (social & non-social) showed a significant treatment effect for three chamber habituation, (H(4) = 12.95, p < 0.05, n,2 = -20); and test, (H(4) = 11.22, p < .05, q2 = -17) with the non-injected group showing significantly more total cylinder exploration time than vehicle on the three chamber habituation trial, (0(27) = 2.81, p < .05); as well as on the test trial,

(0(27) = 3.12, p < .05). This appears to reflect greater investigation of the non- social, but not of the social stimulus in the non-injected mice.

41 Total social behaviour time (figure 5A) had a significant main effect of treatment on the three chamber habituation trial, (F(4,62) = 2.89, p < .05, rj2 = -16), but not the test trial. Exploration time of the non-social cylinder showed a significant main effect of treatment on the test trial, (F(4,62) = 7.77, p < .001, q2 = -33) which was explained by the non-injected group investigating the non-social cylinder significantly more than the vehicle group, (q(27) = 7.35, p < .001). The time spent by the experimental mouse in the chamber that at test contained the target social stimulus was examined and no statistical significances were found for either trial.

Total activity time (figure 5B) showed significant effects of treatment at test, (H(4)

= 17.81, p< 0.001, n2 = -27), with the non-injected group showing significantly higher activity time than vehicle, (0(27) = 3.50, p < .05).

In addition to total activity, total number of chamber entries was assessed as a measure of activity. Significant main effects of treatment were found for both three chamber habituation, (F(4,62) = 3.77, p < .01, r\2 = .20), and test, (F(4,62) =

3.16, p < .05, n2 = -17). The significant effect on the test trial was due to the non- injected group having significantly more chamber entries than vehicle, (g(27) =

4.17, p<.05).

42 Total vertical movement time for all chambers (figure 5C) showed a significant effect of treatment for both three chamber habituation, (F(4,62) = 5.57, p < .001,

H2 = .26), and test, (F(4,62) = 5.55, p < .001, if = -26). The non-injected group had significantly less vertical movement time than vehicle, (q(27) = 5.69, p < .01) during habituation. Vertical movement time in the social chamber was significantly affected by treatment for both three chamber habituation, (F(4,62) =

4.96, p < .01, n2 = -24), and test, (F(4,62) = 2.87, p < .05, rf = .16). Vertical movement time in the non-social chamber showed significant effect for the main factor of treatment for three chamber habituation, (F(4,62) = 3.56, p < .05, n2 =

.19).

Horizontal movement time (figure 5D) showed a significant main effect of treatment for three chamber habituation, (F(4,62) = 2.93, p < .05, r)2 = -16). Total horizontal movement time in the non-social chamber and total horizontal movement in the social chamber both showed no significant statistical results.

Total grooming time (figure 5E) for each trial revealed a significant effect of treatment for test, (H(4) = 13.45, p < .01, rf = .20), due to the fact that the non- injected group had significantly lower grooming time than vehicle, (Q(27) = 3.12, p < .05). Total grooming time in the non-social chamber only for both trials and total grooming time in the social chamber only for both trials had no findings of statistical significance.

43 Treatment significantly affected sitting duration (figure 5F) at test, (H(4) = 21.92,

p < .001, n2 = -33), with the non-injected group showing significantly lower sitting

time than vehicle, (0(27) = 3.03, p < .05) and the 0.05 mg/kg URB597 group

sitting significantly less than both vehicle, (0(25) = 2.91, p < .05); and 0.4 mg/kg

URB597, (0(25) = 3.26, p < .05). Sitting time in the social chamber showed a

significant effect for the test trial, (H(4) = 12.11, p < .05, n2 = .18) with the 0.05

mg/kg URB597 group being significantly less inactive than the 0.4 mg/kg

URB597 group, (Q(25) = 2.92, p < .05) while sitting time in the non-social

chamber gave no statistically significant results.

Social Recognition Test

The results for the social recognition test show a dose-dependent improvement

in the social recognition preference score (figure 3C) with maximal effect at 0.1

mg/kg URB597. There was also a reduced preference score at the high dose of

0.4 mg/kg URB597 relative to the 0.1 mg/kg URB597 dose. The 0.4 mg/kg

URB597 group was the only group to fail to show a significant increase in

preference score from habituation to test. The 0.05 mg/kg URB597 group also

had significantly higher sitting time and lower social investigation than all other

groups.

The social recognition score (figure 3C) from hab-1 through hab-4 showed no

main effects for treatment, trials, or the interaction showing that social

44 preferences remained stable during the habituation phase. Conversely the social recognition score (figure 3C) for the test trial showed a significant main effect for trial (F(1,58) = 37.50, p < .001, rf = .39), and a significant treatment x trial interaction (F(4,58) = 3.06, p < .05, q2 = -17). Significant increases in the social recognition score from social HAB to test were found in non-injected mice, (g(14)

= 3.81, p < .01); and in mice that had received vehicle, (g(11) = 2.90, p < .05);

0.05 mg/kg URB597, (q(11) = 3.37, p < .05); and 0.1 mg/kg URB597, (g(11) =

7.96, p < .001) but not in the 0.4 mg/kg URB597 group which showed no social recognition. Apriori binary comparisons for the test trial revealed a significantly higher 0.1 mg/kg URB597 group than vehicle, (T(12) = 112.00, p< .05) and than

0.4 mg/kg URB597, (/(22) = 3.65, p < .01, d = 1.56) suggesting an improving effect of 0.1 mg/kg URB597 on social recognition.

Total cylinder investigation time (figure 6A), which in this experiment is equivalent to total social behaviour time, showed significant main effects for treatment,

(F(4,58) = 17.47, p < .001, if = -55) and for trial, (F(4,231) = 3.00, p < .05, rf =

.05) and that social hab-3 had significantly less total cylinder investigation time than the test trial, (g(1) = 4.02, p < .05). Overall the non-injected group had significantly more investigation time than the vehicle group, (q(26) = 5.43, p <

.01); and the 0.05 mg/kg URB597 group investigated the two cylinders significantly less than vehicle, (q(23) = 5.87, p< .01), 0.1 mg/kg URB597, (q{23)

= 7.77, p < .001) and 0.4 mg/kg URB597, (g(23) = 5.65, p < .01) groups,

45 suggesting a reduction in motivation to approach the social stimuli in the 0.05 mg/kg treated mice.

Total activity time (figure 6B) showed significant main effects for treatment,

(F(4,58) = 8.70, p < .001, rf = .38) and trial, (F(4,231) = 5.89, p < .001, rf = .09) with the 0.05 mg/kg URB597 group having significantly less total activity time than vehicle, (q(23) = 4.74, p < .05). Overall, the mice showed significantly more total activity time during the first habituation trial than on social hab-3, (g(1) =

4.81, p < .01); social hab-4, (q(1) = 6.22, p < .001); and test, (g(1) = 4.53, p <

.05), indicating heightened arousal when they were first presented with the two social stimuli.

