The ability of the brain to change structurally and functionally with experience is called brain plasticity. High levels of pro-inflammatory cytokines impair normal memory formation and consolidation. To better understand the role of pro-inflammatory cytokines in learning, the contribution of the cytokine interleukin-6 (IL-6) to a motor skill learning task investigated. The Fmr1 Knockout (KO) mouse, an animal model of Fragile X Syndrome, has demonstrated impaired neural plasticity and learning. Fmr1 KO and control wild-type (WT) mice were trained on the dowel and flat beam runways to study motor skill learning and motor activity respectively. The cerebellum from the animals was examined for IL-6 protein using ELISA. No significant differences in the levels of IL-6 in the cerebellum of the Fmr1 KO and WT normal mice were found. The expression of IL-6 was not altered by the behavioural training. These results suggest lack of association between IL-6, and FMRP and motor skill learning. / October 2016

Fragile X syndrome (FXS) is the leading single gene cause of intellectual disability and Autism Spectrum Disorder (ASD). It is caused by epigenetic silencing of the fragile X mental retardation gene (FMR1), causing a loss of Fragile-X Mental Retardation Protein (FMRP). Over the last 2 decades, much has been learned about the pathophysiology related to the loss of FMRP from mouse models of FXS. The recent generation of a rat model of FXS opens the door to: validate phenotypes across mammalian species, address cognitive dysfunction using paradigms that are more difficult to address in mice and explore candidate therapeutics more accurately. This thesis explored the validity of a new rat model for FXS (Fmr1 KO rat). I showed that Fmr1 KO rats exhibit normal spatial navigation memory, social interactions and anxiety levels. On the contrary, when subjects were tested in a battery of spontaneous exploration tasks: object recognition (OR), object-context (OC), object-place (OP), and object-place-context (OPC) recognition, which assess associative memory, Fmr1 KO rats showed a severe deficit in remembering the most complex (episodic-like) associations. Following these results, I sought to explore the development of associative memory from postnatal day 25 (P25) to adulthood (P71). Subjects were tested in the four spontaneous exploration tasks, previously mentioned, 8 times between P25 and P71 to assess the development of their ability to discriminate novel from familiar associations between objects, contexts and places. Fmr1 KO rats’ ability to discriminate novel from familiar object-place (spatial) and object-place-context (episodic-like) associations was significantly impaired (OP was delayed, and OPC ability did not develop). In the last part of this thesis I examined whether early therapeutic intervention with lovastatin can restore the cognitive deficits I observed. Subjects were fed either a diet containing lovastatin (“lovachow”) or an identically looking control diet, between P29 and P64, and tested in the four spontaneous exploration tasks, previously mentioned. Fmr1 KO rats demonstrated a developmental profile of associative memory indistinguishable from that of WT animals. At P64, lovachow was replaced with standard laboratory chow and the animals were tested 1 and 3 months later. Surprisingly, lovastatin treated Fmr1 KO animals maintained the ability to perform the OPC task even at 3 months after the end of treatment, whereas Fmr1 KO animals on control chow showed no improvement with age. The findings of this work indicate that transgenic rats can complement existing mouse models of FXS, providing valuable insights into the effects of FMRP loss on cognitive function. Furthermore, the results from the treatment study show that not only can lovastatin treatment prevent the emergence of cognitive deficits associated with Fragile X Syndrome but also that lovastatin (and perhaps pharmaceutical interventions more generally) may prevent the developmental deficits in neuronal circuit formation which can be maintained into adulthood.

