The cannabinoid partial agonist BAY 59-3704 has been identified as an attractive target to explore structure-activity relationships at cannabinoid receptors for the development of a therapeutic agent for psychostimulant addiction. This thesis will describe the studies associated with the optimization of a palladium-catalyzed oxidative ring closure reaction for the synthesisof dibenzofuran analogues from substituted diaryl ethers. These dibenzofurans are viewed as rigid analogues of BAY 59-3704 and will provide useful information about molecular interactions at cannabinoid receptors. The scope and limitations of the palladium-catalyzed oxidative ring closure reaction as it relates to the synthesis of the target dibenzofuran analogues will be presented.

O sistema canabinóide endógeno tem uma importante função modulatória em muitos processos neurobiológicos, incluindo a neuroproteção, a plasticidade neuronal e a neuroinflamação. Neste estudo nós pré-tratamos os camundongos com um agonista inverso do receptor CB1, para avaliarmos se ocorria a neuroproteção através de respostas adaptativas. Camundongos machos adultos (C57BL/6J) de 3 meses de idade foram pré-tratados durantes 4 dias com injeções diearias de 3 mg/Kg de AM251 (agonista inverso CB1) ou com veículo, intraperitonealmente (i.p.). Vinte e quatro horas depois da última injeção de AM251/Veículo, os animais foram injetados com LPS i.p. (500 µg/kg) ou salina. Foram realizados testes comportamentais e testes bioquímicos com estruturas encefálicas. Nossa hipótese era que o pré-tratamento com o agonista inverso de CB1 iria funcionar como um estressor moderado, levando a uma neuroadaptação. Contudo, o pré-tratamento não ativou vias neuroprotetoras, e sim como atuou como um estresse deletério. / The endogenous cannabinoid system seems to play a modulatory function in many neurobiological processes, including neuroprotection, neuronal plasticity and neuroinflammation. In this study we pretreated the mice with an inverse agonist of the receptor CB1, in order to evaluate this neuroprotection through adaptive responses. Three months-old male mice (C57BL/6J) were pretreated during 4 days with daily injections of 3mg/Kg of AM251 (CB1 inverse agonist) or with vehicle, intraperitoneally (i.p.). Twenty-four hours after the last AM251/Vehicle injection, animals were injected i.p. with LPS (500 µg/kg) or saline. Behavioural and bichoemical assays were performed. Our hypothesis was that the pretreatment with the inverse agonist of CB1 was going to act as a moderate stressor, leading to a neuroadaptation. However CB1 pretreatment potentiated or sensitized the pro-inflammatory signaling pathway. Our pre-treatment worked not as a moderate stressor but as a deleterious stressor.

Doctor of Phylosophy (PhD) / Schizophrenia is a severe, chronic and disabling mental disorder with a worldwide prevalence of approximately 1 %. It is a lifelong illness characterized by psychotic symptoms which typically first appear in late adolescence/early adulthood. The symptoms of schizophrenia are usually categorized as positive (hallucinations and delusions), negative (blunted affect and poverty of speech) and cognitive (memory, attention and executive function impairments). Schizophrenia is thought to arise from an interaction between several susceptibility genes and environmental factors, one of them being the use of cannabis, the most widely used illicit drug in the world. Human population studies show that cannabis use is associated with schizophrenia, and it is now well recognised that cannabis use increases the risk of developing schizophrenia by approximately twofold. The reasons for the association between cannabis and schizophrenia remain controversial and different theories have been proposed to explain the nature of this relationship. According to the self-medication hypothesis of schizophrenia, patients with psychotic disorders use cannabis to alleviate aversive symptoms of the disorder or the side effects associated with antipsychotic medications. Other theories posit that cannabis is a component cause contributing to the development of schizophrenia. Supporting this, an increasing body of evidence shows that cannabis use increases the incidence and severity of psychotic symptoms and that cannabis use most frequently precedes the onset of schizophrenia. As a large majority of cannabis users do not develop schizophrenia, a