Vertical movement time (figure 6C) was also significantly affected by treatment,

(F(4,58) = 9.31, p < .001, rf = .39) and trial, (F(4,231) = 4.15, p < .01, if = -07) with the non-injected group having significantly higher vertical movement time than the vehicle group, (g(26) = 5.75, p < .01) and the 0.1 mg/kg URB597 group having significantly higher vertical movement time than the 0.05 mg/kg URB597 group, (g(23) = 4.91, p < .01). Overall, the mice showed significantly greater vertical movement time on social hab-1 than both social hab-4, (g(1) = 4.07, p <

.05); and test, (q(1) = 5.07, p < .01), again suggesting increased arousal at social hab-1 when they were first exposed to the two social stimuli.

46 Horizontal movement time (figure 6D) was significantly affected by treatment,

(F(4,58) = 17.61, p < .001, rf = .55), with the non-injected group having

significantly lower horizontal movement time (this is the reverse of the vertical

movement time) than the vehicle group, {q(26) = 6.58, p < .001) and the 0.05

mg/kg URB597 group having significantly higher horizontal movement time than

all injected groups: vehicle, (

(g(23) = 7.40, p< .001); and 0.4 mg/kg URB597, (q{23) = 5.02, p< .01).

Grooming (figure 6E) duration showed significant main effects for both treatment,

(F(4,58) = 2.69, p < .05, rf = .16), and trials, (F(4,231) = 3.96, p < .01, n2 = .06)

and that social hab-4 had significantly higher grooming than social hab-1, (q(1) =

4.63, p < .01); and social hab-2, (g(1) = 4.10, p < .05). The 0.1 mg/kg URB597

group, performed significantly more grooming during social hab-4 than social

hab-1, (g(1) = 5.07, p < .01) and test, (Q(1) = 4.56, p < .05). This gradual overall

increase in grooming up to hab-4 in the 0.1 mg/kg URB597 group which declines

again at test seems to parallel a similar but not significant opposite change in

vertical activity (figure 6c).

Treatment significantly affected total digging time as a main factor (F(4,58) =

3.66, p < .05, n2 = -20) and in interaction with trial (F(16,231) = 2.56, p < .01, rj2 =

.15) with the 0.4 mg/kg URB597 group showing significantly more total digging time than both vehicle, (g(23) = 4.05, p < .05); and the 0.05 mg/kg URB597 group, (q(23) = 5.11, p<.01).

47 Sitting time (figure 6F) showed significant main effects for both treatment,

(F(4,58) = 7.03, p < .001, rf = .33), and trials, (F(4,231) = 2.68, p < .05, if = -04) with the 0.05 mg/kg URB597 group sitting for significantly longer than all other injected groups: vehicle, (q(23) = 4.04, p < .05); 0.1 mg/kg URB597, (qr(23) =

5.98, p < .001); and 0.4 mg/kg URB597, (g(23) = 5.27, p < .01). Also, the mice showed significantly lower sitting during social hab-1 than both social hab-4, (g(1)

= 3.95, p < .05); and test, (qr(1) = 4.01, p < .05).

Object Recognition Test

The results for the object recognition test show a lack of learning on the object preference score measure for the 0.05 mg/kg and 0.4 mg/kg URB597 groups while the other groups showed normal learning (figure 3D). This seems to suggest a dose-dependent impairment of object recognition by URB597.

Analysis of object HAB and test trial preference (figure 3D) revealed a significant main effect for the trial factor, (F(1,52) = 35.14, p < .001, n2 = -40) and also showed significant increases in the preference for the novel object at test for non- injected, (/(11) = -6.95, p < .001, d = 2.96); vehicle, (/(11) = -2.86, p < .05, d =

1.22); and 0.1 mg/kg URB597, (t{9) = -3.91, p < .01, d = 1.84) but not in the 0.05 and 0.4 mg/kg URB597 treated mice. There were no significant differences between groups when examining the pair-wise comparisons on the test trial.

48 However, the lack of a significant increase in the preference for the novel object at test in the 0.05 and 0.4 mg/kg treated mice suggests a learning impairment in these two groups.

Analysis of the total object exploration time (figure 7A) showed that both the factors, treatment, (F(4,55) = 7.39, p < .001, if = -35) and trial, (F(4,220) = 5.95, p < .001, n2 = -10) were significant and that there was higher total object investigation by the non-injected group as compared to vehicle, (q(23) = 5.74, p <

.01). The total object investigation time during hab-1 was significantly higher than object hab-3, (g(1) = 5.22, p < .01); and object hab-4, (g(1) = 5.82, p <

.001). As well, the test trial had significantly higher total object investigation time than object hab-4, (q(1) = 4.11, p < .05). This 'u'-shaped pattern suggests a decline in the motivation to investigate the two objects over the habituation phase which is then increased during the test phase when one of the two objects is novel.

The time spent investigating the target object mirrored the pattern of total object exploration time with significant treatment, (F(4,55) = 6.84, p < .001, rf = .33) and trial, (F(4,220) = 7.88, p < .001, rf = .13) factors, and with much higher target object investigation by the non-injected group as compared to vehicle,

(g(23) = 5.57, p < .01). The target object investigation time like the total object investigation time shows a 'u'-shaped time course with the bottom of the 'u' occurring on object hab-4. The test trial had significantly higher object of interest

49 investigation time than object hab-4, (q(1) = 6.71, p < .001); object hab-3, (g(1) =

6.45, p< .001); and object hab-2, (g(1) = 5.07, p< .01). Object hab-1 was

almost significantly higher than object hab-4, (g(1) = 3.83, p = .053). This

habituation/dishabituation curve is common in tests of recognition in mice (see

Choleris et al. 2003; Choleris et al. 2006; Mumby et al. 2007; Palchykova et al.

2006).

Total activity time (figure 7B) across all 5 trials showed a significant main effect for trial, (F(4,220) = 7.62, p < .001, n.2 = .12) and significant decreases across trials in both the non-injected group and the 0.4 mg/kg URB597 groups. For the

non-injected group object hab-3, object hab-4, and test had significantly less activity time than object hab-1: object hab-3, (g(1) = 4.45, p < .05); object hab-4,

(g(1) = 4.32, p < .05); and test, (q(1) = 3.96, p < .05). In the 0.4 mg/kg URB597 group object hab-4, (g(1) = 4.60, p < .05); and test, (g(1) = 4.32, p < .05) had significantly lower activity time than object hab-1. These patterns parallel the 'u'- shaped changes observed in object investigation and vertical activity (below).