The regulation of neural precursor cells (NPCs), which encompass neural progenitor and neural stem cells (NSCs), is fundamental for proper brain development and function. These cells are regulated by orchestrated signalling within their local environment. Aberrant aspects of cell proliferation, differentiation, survival, or integration have been linked to various neurological diseases including Fragile X syndrome (FXS)—a disorder characterized by intellectual and social changes due to the silencing of the gene encoding FMRP. The biology of hippocampal NPCs in FXS during early postnatal development has not been studied, despite high FMRP expression levels in the hippocampus at the end of the first postnatal week. In this thesis, the Fmr1-knockout (KO) mouse model was used to study hippocampal cell biology during early postnatal development. A tissue culture assay, used to study the effect of astrocyte-secreted factors on the proliferation of NSCs, indicated that astrocyte secreted factors from Fmr1-KO brains enhanced the proliferation of wild type, but not Fmr1-KO NSCs (Chapter 3). Next, the proliferation and cell cycle profiles of NPCs in vitro and in vivo studied with immunocytochemistry, Western blotting, and flow cytometry revealed decreased proliferation of NPCs in the Fmr1-KO hippocampus (Chapter 4). Finally, cells isolated from the P7 dentate gyrus and characterized by flow cytometry, showed a reduced proportion of NSCs and an increased proportion of neuroblasts—neuronal committed progenitors—in Fmr1-KO mice. Together, these results indicate that hippocampal NPCs show aberrant proliferation and neurogenesis during early postnatal development. This could indicate stem-cell depletion, increased quiescence, or a developmental delay in relation to lack of FMRP and uncovers a new role for FMRP in the early postnatal hippocampus. In turn, elucidating the mechanisms that underlie FXS will aid in the development of targeted treatments. / Thesis / Doctor of Philosophy (PhD) / Fragile X syndrome is the leading inherited cause of intellectual impairment and autism spectrum disorder. The syndrome is caused by a defect in one gene. This gene has been suggested to play a role in regulating the birth of new brain cells termed neural precursor cells. The importance of neural precursor cells stems from their ability to generate neurons and glia, the main cells in the brain. In this thesis, I focus on studying neural precursor cells from the hippocampus, a brain region important for learning and memory. A mouse model was used to compare neural precursor cells from healthy and Fragile X mice during early postnatal development. I found that neural precursor cells do not divide as much as they should in the Fragile X mouse hippocampus. The results help to determine the causes for learning and memory deficits in Fragile X and potentially open avenues for intervention.

Background: Background of this work was the investigation of the endocannabinoid system (ECS) in the Fmr1 knock- out (KO) mouse. The Fmr1- KO mouse is a mouse model for fragile X syndrome (FXS). FXS is the leading monogenic cause for autism spectrum disorders (ASD) in humans. The Fmr1- KO mouse displays autistic behavior such as an impaired social interaction, repetitive behavior, cognitive deficits, increased anxiety and aggressiveness. Alterations of the ECS have been suggested to play a key role in the etiopathology of a variety of neuropsychiatric disorders. Until today, little has been described about the involvement of the ECS in ASD. Interrogation: 1. Evaluating the manifestation of typical cannabinoid- induced effects in the Fmr1- KO mouse 2. Investigating the influenceability of autistic symptoms with THC treatment in the Fmr1- KO mouse 3. Analyzing the signaling cascade of the stimulated and unstimulated ECS in different brain regions of the Fmr1- KO mouse Material and Methods: Experiments were carried out on adult (12±1 weeks old) male Fmr1- KO and Fmr1- wild- type (WT) mice from the C57BL/6J- (B6)- background. N= 15 mice received THC (10mg/kg bodyweight) and N= 16 received WIN55,212 (3mg/kg bodyweight). 30min after injection, the body temperature was measured and the distance animals moved in an open field during 15min was recorded (locomotion). Then, animals were placed with their forepaws onto a horizontally fixed bar and the time remaining in this position (catalepsy) was measured. Finally animals were placed on a preheated plate and the temperature at which a pain stimulus occurred was determined (testing analgesia). All 4 experiments are called tetrad experiment. Afterwards changes in body temperature, locomotion, catalepsy and analgesia of the animals was evaluated. To explore long-term effects of THC after the tetrad, N= 15 animals were tested in a social interaction test with a female contact mouse, 10 and 20 days after THC treatment. Therefore, the tested mouse and the contact mouse were placed together into a cage and the time mice spent in social interaction (nose, body and anogential sniffing, allogrooming and body contact) was manually quantified during 6min of recorded testing time. Another group of N= 19 received a premedication of rimonabant (Cannabinoid- receptor 1 (CB1) antagonist, 3mg/kg bodyweight) 30min prior to THC treatment. Rimonabant prevents THC from binding to CB1 and therefore allows the assessment of the involvement of CB1 in mediating social behavior. Furthermore the suggestibility of context-dependent fear conditioning with THC treatment has been tested on N= 13 mice. Animals were placed into a conditioning chamber that delivered 6 short electric shocks with a 30sec pause to their paws (conditioning phase). Immediately afterwards mice received THC or placebo. 