gene-environment interaction appears necessary for the development of the disorder. That is, cannabis use may unmask latent schizophrenia in individuals that have a genetic predisposition to the disorder. Family studies provide strong evidence of a genetic contribution to the aetiology of schizophrenia. Several candidate genes are likely involved in the disorder, but this thesis will specifically focus on the neuregulin 1 (NRG1) gene. NRG1 was first proposed as a schizophrenia susceptibility gene in 2002 and linkage studies have since replicated this association in diverse populations around the world. In addition, changes in expression of Nrg1 isoforms and its receptor ErbB4 have been reported in the brain of schizophrenia patients. NRG1 polymorphism has also been associated with cognitive and behavioural differences in schizophrenia patients compared to healthy individuals. Collectively, NRG1 is now recognized as one of the most promising genes that confer an increased risk of developing schizophrenia. The creation of knockout mice lacking a specific gene offers an exciting new approach in the study of mental disorders. While several mutant mice for Nrg1 and ErbB4 receptor have been developed, this thesis focussed on mice that are heterozygous for the transmembrane domain of the Nrg1 gene (named Nrg1 HET mice). These mice exhibit a schizophrenia-like phenotype including hyperactivity that can be used as a reflection of positive symptoms of schizophrenia. Furthermore, they display impairments in social recognition memory and prepulse inhibition (PPI), a model of attentional deficits observed in schizophrenia patients. In addition, the brains of Nrg1 HET contain fewer functional NMDA receptors and more 5-HT2A receptors than wild type-like (WT) animals which is consistent with the neurotransmitters imbalance observed in schizophrenic patients. The phenotype of Nrg1 HET mice is age-dependent, another aspect that mirror the late adolescent/early adulthood onset of schizophrenia symptoms. The present thesis aimed at developing an animal model of genetic vulnerability to cannabinoid-precipitated schizophrenia by utilising Nrg1 HET mice to observe if these animals show an altered behavioural and neuronal response to cannabinoid exposure. We hypothesise that Nrg1 deficiency will alter the neurobehavioural responses of animals to cannabinoids. The experiments detailed within the first research chapter (Chapter 2) aimed at examining the behavioural effects of an acute exposure to the main psychoactive constituent of cannabis, Δ9-tetrahydrocannabinol (THC), in Nrg1 HET mice using a range of behavioural tests of locomotion, exploration, anxiety and sensorimotor gating. Relative to WT control mice, Nrg1 HET mice were more sensitive to both the locomotor suppressant action of THC, as measured in the open field test, and to the anxiogenic effects of THC in the light-dark test, although the effects in this procedure may be confounded by the drug-free hyperactive phenotype of Nrg1 HET mice. Importantly, Nrg1 HET mice expressed a greater THC-induced enhancement in PPI than WT mice. Taken together, the data presented in Chapter 2 show that a deficiency in a schizophrenia susceptibility gene Nrg1 enhanced the behavioural impact of THC. After having established a link between Nrg1 deficiency and increased sensitivity to the behavioural effects of cannabinoids in Chapter 2, Chapter 3 assessed the neuronal activity underlying the effects of an acute THC exposure on Nrg1 HET mice by using c-Fos immunohistochemistry. In the ventral part of the lateral septum (LSV), THC selectively increased c-Fos expression in Nrg1 HET mice with no corresponding effect being observed in WT mice. In addition, a non-significant trend for THC to promote a greater increase in c-Fos expression in Nrg1 HET mice than WT mice was observed in the central nucleus of the amygdala, the bed nucleus of the stria terminalis and the paraventricular nucleus of the hypothalamus. Consistent with Nrg1 HET mice exhibiting a schizophrenia-related phenotype, these mice expressed greater drug-free levels of c-Fos in the shell of the nucleus accumbens and the LSV. Interestingly, the effects of genotype on c-Fos expression, drug-free or following THC exposure, were only observed when animals experienced behavioural testing prior to perfusion. This suggests that an interaction with stress was necessary for