Vertical movement time (figure 7C) showed significant main effects for both the factors, treatment, (F(4,55) = 2.98, p < .05, n2 = -18) and trial, (F(4,220) = 5.48, p

< .001, n2 = -09) and had significantly lower vertical time at object hab-4 than at object hab-1, (g(1) = 5.24, p< .01); object hab-2, (g(1) = 5.01, p< .01), and object hab-3, (qr(1) = 5.11, p< .01). The vehicle group also had a significant

50 decrease in vertical movement time from object hab-3 to object hab-4, (g(1) =

4.17, p<.05).

Horizontal movement time analysis (figure 7D) revealed a significant main effect for the treatment factor, (F(4,55) = 12.31, p < .001, if = .47) with the non-injected group being significantly lower than vehicle (q(23) = 7.71, p < .001).

Grooming (figure 7E) and sitting time (figure 7F) showed significant effect for trial

(grooming; F(4,220) = 3.45, p < .01, if = 06 and sitting; F(4,220) = 3.75, p < .01,

H2 = .06). The hab-1 trial had significantly less sitting time than test, (g(1) = 4.13, p < .05) and more grooming time than object hab-3, (g(1) = 4.93, p < .01).

Sit on object time was significant for both the treatment, (F(4,55) = 5.97, p < .001,

H2 = .30), and trial, (F(4,220) = 3.00, p < .05, n2 = -05), with the non-injected group spending more time sitting on the objects than the vehicle group (g(23) =

5.47, p < .01). This increased sit on object time partly explains the significantly lower horizontal movement time in the non-injected group. The object hab-3 trial had significantly more sit on object time than object hab-1, (g(1) = 4.58, p < .05) and this was largely explained by the non-injected group which had significantly higher sit on object time than vehicle on object hab-3, (q(23) = 4.82, p < .01).

Object Placement Test

51 In the present experiment object placement as measured by the object

placement preference score appears to be impaired by URB597 as no significant

increases from habituation to test was found in the treated mice (figure 3E) while

the vehicle dose did show an increasing trend on the object placement

preference score. This effect appears specific for the placement score as other

behaviours were largely unaffected by URB597.

Apriori comparisons revealed a trend for the vehicle treated group to show

increased preference for the novel object at test, (/(11) = -1.92, p = .08, d= .82)

(figure 3E). In all other groups there was no increase in the preference score

from object HAB to test, indicating that the vehicle treated group was the only

one to show spatial learning, albeit marginal, in this task.

The actual time spent investigating the target object (figure 8A) gave a significant

treatment effect, (F(4,55) = 7.38, p < .001, n,2 = -35), showing greater target

object investigation in the non-injected group relative to vehicle, (g(23) = 5.50, p

< .01). The results for the total object exploration time closely mimicked the

results for the time spent investigating the target object. The treatment factor was significant, (F(4,55) = 6.70, p < .001, q2 = -33) with the non-injected group

showing more total object exploration time than vehicle, (q(23) = 4.92, p < .01).

Total activity time (figure 8B) changed over trials, (F(4,220) = 2.97, p < .05, rf =

.05) with activity during the object hab-4 trial being significantly lower than at hab-

52 1, (g(23) = 3.86, p < .05); and hab-2, (g(23) = 3.97, p < .05). This decrease in

activity over trials was similarly seen in vertical movement time (figure 8C), trials,

(F(4,220) = 3.18, p < .05, rf = .05), treatment, (F(4,55) = 2.44, p = .057, n.2 = -15),

with the object hab-2 trial being significantly higher than in hab-4, (g(23) = 4.18, p

< .05) and in the test trial, (q(23) = 4.41, p < .05).

There was a significant treatment effect for horizontal movement time (figure 8D),

(F(4,55) = 5.17, p < .001, (f = .27), with the non-injected group having

significantly lower horizontal movement time than vehicle, (g(23) = 5.17, p < .01).

Total digging time had a significant trial, (F(4,220) = 8.54, p< .001, rf = .13) as

well as treatment x trial factor interaction, (F(16,220) = 1.79, p < .05, n,2 = .12).

Overall, both the object hab-4 trial and the test trial were significantly lower than

both the hab-1 (g(23) = 6.04, p < .001) and hab-2 (g(23) = 6.18, p< .001) trials.

The test trial was lower than hab-1, (g(23) = 5.48, p < .01); and hab-2, (g(23) =

5.62, p < .001). The significantly lower digging times on object hab-4 and the test

trial are due to significant decreases in digging across trials by the non-injected, the 0.1 mg/kg URB597 and the 0.4 mg/kg URB597 groups. For the non-injected

group, object hab-1 was significantly higher than test, (g(1) = 6.47, p < .001); than object hab-4, (g(1) = 6.12, p< .001); and object hab-3, (g(1) = 5.52, p<

.001). For the 0.1 mg/kg URB597 group, object hab-4 was significantly lower than object hab-2, (g(1) = 4.71, p < .01); and object hab-1, (g(1) = 4.62, p < .05).

53 For the 0.4 mg/kg URB597 group, test was significantly lower than object hab-2,

(0(1) = 4.39, p<.05).

The factor of trials was significant for grooming (figure 8E), (F(4,220) = 4.95, p <

.001, n2 = -08) with grooming time spiking at object hab-4 which had significantly

higher grooming time than object hab-1, (q(23) = 5.62, p < .001); and object hab-

2, (g(23) = 5.14, p < .01). Sitting time (figure 8F) showed no significant effects.

Total sit on object duration showed a significant treatment effect (F(4,55) = 2.98, p < .05, q2 = -18). The lower horizontal movement time seen in the non-injected group appears to be offset to some extent by the higher sit on object time and vertical movement time.

Chocolate Chip Test

The independent samples t-test run on the latency times for chocolate chip test

(figure 9) found no significant difference between treatment groups.

Olfactory Recognition Test

In the olfactory recognition test it was found that there was no difference in stimulus investigation at test (figure 10A) between the 0.1 mg/kg URB597 dose and vehicle . There does appear to be an experimental design issue as a

54 decrease in stimulus investigation across habituation trials followed by an increase at test was expected in this paradigm since mice typically express a preference and increased exploration for a novel olfactory stimulus (Smith et al.

2009). However, only a trend towards an increased stimulus investigation was found at test in the vehicle group.

