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.

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.

The enzyme dimethylarginine dimethylaminohydrolase 1 (DDAH1) plays a pivotal role in the regulation of nitric oxide levels by degrading the main endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA). Growing evidence highlight the potential implication of DDAH/ADMA axis in the etiopathogenesis of several neuropsychiatric and neurological disorders, yet the underlying molecular mechanisms remain elusive. In this study, we sought to investigate the role of DDAH1 in behavioral endophenotypes with neuropsychiatric relevance. To achieve this, a global DDAH1 knockout (DDAH1-ko) mouse strain was employed. Behavioral testing and brain region-specific neurotransmitter profiling have been conducted to assess the effect of both genotype and sex. DDAH1-ko mice exhibited increased exploratory behavior toward novel objects, altered amphetamine response kinetics and decreased dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) level in the piriform cortex and striatum. Females of both genotypes showed the most robust amphetamine response. These results support the potential implication of the DDAH/ADMA pathway in central nervous system processes shaping the behavioral outcome. Yet, further experiments are required to complement the picture and define the specific brain-regions and mechanisms involved.

The prevalence of obesity is growing worldwide partly due to an increase in consumption of diets high in fat. Obesity is known as a risk factor for developing Alzheimer’s disease (AD) later in life. Both obesity and AD are associated with cognitive deficits and experimental high-fat diets can impair memory in cognitively normal rodents but also worsen memory deficits in AD mouse models. What is still unclear is the molecular mechanisms behind the detrimental effects of a high-fat diet on memory and if sex can influence its effect. Data in this thesis demonstrated that compared to females, male control non-transgenic (Non-Tg) mice had earlier deficits in memory after a high-fat diet that were associated with hyperinsulinemia. However, female Non-Tg mice were more vulnerable to ultrastructural changes in mitochondria morphology and loss of synapses after 6 months of a high-fat diet, changes that were similar to those observed in control-fed female triple-transgenic mice (3xTgAD). Finally, the memory deficits observed after a high-fat diet in cognitively normal mice were not associated with obesity and adiposity, as treatment with resveratrol (RSV) an anti-obesogenic compound, attenuated body weight gain and adipose tissue but failed to reverse memory impairment. In control fed 3xTgAD mice, RSV rescued memory deficits. In all experiments a high-fat diet had no detectable effect on cognitive impairment in 3xTgAD mice. In conclusion, the present thesis demonstrates that the sex-dependent differences in the effect a high-fat diet on memory are likely due to hyperinsulinemia and mitochondrial impairment and do not depend on obesity phenotype. These results demonstrate the importance of gender when studying both obesity and AD and are relevant for future clinical trials.