the promotion of these effects. As the risk of developing psychosis in vulnerable individuals increases with the frequency of cannabis use, Chapter 4 assessed the effects of repeated exposure to cannabinoids on Nrg1 HET mice. As THC was not available at the time, the synthetic analogue of THC, CP 55,940, was used in this experiment. Behavioural testing showed that tolerance to CP 55,940-induced hypothermia and locomotor suppression developed more rapidly in Nrg1 HET mice compared to WT mice. On the contrary, tolerance to the anxiogenic-like effect of CP 55,940 in the light-dark test was observed in WT mice, however no such tolerance occurred to this effect in Nrg1 HET mice. Similarly, no tolerance developed to CP 55,940-induced thigmotaxis in Nrg1 HET mice as measured in the open field. For PPI, on the first day of exposure opposite effects were observed, with CP 55,940 treatment facilitating PPI in Nrg1 HET mice and decreasing it in WT mice. However, the differential effect of CP 55,940 on PPI was not maintained with repeated testing as both genotypes became tolerant to the effects of the cannabinoid on sensorimotor gating. In addition, a selective increase in Fos B/ΔFos B expression, a marker of longer-term neuronal changes, was observed in the LSV of Nrg1 HET mice following chronic CP 55,940 exposure, with no corresponding effect seen in WT mice. These results collectively demonstrate that the neuregulin system is involved in the neuroadaptive response to repeated cannabinoid exposure. One of the main schizophrenia endophenotypes observed in human studies are cognitive impairments of higher executive functions. Thus Chapter 5 aimed to develop a procedure to allow evaluation of cannabis-induced working memory deficits in mice. Few studies have investigated the effects of chronic cannabinoid exposure on memory performance and whether tolerance occurs to cannabinoidinduced memory impairment. Here we studied the effects of repeated exposure to THC on spatial memory and the expression of the immediate early gene zif268 in mice. One group of animals were not pre-treated with THC while another group was given 13 daily injections of THC prior to memory training and testing in the Morris water maze. Both groups were administered THC throughout the memory training and testing phases of the experiment. THC decreased spatial memory and reversal learning, even in animals that received the THC pre-treatment and were tolerant to the locomotor suppressant effects of the drug. Zif268 immunoreactivity was reduced in the CA3 of the hippocampus and in the prefrontal cortex only in non pre-treated animals, indicating that while tolerance to the effects of cannabinoids on neuronal activity arose, cannabinoid-promoted memory impairment in these animals persisted even after 24 days of exposure. Taken together these data demonstrate that the spatial memory impairing effects of THC are resistant to tolerance following extended administration of the drug. Such a model could be applied to Nrg1 HET mice in future studies to observe if cannabinoid-induced working memory impairments and the development of tolerance to this effect are altered relative to WT mice. In conclusion, this thesis provides the first evidence that partial deletion of the schizophrenia susceptibility gene Nrg1 modulates the neurobehavioural actions of acutely and chronically administered cannabinoids. Nrg1 HET mice appear more sensitive to the acute neurobehavioural effects of cannabinoids. Notably, acutely administered THC facilitated attentional function by increasing PPI in Nrg1 HET mice. However, with repeated cannabinoid administration this acute benefit was lost. The Nrg1 HET mice displayed a long-lasting anxiogenic profile that was resistant to tolerance. Conversely, Nrg1 HET mice developed tolerance to the locomotor suppressant and hypothermic effects of cannabinoids more rapidly than WT mice, indicating a distorted neuroadaptive response in these animals. Another major finding of this thesis is that the lateral septum appears to be an important brain region dysregulated by cannabinoids in Nrg1 HET mice. Cumulatively, this research highlights the fact that neuregulin 1 and cannabinoid systems appear to interact in the central nervous system. This may ultimately enhance our understanding of how gene-environment interactions are responsible for cannabis-induced development of schizophrenia.