Odour cylinder investigation significantly changed over the habituation trials

(figure 10A), (F(3,81) = 2.91, p< .05, rf = -10) which was due to a near significant decrease in cylinder investigation time from the olfactory hab-3 trial to olfactory hab-4, (g(1) = 3.65, p = .055). There was also a trend for a significant increase in cylinder investigation (figure 10A) from habituation to test, (F(1,27) =

3.26, p = .082, n2 = -11) which was due to a nearly significant increase in cylinder investigation time by the vehicle group from olfactory hab-4 to test, (g(1) = 2.68, p

= .069). No effect of the order of presentation of the vanilla and almond scents was found. The overall habituation/dishabituation pattern is consistent with other similar paradigms (see Choleris 2003). However, the lack of this pattern in most groups except for the trend in the vehicle treated mice indicates that the task as run in the present study was not sufficient to reliably detect olfactory recognition.

Total activity time (figure 10B) had a significant treatment factor, (F(2,27) = 5.00, p < .05, n2 = -27) due to the vehicle, (g(21) = 3.85, p < .05); and 1.0 mg/kg

URB597, (g(18) = 3.79, p < .05) groups had significantly less activity time than the 0.1 mg/kg URB597 group.

55 Sitting time (figure 10F) had a significant main effect for treatment, (F(2,27) =

6.03, p < .01, if = -31) with the vehicle, (q^) = 3.88, p < .05); and 1.0 mg/kg

URB597 groups, (g(18) = 4.46, p < .05) sitting for longer than the 0.1 mg/kg

URB597 group. The results for sitting time and activity time may explain one another since the time spent by the test groups in active behaviour is reversed for sitting behaviour.

The trial factor for vertical movement time (figure 10C) was significant, (F(4,108)

= 2.94, p < .05, n2 = 10) and showed that olfactory hab-4 was almost significantly lower than olfactory hab-2, (qr(1) = 3.80, p = .062).

Horizontal movement time (figure 10D), total grooming time (figure 10E), total digging time, object directed digging time and object non-directed digging time revealed no significant findings.

Summary of Results

0.1 mg/kg URB597 Social Recognition Score f

- Facilitated relative to vehicle

Object Placement Score <-•

- Possible impairment relative to vehicle

Olfactory Stimulus Investigation <-•

56 - No effect or possible impairment

0.4 mg/kg URB597 Social Approach Home Cage Score l

- Impaired relative to vehicle

Social Recognition Score [

- Impaired related to 0.1 mg/kg URB597 dose

Object Recognition and Placement Scores «->

- Possible impairment relative to vehicle

0.05 mg/kg URB597 Object Recognition and Placement Scores <->

- Possible impairment relative to vehicle

Vehicle Object Placement Score <->

- Possible improvement relative to non-injected

Vertical Movement J,

- Impaired relative to non-injected

Horizontal Movement, Grooming, and Sitting |

- Facilitated relative to non-injected

Discussion

Main Findings

The doses of URB597 used in this thesis have resulted in a variety of effects across the behavioural tests examined herein. The data shows that CD-1 mice

57 given 0.1 mg/kg URB597 one hour pre-test have improved social recognition

(figure 3C) as measured by the social recognition preference score. Social

approach in both novel (figure 3B) and familiar (figure 3A) environments as well

as object recognition (figure 3D) and olfaction (figure 10A) is unaffected relative

to vehicle at this dose. The improving effects of the 0.1 mg/kg URB597 dose on

social recognition were not a result of an olfactory facilitation in general olfactory

ability (figure 9) or in olfactory discrimination (figure 10a). Taken together these

results with URB597 at 0.1 mg/kg indicate a specific effect on social recognition.

A second main result of the present thesis is that the high dose of 0.4 mg/kg

URB597 impaired social (figure 3C) and object recognition (figure 3D) as well as

social preference in the home cage social approach test (figure 3A) without

affecting activity for these tests (figures 4B, 6B, and 7B). Spatial recognition of a

novel placement of a familiar object also appears to be impaired at all URB597 doses relative to vehicle (figure 3E). These effects appear to be relatively specific to recognition learning and aspects of social motivation as activity is

unaffected.

The low dose of 0.05 mg/kg URB597 impaired a number of behaviours including social investigation (figure 6A) in the social recognition test, the object

recognition preference score (figure 3D), and percent change in social approach from habituation to test in the home cage. These impairing effects may extend to spatial recognition in the object placement test; however the 0.05 mg/kg dose

58 was not significantly different from other URB597 doses (figure 3E). Because the

0.05 mg/kg URB597 dose was not used in the olfactory recognition test, it is

unclear whether these impairing effects were olfactory-mediated. However, the

fact that similar impairments were seen at both the lower 0.05 and higher 0.4

mg/kg URB597 doses makes olfactory-mediation an unlikely explanation.

Taken together, these results reveal a dose-dependent pattern for social

recognition and suggest it for object recognition with a maximal URB597 effect at

0.1 mg/kg (figures 3C, 3D). Indeed, the main finding of this thesis, that social

recognition is facilitated at the middle dose of 0.1 mg/kg URB597 is the dose at which other researchers report significant findings of anxiety and exploration

related effects (Gobbi et al. 2005; Naidu et al. 2007; Patel & Hillard 2006). To the best of our knowledge, this is the first study investigating the effects of

URB597 on social recognition however the dose-response found in our results is consistent with Trezza & Vanderschuren's (2008a) results which indicated a max

URB597 facilitation effect for social play in rats at 0.1 mg/kg. Social exploratory behaviour and locomotor activity were not affected at this dose in their study suggesting a specific effect just like our social recognition results. Thus the inverted 'U'-shaped dose response that our results on social recognition show with maximal effects at the middle dose is consistent with the literature.

Research has shown that acute treatment with a CB1 agonist, WIN 55,212-2 has impairing effects on social recognition (Schneider et al. 2008), while a CB1

59 antagonist, SR 141716A had improving effects and reversed the effects of an

agonist (Terranova et al. 1996). Hence, these studies agree in showing that the

generalized activation of all CB1 receptors inhibits social recognition. Conversely

in the present study, URB597, an indirect agonist that activates predominantly

already active synapses, shows improving effects on social recognition at the 0.1

mg/kg dose. Therefore our results, together with those of Terranova et al. (1996)

and Schneider et al. (2008) show that while the acute activation of those

synapses that are active during social recognition improve this type of learning, the generalized acute activation of all CB1 receptors in the brain impairs it. This

impairing effect is possibly due to the action of cannabinoid agonists acting on

areas of the brain that are not specifically involved in social recognition, but that

may still affect it by influencing other cognitive mechanisms that are needed for social recognition such as those involved in memory formation, attention, and sensory processing (reviewed in Insel & Fernald 2004).