Many common cancers, including breast, prostate and lung, have a predilection to metastasize to the bone, bringing not only bone destruction but severe pain. Although novel chemotherapeutic agents have increased life expectancy, patients are experiencing higher incidences of fracture, pain and drug-induced side effects; furthermore, recent findings suggest that patients are severely under-treated for their cancer pain. Strong analgesics, namely opiates, are the first-line therapy in alleviating cancer-related pain despite severe side effects including enhanced bone destruction with sustained administration. Bone resorption is primarily treated with bisphosphonates, which can bring highly undesirable side-effects including nephrotoxicity and osteonecrosis of the jaw. Thus novel therapeutics are needed to treat the pain of metastatic cancer patients. Animal models of cancer-induced bone pain (CIBP) have revealed that the neurochemistry of cancer has distinctive features from other chronic pain states. These include factors released from the cancer cells, tumor activated macrophages and increased osteoclast degredation of bone within the bone microenvironment, all acting to sensitize free nerve endings.One possibility of inhibiting cancer-mediated pain inducing factors includes agonism of the Cannabinoid 2 receptor agonists. Cannabinoid CB2 receptor-specific agonists have been shown to reduce bone loss and stimulate bone formation in a model of osteoporosis. CB2 agonists produce analgesia in both inflammatory and neuropathic pain models. Notably, mixed CB1/CB2 agonists also demonstrate a reduction in ErbB2-driven breast cancer progression. Osteolytic sarcoma within the femur produced spontaneous and touch evoked behavioral signs of pain within the tumor-bearing limb. The systemic administration of AM1241 both acutely and for 7 days significantly attenuated spontaneous and evoked pain in the inoculated limb. Sustained AM1241 significantly reduced bone loss and decreased the incidence of cancer-induced bone fractures. In addition, CB2 agonists significantly reduce breast cancer-induced bone pain, bone loss and breast cancer proliferation in part via cytokine/chemokine suppression. Studies utilized the spontaneously-occurring syngenic murine mammary cell line (66.1) implanted and sealed into the femur intramedullary space. Measurements were made of spontaneous pain, bone loss and cancer proliferation. The central and systemic administration of the CB2 agonist JWH015 for seven days significantly attenuates pain. Pharmacological characterization with cannabinoid 1 and 2 antagonists demonstrates that the effects JWH015 on pain were mediated by the CB2 receptor. We and others have found that bone induced cancer pain increases the expression of GFAP and Iba1 in the lumbar spinal cord which are markers of astrocytes and microglia respectively, compared to control animals. After administration of JWH015 (i.t), the release of spinal pro-inflammatory cytokines, IL-6 and TNFá, are reduced suggesting that modulation of glial cytokines may be one mechanism by which CB2 agonists can attenuate pain centrally. On the other hand, systemic administration of JWH015 reduces cancer-induced elevation of cytokines in the tumor microenvironment, suggesting a mechanism by which CB2 agonist is attenuating pain peripherally. Additionally, systemic administration improves bone modification, as demonstrated via micro-computed tomography and bone serum markers while decreasing femoral tumor burden. In vitro, JWH015 reduced cancer cell proliferation and other inflammatory mediators shown to promote pain, bone loss and proliferation. These results suggest CB2 agonists as a novel treatment for breast cancer-induced bone pain, where disease modifications include a reduction in bone loss, suppression of cancer growth, attenuation of severe bone-pain and increased survival without the major side effects of current therapeutic options. Another future therapeutic option for metastatic bone cancer pain may include cathepsin inhibitors. Cysteine cathepsins (B, C, F, H, K, L, O, L2/V, W, X/Z) are highly expressed in many human cancers and have been associated with poor patient prognosis. In the RIP1-Tag2 transgenic model of pancreatic cancer, mice treated with VBY-825, a reversible inhibitor of cathepsins S, B, V, L, K showed a significant reduction in tumor incidence and growth. Here we demonstrate the cathepsin inhibitor VBY-825 reduces cancer-induced pain behaviors. Additionally, tumor bearing animals treated with VBY-825 demonstrate a reduction in bone resorption, possibly mediated through a reduction in osteoclast activity. These results indicate that a cathepsin inhibitor targeting multiple cathepsins, such as VBY-825, could be a novel therapeutic for bone metastases.Part of the failure to palliate cancer pain is due to a poor understanding of the etiology of cancer pain. Preclinical studies have just begun to scratch the surface on how such cancers may interact with the bone microenvironment to result in pain and bone loss. Further studies are desperately needed at both the preclinical and clinical level to determine the unique molecular profile of cancer pain that may lead to the development of superior therapeutics for CIBP. The studies presented herein provide preclinical evidence that warrant the investigation of these compounds in the clinic as treatment for cancer-induced bone pain.