Several other experimental factors could explain why our results suggest that the cannabinoid system can promote social recognition while others' results indicate that it is inhibitory towards social recognition and memory. These factors include the animal species used, memory phase in which drug injection is given, experimental methodology and the possible URB597-mediated activation of other

receptors. The first possible explanation, species differences between studies, seems an unlikely explaining factor as Terranova et al. (1996) found similar experimental results with both rats and mice. The timing of the CB1 generalized

60 agonist, which was pre-acquisition in Schneider et al. (2008), and during consolidation in Terranova et al. (1996) produced results that are consistent with each other, suggesting that the memory phase in which the drug injection is given is a possible but unlikely explanation for the different URB597 results.

There are also differences in methodologies between Terranova et al.'s (1996) study which involved a repeated habituation procedure and our own which utilized a repeated habituation followed by a dishabituation choice test. It has been shown by Borelli et al. (2009) through Fos-immunoreactivity that repeated habituation and habituation-dishabituation procedures have significant neural activation differences. However, the different behavioural protocols used cannot explain the results with the generalized agonist WIN 55,212-2 and the blockage of its impairing effects by SR 141716A found by Terranova et al. (1996) suggesting these also cannot explain our findings. And while the levels of other

FAEs are being increased by URB597 (Fegley et al. 2005), as well as some of anandamide's effects being mediated by TRPV1 (Zygmunt et al. 1999), the blockage of WIN 55,212-2's effects by SR 141716A makes the meaningful activation of non-cannabinoid receptors unlikely.

Overall these results suggest that URB597 at 0.1 mg/kg when acutely administered facilitates social recognition while direct agonists impair it. Other factors while possibly contributing to our findings cannot fully explain them.

Because the experimental conditions and procedures of one study are rarely fully duplicated, further research could be conducted to see whether both URB597

61 and SR 141716A enhance social recognition to the same extent when tested in

the same behavioural paradigm.

The mechanisms of action of the social recognition enhancing effects of the 0.1

mg/kg dose of URB597 might be found in the results of Trezza & Vanderschuren

(2008a). They found that the dose-dependent enhancing effects of morphine on social play are attenuated by SR 141716A, WIN 55,212-2's inhibitory effects on social play are blocked by naloxone and URB597's enhancing effect on social

play can be augmented by morphine and blocked by naloxone. Thus we can

expect there to be crosstalk between the cannabinoid and opioid systems for social behaviour. And since u-opioid receptors but not d- or K-opioid receptors enhance social play (Vanderschuren et al. 1995) and the opioid heroin can enhance social recognition (Levy et al. 2009) it may be the case that a common social signalling cascade is activated through both the u-opioid receptors and

CB1 receptors.

Two main brain systems have been identified as the site of this social signalling cascade and the mechanisms of action and may be involved in the observed effects of URB597 on social recognition. One possibility is endocannabinoid mediated effects in the anterior piriform cortex, the CA1 and CA3 regions of the anterior dorsal hippocampus, anterior and posterior dentate gyrus, and perirhinal cortex, all of which have been implicated in individual recognition within an aggressive context (Lai et al. 2005) and have dense CB1 receptor localisation

62 (Svizenska et al. 2008). Other research suggests that the perirhinal-entorhinal cortex is critical for individual recognition memory in a sexual context but not in an olfactory recognition test (Petrulis & Eichenbaum 2003). These researchers have shown in a habituation-discrimination test with male hamsters using glass plates with vaginal secretions that while the perirhinal-entorhinal cortex is needed for acute recognition, the hippocampus is not. Borelli et al. (2009) using Fos immunoreactivity found no significant activation in the hippocampus in a social recognition paradigm.

Another brain area that has been implicated in recognition is the amygdala, which also has a dense CB1 distribution (Viveros et al. 2005) and is activated by cannabinoid agonists (Phan et al. 2008). Research by Choleris et al. (2007) and

Ferguson et al. (2001) suggests that the oxytocin system in the amygdala is critical in social recognition during the acquisition but not consolidation phase of social recognition. Oxytocin gene expression primarily occurs in the magnocellular neurons of the hypothalamic paraventricular and supraoptic nuclei

(reviewed in Gimpl & Fahrenholz 2001). And indeed a study looking at Fos- immunoreactive cells in male mice after a social recognition test involving familiar and novel male mice revealed that the primary structures involved were amygdalar and hypothalamic nuclei (paraventricular hypothalamic nucleus, lateral hypothalamic nucleus and dorsal-medial part of ventral medial hypothalamic nucleus) with specific connections to olfactory and emotional systems (Borelli et al. 2009). Borelli et al.'s (2009) results pointed specifically to

63 the basalateral nucleus of the amygdala and the medial amygdala regions but not the central amygdaloid nucleus and this is supported by previous research showing that the same two amygdalar regions are involved in social interaction

(Fleming et al. 1994).

No one study has clearly pieced together all the brain components needed for social recognition as tested in this thesis, and thus it is necessary to examine several possible explanations. It is possible that the brain systems identified by

Lai et al. (2005) may work in conjunction with amygdalar regions forming the neural basis of the social recognition results. However, the study by Lai et al.

(2005) may alternately be tapping into a type of memory involving aggressive and sexual responses to familiar and novel stimuli that can be partially disassociated from other forms of social recognition memory. The neurological structures involved in Lai et al. (2005)'s study on aggression are similar to those for individual sex odour recognition involving the piriform and entorhinal cortices along with the cortical amygdala (reviewed in Petrulis et al. 2009). The social recognition test as it was conducted in this thesis is unlikely to have a strong aggression component because of the use of castrated stimuli. There was no sexual component in any of the tests conducted herein. Social memory, for a familiar conspecific, may have a neural basis distinct from other types of memory such as non-social olfactory and acoustic recognition and spatial memory

(Ferguson et al. 2000). It may be that the pathway for sexual and aggressive recognition memories also utilizes some unique neural structures or components

64 thereof, explaining the different structures implicated in the model of recognition by Lai et al. (2005) as opposed to those suggested by others (Choleris et al.

2007; Ferguson et al. 2001).

The neurobiological structures that underpin social recognition are known to have some sex-based differences. For instance, lesions of the medial amygdala in female hamsters do not impair individual odour discrimination (Petrulis &

Johnston 1999) and male hamsters need the vomeronasal organ to discriminate individual odours whereas female hamsters do not (Johnston & Peng 2000;

Petrulis et al. 1999). Sex differences in social recognition in both rats and mice are also seen with the hypophyseal neuropeptide, vasopressin which when antagonized blocks social recognition in males but not females (Dantzer 1998).

Since CB1 receptor binding also varies by sex (Rodriguez de Fonseca et al.

1993; Rodriguez de Fonseca et al. 1994), it is possible that sexual dimorphisms could affect results and thus further experimentation will be required to assess whether the present findings can be extended to female mice.