Resveratrol has been reported as a potential anticancer agent but cannot be used as an antitumor drug due to its chemical and metabolic instability. We have designed and synthesized 184 novel compounds related to resveratrol in an attempt to produce more potent and drug-like molecules. We have identified a tetrazole analog of resveratrol, ST-145(a) as a lead anticancer agent from the resveratrol analog series of compounds with a GI50 value of less than 10nM against almost all the human cancer cell lines in the National Cancer Institute’s screening panel. In a separate study, we tested the hypothesis that the limited bioavailability of resveratrol, can be improved by synthesizing analogs which would be glucuronidated at a lower rate than resveratrol itself. We demonstrated that ST-05 and ST-12(a) exhibit lower glucuronidation profiles when compared to resveratrol and that these synthesized stilbenoids likely represent useful scaffolds for the design of efficacious resveratrol analogs. We have also initiated a new discovery program to identify selective CB1 and CB2 receptor ligands from a library of novel stilbene scaffolds structurally related to the resveratrol molecule. From the screened resveratrol analogs, two compounds were identified as selective CB2 and CB1 ligands. Compound ST-179 had 47-fold selectivity for CB2 (Ki = 284 nM) compared to CB1, while compound ST-160 was 2-fold selective for CB1 (Ki = 400 nM) compared to the CB2 receptor. These structural analogs have the potential for development as novel cannabinoid therapeutics for treatment of obesity and/or drug dependency. Combretastatin A4 (CA-4) is one of the most potent antiangiogenic and antimitotic agents of natural origin. However, CA-4 suffers from chemical instability due to cis-trans isomerism in solution. To circumvent this problem, we have developed a facile procedure for the synthesis of novel 4,5-diaryl-2H-1,2,3-triazoles as CA-4 analogs to constrain the molecule to its cis-configuration. Twenty three triazoles were prepared as CA-4 analogs and submitted for anticancer screening. Among these CA-4 analogs, ST-467 and ST-145(b) can be considered as lead anticancer agents from this series, and further investigation against various cancer cell types in vivo with this class of compound may provide novel therapeutic avenues for treatment.

Doctor of Phylosophy (PhD) / Schizophrenia is a severe, chronic and disabling mental disorder with a worldwide prevalence of approximately 1 %. It is a lifelong illness characterized by psychotic symptoms which typically first appear in late adolescence/early adulthood. The symptoms of schizophrenia are usually categorized as positive (hallucinations and delusions), negative (blunted affect and poverty of speech) and cognitive (memory, attention and executive function impairments). Schizophrenia is thought to arise from an interaction between several susceptibility genes and environmental factors, one of them being the use of cannabis, the most widely used illicit drug in the world. Human population studies show that cannabis use is associated with schizophrenia, and it is now well recognised that cannabis use increases the risk of developing schizophrenia by approximately twofold. The reasons for the association between cannabis and schizophrenia remain controversial and different theories have been proposed to explain the nature of this relationship. According to the self-medication hypothesis of schizophrenia, patients with psychotic disorders use cannabis to alleviate aversive symptoms of the disorder or the side effects associated with antipsychotic medications. Other theories posit that cannabis is a component cause contributing to the development of schizophrenia. Supporting this, an increasing body of evidence shows that cannabis use increases the incidence and severity of psychotic symptoms and that cannabis use most frequently precedes the onset of schizophrenia. As a large majority of cannabis users do not develop schizophrenia, a gene-environment interaction appears necessary for the development of the disorder. That is, cannabis use may unmask latent schizophrenia in individuals that have a genetic predisposition to the disorder. Family studies provide strong evidence of a genetic contribution to the aetiology of schizophrenia. Several candidate genes are likely involved in the disorder, but this thesis will specifically focus on the neuregulin 1 (NRG1) gene. NRG1 was first proposed as a schizophrenia susceptibility gene in 2002 and linkage studies have since replicated this association in diverse populations around the world. In addition, changes in expression of Nrg1 isoforms and its receptor ErbB4 have been reported in the brain of schizophrenia patients. NRG1 polymorphism has also been associated with cognitive and behavioural differences in schizophrenia patients compared to healthy individuals. Collectively, NRG1 is now recognized as one of the most promising genes that confer an increased risk of developing schizophrenia. The creation of knockout mice lacking a specific gene offers an exciting new approach in the study of mental disorders. While several mutant mice for Nrg1 and ErbB4 receptor have been developed, this thesis focussed on mice that are heterozygous for the transmembrane domain of the Nrg1 gene (named Nrg1 HET mice). These mice exhibit a schizophrenia-like phenotype including hyperactivity that can be used as a reflection of positive symptoms of schizophrenia. Furthermore, they display impairments in social recognition memory and prepulse inhibition (PPI), a model of attentional deficits observed in schizophrenia patients. In addition, the brains of Nrg1 HET contain fewer functional NMDA receptors and more 5-HT2A receptors than wild type-like (WT) animals which is consistent with the neurotransmitters imbalance observed in schizophrenic patients. The phenotype of Nrg1 HET mice is age-dependent, another aspect that mirror the late adolescent/early adulthood onset of schizophrenia symptoms. The present thesis aimed at developing an animal model of genetic vulnerability to cannabinoid-precipitated schizophrenia by utilising Nrg1 HET mice to observe if these animals show an altered behavioural and neuronal response to cannabinoid exposure. We hypothesise that Nrg1 deficiency will alter the neurobehavioural responses of animals to cannabinoids. The experiments detailed within the first research chapter (Chapter 2) aimed at examining the behavioural effects of an acute exposure to the main psychoactive constituent of cannabis, Δ9-tetrahydrocannabinol (THC), in Nrg1 HET mice using a range of behavioural tests of locomotion, exploration, anxiety and sensorimotor gating. Relative to WT control mice, Nrg1 HET mice were more sensitive to both the locomotor suppressant action of THC, as measured in the open field test, and to the anxiogenic effects of THC in the light-dark test, although the effects in this procedure may be confounded by the drug-free hyperactive phenotype of Nrg1 HET mice. Importantly, Nrg1 HET mice expressed a greater THC-induced enhancement in PPI than WT mice. Taken together, the data presented in Chapter 2 show that a deficiency in a schizophrenia susceptibility gene Nrg1 enhanced the behavioural impact of THC. After having established a link between Nrg1 deficiency and increased sensitivity to the behavioural effects of cannabinoids in Chapter 2, Chapter 3 assessed the neuronal activity underlying the effects of an acute THC exposure on Nrg1 HET mice by using c-Fos immunohistochemistry. In the ventral part of the lateral septum (LSV), THC selectively increased c-Fos expression in Nrg1 HET mice with no corresponding effect being observed in WT mice. In addition, a non-significant trend for THC to promote a greater increase in c-Fos expression in Nrg1 HET mice than WT mice was observed in the central nucleus of the amygdala, the bed nucleus of the stria terminalis and the paraventricular nucleus of the hypothalamus. Consistent with Nrg1 HET mice exhibiting a schizophrenia-related phenotype, these mice expressed greater drug-free levels of c-Fos in the shell of the nucleus accumbens and the LSV. Interestingly, the effects of genotype on c-Fos expression, drug-free or following THC exposure, were only observed when animals experienced behavioural testing prior to perfusion. This suggests that an interaction with stress was necessary for the promotion of these effects. As the risk of developing psychosis in vulnerable individuals increases with the frequency of cannabis use, Chapter 4 assessed the effects of repeated exposure to cannabinoids on Nrg1 HET mice. As THC was not available at the time, the synthetic analogue of THC, CP 55,940, was used in this experiment. Behavioural testing showed that tolerance to CP 55,940-induced hypothermia and locomotor suppression developed more rapidly in Nrg1 HET mice compared to WT mice. On the contrary, tolerance to the anxiogenic-like effect of CP 55,940 in the light-dark test was observed in WT mice, however no such tolerance occurred to this effect in Nrg1 HET mice. Similarly, no tolerance developed to CP 55,940-induced thigmotaxis in Nrg1 HET mice as measured in the open field. For PPI, on the first day of exposure opposite effects were observed, with CP 55,940 treatment facilitating PPI in Nrg1 HET mice and decreasing it in WT mice. However, the differential effect of CP 55,940 on PPI was not maintained with repeated testing as both genotypes became tolerant to the effects of the cannabinoid on sensorimotor gating. In addition, a selective increase in Fos B/ΔFos B expression, a marker of longer-term neuronal changes, was observed in the LSV of Nrg1 HET mice following chronic CP 55,940 exposure, with no corresponding effect seen in WT mice. These results collectively demonstrate that the neuregulin system is involved in the neuroadaptive response to repeated cannabinoid exposure. One of the main schizophrenia endophenotypes observed in human studies are cognitive impairments of higher executive functions. Thus Chapter 5 aimed to develop a procedure to allow evaluation of cannabis-induced working memory deficits in mice. Few studies have investigated the effects of chronic cannabinoid exposure on memory performance and whether tolerance occurs to cannabinoidinduced memory impairment. Here we studied the effects of repeated exposure to THC on spatial memory and the expression of the immediate early gene zif268 in mice. One group of animals were not pre-treated with THC while another group was given 13 daily