Another main finding of this thesis was the apparent impairing effects of the high dose of 0.4 mg/kg URB597 on recognition memory; both social and non-social

(e.g. object). This is indicated by the fact that the 0.4 mg/kg URB597 treated mice did not show good recognition for either social or object stimuli. The recognition impairments at the 0.4 mg/kg URB597 dose do not appear to be a

65 function of activity nor is the group significantly different from vehicle on any other behavioural measure.

The impairing effects of pre-acquisition acute administration of URB597 at 0.4 mg/kg on object recognition found in the present study are in agreement with the findings showing that acute pre-acquisition activation of the cannabinoid system with both an anandamide analogue, WIN 55,212-2 and the CB1 agonist, CP

55,940, impaired short-term object recognition memory (Kosiorek et al. 2003;

Schneider et al. 2008). Similarly, acute systemic activation of the cannabinoid system using the cannabinoid agonist WIN 55,212-2 during memory consolidation dose-dependently impaired object recognition in rats (Baek et al.

2009), suggesting that the cannabinoid system is involved in both the acquisition and the consolidation of object recognition memory. By contrast, chronic activation of the cannabinoid system was shown to impair object recognition in adolescent (O'Shea et al. 2004, 2006) but not in adult rats (Higuera-Matas et al.

2009; Schneider and Koch 2003), suggesting adaptation of the system to chronic stimulation in adults. Hippocampal involvement in these effects has been suggested, with intra-hippocampal administration of WIN 55,212-2 or the CB1 agonist ACEA during consolidation impairing retention of long-term object recognition memory (Clarke et al. 2008). These effects were reversed by a CB1 receptor antagonist, suggesting that hippocampal CB1 receptors are involved in long term memory for objects (Clarke et al. 2008). Short-term object memory, instead, seems to not depend upon hippocampal cannabinoid activation, with

66 intra-hippocampal injection of the cannabinoid agonist WIN 55,212-2 prior to acquisition (Suenaga & Ichitani 2008) or during memory consolidation (Clarke et al. 2008) having no effects on short-term object recognition. These results are consistent with the notion that long-term object recognition memory is

hippocampal-dependent while short-term memory for objects is not hippocampal- dependent but rather requires the perirhinal-entorhinal cortex (Petrulis &

Eichenbaum 2003). However, a number of other studies show that object recognition is spared after hippocampal lesions (Broadbent et al. 2004; Forwood et al. 2005; Winters et al. 2004), while it is lost only after selective perirhinal cortex lesions (Winters et al. 2004; Winters & Bussey 2005) that affected acquisition, consolidation and retrieval of memory, suggesting that the perirhinal cortex is involved in all stages of object memory formation (Winters & Bussey

2005). This may rely partially upon cannabinoid mechanisms since this area expresses high densities of CB1 receptors (Liu et al. 2003). The hippocampus could have an indirect role through cannabinoid activation of N-methyl-D- aspartate (NMDA) receptors since impaired object recognition has been found in mice with NMDA receptor deletion specific to the CA1 hippocampal subregion

(Rampon et al. 2000). However this object recognition impairment may be more of a general cognition impairment since mice with genetically deleted NMDA receptors have disrupted social transmission of food preference, contextual fear conditioning and spatial memory in a water maze task (Rampon et al. 2000;

Tsienetal. 1996).

67 These studies on object recognition memory provide some clues as to a) why the

0.4 mg/kg URB597 dose results in impaired recognition memory and b) where the neurobiological location of a recognition impairment might be occurring. For short-term recognition memory it seems that impairments occur with cannabinoid agonist injections regardless of whether the injection is given pre-acquisition or during consolidation and that the hippocampus is not involved. Our injections were systemic (not intra-hippocampal) and pre-acquisition and the recognition paradigms involved short-term memory so our impairing results are in agreement with those showing impairing effects of cannabinoid activation on recognition memory. However, URB597-mediated increases in anandamide at active synapses (Fegley et al. 2004; Scherma et al. 2008; Trezza & Vanderschuren

2008b) caused memory enhancement when given pre-acquisition (Mazzola et al.

2009; Trezza & Vanderschuren 2008a) but not when given during consolidation or prior to a retention test (Mazzola et al. 2009). This memory enhancement is specific to anandamide's potentiation at active synapses since systemic direct infusions of anandamide result in memory impairment (Mallet & Beninger 1998).

It may be the case that there is a very narrow range of URB597 that is optimal for memory enhancement. If URB597 levels are too high, cannabinoid system activation becomes more like activation via a direct CB1 agonist either because anandamide tone would reach high levels even at synapses that initially had low levels of activity or because anandamide may disperse from active synapses and also reach low-active sites. Conversely, if URB597 levels are not too high, there may not be enough FAAH inhibition to increase the anandamide-mediated

68 activity of otherwise low active synapses to functionally meaningful levels. Thus, the mechanism through which the impairing effects of the 0.4 mg/kg dose of

URB597 in the present study acted could be a function of an increase in activation in neural pathways which impair one another rather than in the signal at potentiated synapses (reviewed in Riedel & Davies 2005) since the inhibitory or excitatory effect of cannabinoid retrograde messengers on synaptic plasticity depends on a fine balance between production and degradation of the endocannabinoid (Hashimotodani et al. 2007a; Hashimotodani et al. 2007b).

An alternate explanation of our impairing effects is that the 0.4 mg/kg dose of

URB597 may have, through increased and more widespread inhibition of FAAH, sufficiently increased levels of FAEs other than anandamide in brain areas where they are active. While FAAH is the primary catabolic agent for anandamide

(Fegley et al. 2005), it also degrades other FAEs like oleamide, OEA or PEA

(Cravatt et al. 1995). OEA and PEA are ligands for alpha-type peroxisome proliferator-activated nuclear receptors (PPAR-alpha). Enhanced memory acquisition has been shown in a passive-avoidance task in rats using either

URB597 or an agonist (WY14643) for PPAR-alpha (Mazzola et al. 2009). The effects of both drugs were blocked by PPAR-alpha antagonist MK886. This suggests that at least some of URB597's memory enhancing effects and impairing effects are mediated through the PPAR-alpha. To confirm that our findings are indeed specific to increased anandamide levels acting through CB1 receptors our tests could be repeated using the CB1 inverse agonist SR

69 141716A or the PPAR-alpha antagonist MK886 to verify if either can block the effects of URB597.

Another finding for the high dose of 0.4 mg/kg URB597 in the present thesis was that it impaired social motivation in the social approach home cage test. Similar findings of reduced social behaviour have been seen in mice with genetic CB1 deletion in a social interaction test (Haller et al. 2004) which could be due to these CB1 KO mice having increased anti-social behaviours such as aggression in a resident-intruder test and anxiogenesis in the light-dark box (Martin et al.