injections of THC prior to memory training and testing in the Morris water maze. Both groups were administered THC throughout the memory training and testing phases of the experiment. THC decreased spatial memory and reversal learning, even in animals that received the THC pre-treatment and were tolerant to the locomotor suppressant effects of the drug. Zif268 immunoreactivity was reduced in the CA3 of the hippocampus and in the prefrontal cortex only in non pre-treated animals, indicating that while tolerance to the effects of cannabinoids on neuronal activity arose, cannabinoid-promoted memory impairment in these animals persisted even after 24 days of exposure. Taken together these data demonstrate that the spatial memory impairing effects of THC are resistant to tolerance following extended administration of the drug. Such a model could be applied to Nrg1 HET mice in future studies to observe if cannabinoid-induced working memory impairments and the development of tolerance to this effect are altered relative to WT mice. In conclusion, this thesis provides the first evidence that partial deletion of the schizophrenia susceptibility gene Nrg1 modulates the neurobehavioural actions of acutely and chronically administered cannabinoids. Nrg1 HET mice appear more sensitive to the acute neurobehavioural effects of cannabinoids. Notably, acutely administered THC facilitated attentional function by increasing PPI in Nrg1 HET mice. However, with repeated cannabinoid administration this acute benefit was lost. The Nrg1 HET mice displayed a long-lasting anxiogenic profile that was resistant to tolerance. Conversely, Nrg1 HET mice developed tolerance to the locomotor suppressant and hypothermic effects of cannabinoids more rapidly than WT mice, indicating a distorted neuroadaptive response in these animals. Another major finding of this thesis is that the lateral septum appears to be an important brain region dysregulated by cannabinoids in Nrg1 HET mice. Cumulatively, this research highlights the fact that neuregulin 1 and cannabinoid systems appear to interact in the central nervous system. This may ultimately enhance our understanding of how gene-environment interactions are responsible for cannabis-induced development of schizophrenia.

Synthetic cannabinoids were first created in a pharmaceutical setting where scientists were studying marijuana. Researchers were trying to develop medically beneficial marijuana analogs. The compounds, however, were found to give physiological effects that were more potent than marijuana. Presently, synthetic cannabinoids have become a psychoactive drug of abuse, sold in head shops and over the Internet. New compounds are constantly being synthesized, which makes analysis of the drugs difficult. Solid phase extraction (SPE) is a well-studied method used in toxicological analysis to extract drugs and their metabolites from biological fluids. This sample preparation method is necessary to isolate the desired components of a sample for analysis by gas chromatography and mass spectrometry (GC/MS). This study sought to compare four brands of commercially available SPE cartridges using a procedure from United Chemical Technologies (UCT) for the simultaneous extraction of the three synthetic cannabinoid metabolites, JWH-018 N-(4-hydroxypentyl), JWH-122 N-(5-hydroxypentyl), and JWH-250 N-(5-hydroxypentyl), from urine. The cartridges from UCT, Thermo Scientific, Agilent Technologies, and SiliCycle were evaluated to determine how they performed throughout the SPE procedure. A recovery efficiency study was conducted to measure the amount of extracted metabolites from the urine. The responses of the quantification ion of the metabolites from an extracted urine sample were compared to a neat sample and the percent recovery was calculated. A within-run precision study was also utilized to measure the reproducibility of the cartridges, which was determined by the coefficient of variation (CV) of the different brands. The outcome of this research led to a development of a GC/MS method for detection of the three metabolites, creation of calibration curves for quantification, use of SPE for the extraction of the metabolites from urine, and the quantification of the extracted compounds to determine the efficacy and consistency of four brands of SPE cartridges. Method optimization was able to minimize the interday variations seen in the results of aliquots of the same samples. Optimal parameters include initial validation of the GC/MS method, a clean liner for the analysis of synthetic cannabinoid metabolites, using a GC column with a high temperature limit, and derivatization of the extracts before injection into the GC. While this study shows it is possible to use GC/MS for the analysis of these metabolites, LC/MS does not have the same restrictions because a liner, temperature elution, and derivatization of the analytes are not utilized. It was determined from the results of these studies that SiliCycle had the most reproducible and efficient cartridges. SiliCycle cartridges had a consistent and fast flow rate with a percent recovery efficiency within ±20% of the actual value. The results from SiliCycle were followed by cartridges from UCT, Thermo Scientific, and Agilent brands, respectively.

High density electroencephalographic (EEG) measures were used to assess the effects of acute delta 9-tetrahyrdrocannabidol (THC) administration on extinction of conditioned fear responses. Fear conditioning was initiated using a differential classical conditioning paradigm that paired an aversive unconditioned stimulus (shock) with a signaling stimulus (CS+), whereas another stimulus served as a safety signal (CS-). Evoked potentials, induced event-related spectral perturbations (ERSP), and associated intertrial coherence (ITC) measures were used to quantify the acquisition and extinction of conditioned fear responses. Participants (N = 10 males) exhibited conditioning to the CS+ across fear acquisition training, as reflected by greater late positive (posterior sites) and late negative (anterior sites) potential amplitude to the CS+ relative to the CS-. Acute administration of THC facilitated extinction of the conditioned response to the CS+ relative to placebo, as reflected by greater LPP and LNP amplitude to the CS+ relative to the CS- in the placebo, but not THC condition. ERSP analyses suggest the lack of difference between CS+ and CS- ERP amplitude may be partially explained by a shifting of attention from external stimuli to internal processing in the THC condition. However, relative to placebo, THC administration also increased the amplitude of some measures of the conditioned response (LNP) to the CS-, suggesting a generalization of fear or lack of discrimination in this condition.

Cannabinoid receptors (CBR) are the most abundant G-protein coupled receptors in the mammalian brain with the highest densities within the cerebellum (Herkenham et al., 1990). Cannabinoid manipulations have been reported to cause deficits in cerebellar-dependent learning (Kishimoto and Kano, 2006; Skosnik et al., 2007; Steinmetz and Freeman, 2010; 2013). Cannabinoid receptors-1 (CB1R) have been hypothesized to be important in the establishment of long-term depression within the cerebellar cortex (Levenes et al., 1998; Safo and Regehr, 2005). However, all investigations in vivo have used global manipulations and have not attempted to localize or characterize these receptors during cerebellar-dependent learning. Chapter 2 systematically examined localization within the cerebellar cortex of cannabinoid effects on eyeblink conditioning, a type of cerebellum-dependent learning. Local infusions into a specific portion of the cerebellar cortex, the eyeblink conditioning microzone, resulted in deficits in learning similar to systemic injections. Additionally, infusions of cannabinoids into the eyeblink conditioning microzone, and no other parts of the cerebellar cortex or deep nuclei, were responsible for the deficits. Finally, tetrode recordings were made in Purkinje cells while receiving either CBR agonist or vehicle injections prior to training. Fewer Purkinje cells exhibited learning-related decreases in activity when the rat was administered a CBR agonist as compared to when it was injected with the vehicle. The CBR administered Purkinje cells also showed earlier onsets and smaller amplitudes in their learning-related activity. Purkinje cells that show a learning-related increase in activity were not affected by cannabinoid administration. The impairment in Purkinje cell plasticity was not observed after the rats reached asymptotic levels of learning. These results indicate that CBR agonist administration disrupts the induction of plasticity within the cerebellar cortex and this may account for the behavioral deficit in eyeblink conditioning. Chapter 3 examined whether infusions of the CBR agonist into the cerebellar cortex impaired forebrain-dependent learning as well as forebrain-independent associative learning. Similar to subcutaneous injections, forebrain-dependent trace eyeblink conditioning was unimpaired, whereas forebrain independent delay eyeblink conditioning was impaired. These findings provide evidence that plasticity mechanisms that are modulated by cannabinoids do not play a significant role in trace eyeblink conditioning. Finally, in Chapter 4 the role of CBRs and endocannabinoids during memory consolidation were examined. CBR and endocannabinoid manipulations prior to training resulted in impaired eyeblink conditioning. However, a CBR agonist or a drug increasing endocannabinoid levels resulted in enhanced consolidation when administered 1 hour post-training. In contrast, a CBR antagonist or an endocannabinoid decreasing drug resulted in impairments 1 hour post-training. Thus, CBRs and endocannabinoids appear to be important in learning and consolidation of cerebellar-dependent learning.

No description available.