2002). Direct CB1 agonists administered acutely have been shown to be inhibitory towards time spent in social interaction, CP 55,940 (Genn et al. 2004), social play, WIN 55,212-2 (Trezza & Vanderschuren 2008a) and social interaction with low-dose A-9-THC (1 mg/kg) (Malone et al. 2009), as well as chronically in social interaction with CP 55,940 and WIN 55,212-2 (O'Shea et al.

2006; Schneider & Koch 2005). Since both CB1 receptor activation and its deletion cause social impairments it is possible that there is a very specific range of activation that allows for normal or enhanced social function. Further research is needed to examine the influence of anti-social behaviours on the results of social tests performed in this thesis. For instance, the effects of URB597 on aggression could be examined by means of a social interaction test in the experimental mouse's home cage in which the experimental mouse has been isolated and the intruder mouse is a castrated stimulus comparable to those used in the social approach and recognition paradigms.

70 The impairment of social approach preference score with the 0.4 mg/kg URB597 dose in the social approach home cage test does not extend to social approach in the three-chamber apparatus (figure 3B) which suggests an effect of the novel environment on social approach. The elimination of the social approach impairment in the novel environment that was witnessed in the home cage may be a function of decreased aggression (Haller et al. 2004) combined with increased anxiety (Bourin & Hascoet 2003) in the novel environment. It may be that increased anxiety-mediated stimulation of the CB1 receptors in the novel environment compensates for the social approach impairment seen with the 0.4 mg/kg URB597 dose in the home cage. The importance of CB1 receptors in mediating anxiety in novel environments is seen when testing social interaction, a behavioural measure related to social approach, and which is impaired in CB1

KO mice in a novel environment (Urigiien et al. 2004). Further investigation may be warranted to assess whether other novel environmental stimuli such as altered lighting can compensate for cannabinoid-induced social approach impairments.

In the present study, the 0.05 mg/kg URB597 dose like the 0.4 mg/kg dose showed a decreased social motivation, but on slightly different parameters than the 0.4 mg/kg dose. The 0.05 mg/kg URB597 dose failed to affect social approach and social recognition (figures 3A, 3B, 3C), but decreased social motivation on the social recognition test as indicated by a reduction in social

71 behaviour (figure 6A). Although social approach was not affected at the 0.05 mg/kg URB597 dose, the percent change in social approach from habituation to test in the home cage was significantly lower as was the time spent in the social chamber of the three chamber apparatus (although the significant percent change may be due to a higher baseline, figure 3A). The reduced social investigation, time spent in social proximity and percent change in social approach seem to support a social impairment.

Schneider et al. (2008) reported that i.p. injections of cannabinoid agonist WIN

55,212-2 in an acute social recognition test revealed a reduced initial exploration of the social stimulus at habituation. This is comparable to our findings that social behaviour was reduced across all trials for 0.05 mg/kg URB597.

Schneider et al. (2008) also found reduced social behaviour in a social interaction test on the test trial and that social recognition was impaired with injections of WIN 55,212-2. Schneider et al.'s (2008) results do not appear to be explained by changes in overall activity, nor does the 0.05 mg/kg URB597 social behaviour impairment in the present study, which while lower than injected groups, was not significantly different (figure 6B). Horizontal movement time for the 0.05 mg/kg URB597 group was also higher on this test, arguing against an activity-mediated effect.

It may be that the low URB597 dose of 0.05 mg/kg activates too few of the synapses that promote social recognition and thus its activation of the

72 cannabinoid system follows a similar pattern of reduced social exploration and behaviour that Schneider et al. (2008) found with a cannabinoid direct agonist.

Indeed, the reduced social investigation may be part of wider memory impairment at this dose as suggested by the 0.05 mg/kg URB597 object recognition and placement impairments which are not explained by activity differences (figures 7B, 8B).

The object placement test preference score results (figure 3E) are difficult to interpret since in none of the URB597 treated groups was there an increase in the preference score from habituation to test and even in the vehicle control group the change in preference score was only a statistical trend. The reason may lie in the test methodology. As other researchers have found (Suenaga &

Ichitani 2008), recognition of a new place is more difficult than recognition of a new object. In order to improve mouse performance in the object placement test, these experimenters shortened their object placement inter-stimulus delay time to 5 minutes as compared to 20 minutes for object recognition. They also used more habituation exposures for object place recognition. We used 15 minute inter-stimulus delay times so perhaps shortening these would have led to better performance by the mice in this test. A further improvement would be to use objects with a greater tactile saliency to increase their investigation time since in many cases (for both object recognition and placement tests) the total object investigation time was below forty seconds in the five minute test. Suenaga &

73 Ichitani (2008) had similar total object investigation times on both object tests

however their test trial was only 3 minutes long.

Caveats in our paradigm withstanding, the absence of any changes from

habituation to test in all URB597 treated mice suggests that URB597 is impairing

performance for the object placement paradigm relative to vehicle at all doses.

This would be in agreement with Egashira et al.'s (2002) findings that injection of

A-9-THC in each of the dorsal hippocampus, the ventral hippocampus and the

dorsomedial thalamus nucleus impaired spatial memory in a manner similar to

what was found with hippocampal lesions (Winters et al. 2004). Indeed the

dentate gyrus is known to play a critical role in forming distinct representations

and discriminating them from similar contexts previously encountered (McHugh

et al. 2007). The hippocampus is also rich in CB1 receptors as well as FAAH

(Egertova et al., 1998). As well, administration of both A-9-THC and the synthetic

A-9-THC analogue HU-210 have been shown to disrupt or reduce hippocampal

cell firing in rats perhaps explaining the origin of spatial memory deficits (Heyser

et al. 1993; Robinson et al. 2007). This is consistent with the findings that object

placement tasks are impaired by direct-acting CB1 agonists (A-9-THC, HU-210,

WIN 55,212-2) as are other spatial tasks such as water maze tasks (Cha et al.

2007; Robinson et al. 2007; Suenaga & Ichitani 2008). It may be that the

URB597 effects witnessed at the present doses are comparable to effects of direct-agonists for spatial memory and thus act in an impairing manner.

74 Injection Effects

When compared to the non-injected mice, the vehicle group in this thesis (2-

HPBCD) had reduced investigation of social (figures 4A, 6A) and object stimuli

(figures 7A, 8A) as well as vertical exploration (figures 5C, 6C), while having increased horizontal exploration (figures 4D, 6D, 7D, 8D). Interestingly, except for the three chamber test as noted below, there was no difference between vehicle and non-injected groups on a behavioural measure of sitting, indicating that vehicle effects were not on overall levels of mouse activity. Overall, it seems that the vehicle injected mice shift their behaviour from task oriented behaviour to non-task related activity. Different studies have found that vertical movement time, often labelled as escape behaviour, increases with increased stress when testing was done in a novel environment (Chuang & Lin 1994; Brinks et al. 2007).

If this is the case, the fact that in most paradigms we observed a lower vertical activity in the vehicle treated mice would suggest that injections have anxiety reducing effects. Alternatively, in our paradigms conducted in the mouse's home cage, increased stress might result in higher horizontal activity, perhaps a reflection of heightened territorial protection, rather than escape behaviour. This latter explanation would be consistent with our results showing increased horizontal activity for the vehicle group in the home cage. Also consistent with this explanation are the results of the social approach three chamber apparatus test (a novel environment) where no difference in horizontal movement was found between vehicle and non-injected groups, (figure 5D) This test was also

75 the only test to have increased sitting (figure 5F) and increased activity (figure

5B) in the vehicle group relative to the non-ininjected group. Taken together these findings might suggest an increased fear-related anxiety response induced by a vehicle injection in the larger novel environment (Nikaido & Nakashima

2009). Handling, sham injections and i.p. injections have been shown to induce a stressed and anxious profile in mice, increasing latencies in an elevated plus- maze, a common measure of anxiety (Lapin 1995). Indeed, a simple i.p. injection of saline in inbred mice has been shown to increase expression of Fos protein and Fos-related antigens, both of which are associated with a stress response (Ryabinin etal. 1999).

The significantly different results seen between non-injected and vehicle groups could be explained by stress effects as mentioned above, or by the vehicle having its own unique effects which might be implicated. The cyclodextrin family of vehicles has only been shown to have rare and non-behavioural effects such as causing pulmonary edema, agitation (Carpenter et al. 1995) and lowering cholesterol in serum (Irie et al. 1992). Behavioural studies in rodents utilizing

URB597 have used a variety of other vehicles such as 5% Tween-80, 5% polyethylene glycol (PEG) in saline (Trezza & Vanderschuren 2008b), 10%

DMSO in saline (Micale et al. 2009), 5% PEG400, 5% Tween-80 in saline

(Cippitelli et al. 2008), 5% DMSO, 5% ethanol in saline (Moreira et al. 2008) and

10% DMSO, 10% emulphor in saline (Rademacher & Hillard 2007) as well as

45% 2-HPBCD in sterile water (Rock et al. 2008). However 2-HPBCD has been

76 examined for vehicle use and was determined to be largely (90%) excreted into

urine in 4 hours (Pitha et al. 1994) and compatible with brain and spinal tissue

(Yaksh et al. 1991) and its reported effects (Carpenter el al. 1995; Irie et al.

1992) are not on behavioural measures. Conversely, some of the other vehicles

used in studies with URB597 have demonstrated behavioural effects such as

changes in defensive burying with i.p. saline injection (Saldivar-Gonzalez et al.

1997) and decreased locomotor activity with Tween-20, Tween-80 and dimethyl

sulphoxide (Castro et al. 1995). These and other effects of other vehicles together with the milder effects of 2-HPBCD are the main reasons for our choice of vehicle. However, non stress-related vehicle effects of 2-HPBCD cannot be definitively ruled out in the present studies without retesting with the substitution of an alternate vehicle.

Overall Conclusions

From the present data it can be concluded that selective agonism of active synapses of the cannabinoid system by a relatively low dose (0.1 mg/kg

URB597), can promote social recognition, thus providing the basis for appropriate social interactions. Conversely, high doses (0.4 mg/kg URB597), that likely stimulate synapses that would otherwise have low levels of activity seem to have impairing effects on a number of learning paradigms such as object or social recognition as well as on social approach. These impairing effects might also be on spatial learning suggesting that they are generalized to other cognitive functions but not to general activity or arousal. When all doses

77 are considered together it appears that the social behaviour and memory effects seen in male mice are sensitive to relatively small changes in drug dose and that optimal levels of cannabinoids could have a facilitating effect on social recognition while deviation from these can result in a variety of memory and motivational impairments.

Outlook and Implications

Given the limitations of social behavioural animal models in providing results that can be meaningfully interpreted for a human population (reviewed in Gould &

Gottesman 2006), it might be difficult to extrapolate the present results and the potential use of URB597 to humans. For instance, humans primarily use visual cues for social recognition while mice use olfactory cues (Ricceri et al. 2007).

Also, a common concern with drugs that act on the cannabinoid system is hallucinogenic effects as noted by Pertwee (1999), although this particular concern does not apply to URB597 since it is not known to have any hallucinogenic effects, reinforcing properties or risk of triggering a relapse

(Trezza & Vanderschuren 2008a; Justinova et al. 2008). Typical and potentially problematic signs of CB1 receptor activation are also overcome, such as catalepsy, hypothermia or feeding (reviewed in Chaperon & Thiebot 1999), which do not appear to be elicited by URB597 (Kathuria et al. 2003). This drug does however have interactions with carboxylesterases in liver which could limit its

78 effectiveness and thus a new generation of FAAH inhibitors is already in

development (Clapper et al. 2009).

As we show here, moderate to low doses of URB597 can improve social

recognition, a behaviour that is key to the performance of appropriate social

skills. URB597 has also been shown to enhance social play behaviour in

adolescent rats (Trezza & Vanderschuren 2008a) and appears to be one of the

few synthetic agonists targeting the cannabinoid system that can elicit social

behavioural changes. While we found that social recognition was dose-

dependently enhanced, URB597's effects appear to be wide ranging with

recognition memory being enhanced or impaired depending on the dose, test or

both. Some of these effects may prove to be of substantial human benefit and

outweigh associated drug-induced detriments. For instance, it has been reported that URB597 may provide the means for extinguishing maladaptive behaviours

because anandamide appears to play a facilitatory role in extinction in spatial

memory and conditioned aversion through the CB1 receptor (Manwell et al.

2009; Varvel et al. 2007). From a human usage standpoint the outlook of

URB597 is favourable since sub-chronic repeated dose studies in rats and

monkeys have not exhibited systemic toxicity (Piomelli et al. 2006). To this end it seems appropriate to further investigate what social and non-social behaviours could be manipulated using URB597. This will help to provide a better

understanding of the social implications of cannabinoids as well as possible future avenues of research for growing areas like autism. In fact chronic

79 cannabinoid administration in addition to neonatal lesions in the medial prefrontal cortex has been proposed as a model for studying impaired social functioning comparable to autism (Schneider & Koch 2005) suggesting that cannabinoid studies have much to contribute to clinical research of social disorders.

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99 Figure 2: Schematic of the mouse home cage including plexiglass cylinders. Shown is the side view (A), end view (B), top view (C), and the 3D view (D). All measurements are in centimeters. Not to scale.

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108