24h later contextdependent fear was evaluated by quantification of the time mice spent freezing in the conditioning-chamber (fear) without receiving foot shocks. Intraneuronal signaling of the ECS was analyzed with N= 29 animals using western blots. Quantities of phosphorylated (“activated”) protein kinases (ERK, AKT and S6) from different brain homogenates (hippocampus, striatum, cortex and cerebellum) were therefore measured after THC or placebo injection (30 minutes prior to sacrificing). Results: Cannabinoids induced hypothermia, hypolocomotion, analgesia and catalepsy in WTmice. These effects were significantly less detectable in Fmr1- KO mice. Effects of both cannabinoids, THC and WIN55,212, were comparable with a slightly greater but not significant efficiency of THC. THC treated WT- mice exhibited further reduced social interaction 10 days after treatment, an effect that was partially prevented by premedication with rimonabant. THC increased social interaction in Fmr1- KO mice comparable to the level of untreated WT- mice. THC had no effect on behavior of WT- mice in context-dependent fear conditioning. Fmr1- KO mice showed significant less contextdependent fear conditioning compared to WT- mice. THC facilitated the recognition of an anxiety-correlated context in Fmr1- KO mice comparable to untreated WT- mice. In western blots significant changes in the THC- induced signaling cascade were detectable and depending on genotype, brain-region and analyzed protein-kinase. In the hippocampus there were no changes in untreated Fmr1- KO mice compared to WT- mice. THC had no effect on activation of protein-kinases in WT- and Fmr1- KO mice. In the striatum there were no changes in untreated Fmr1- KO mice compared to WTmice. THC significantly increased activity of ERK, AKT and S6 in WT-mice and not in Fmr1- KO mice. In the cortex of untreated Fmr1- KO mice AKT showed a significantly increased activity compared to WT- mice. THC significantly increased AKT activity in WT- mice without having an effect on KO- mice. In the cerebellum there were no changes in untreated Fmr1- KO mice compared to WT- mice. THC significantly increased ERK- activity in Fmr1- KO mice but had no effect on protein kinase activity in WT- mice. Conclusion: We observed physiological cannabinoid effects in WT- mice after treatment with THC and WIN55,212. These effects are significantly attenuated in Fmr1- KO mice. This may be interpreted as a desensitization of the ECS in the Fmr1- KO mouse. At the same time it was demonstrated that THC has the potential to improve context dependent memory consolidation and to increase social interaction in the Fmr1- KO mouse. In particular the influence of THC on impaired social interaction should be a target of further investigations to find possible therapeutic options for this typical symptom of Autism. Underlying molecular mechanisms remain unclear and the analysis of THC stimulated intraneuronal signaling gave no clear indication of possible molecular alterations in the Fmr1- KO mouse.

Background: Background of this work was the investigation of the endocannabinoid system (ECS) in the Fmr1 knock- out (KO) mouse. The Fmr1- KO mouse is a mouse model for fragile X syndrome (FXS). FXS is the leading monogenic cause for autism spectrum disorders (ASD) in humans. The Fmr1- KO mouse displays autistic behavior such as an impaired social interaction, repetitive behavior, cognitive deficits, increased anxiety and aggressiveness. Alterations of the ECS have been suggested to play a key role in the etiopathology of a variety of neuropsychiatric disorders. Until today, little has been described about the involvement of the ECS in ASD. Interrogation: 1. Evaluating the manifestation of typical cannabinoid- induced effects in the Fmr1- KO mouse 2. Investigating the influenceability of autistic symptoms with THC treatment in the Fmr1- KO mouse 3. Analyzing the signaling cascade of the stimulated and unstimulated ECS in different brain regions of the Fmr1- KO mouse Material and Methods: Experiments were carried out on adult (12±1 weeks old) male Fmr1- KO and Fmr1- wild- type (WT) mice from the C57BL/6J- (B6)- background. N= 15 mice received THC (10mg/kg bodyweight) and N= 16 received WIN55,212 (3mg/kg bodyweight). 30min after injection, the body temperature was measured and the distance animals moved in an open field during 15min was recorded (locomotion). Then, animals were placed with their forepaws onto a horizontally fixed bar and the time remaining in this position (catalepsy) was measured. Finally animals were placed on a preheated plate and the temperature at which a pain stimulus occurred was determined (testing analgesia). All 4 experiments are called tetrad experiment. Afterwards changes in body temperature, locomotion, catalepsy and analgesia of the animals was evaluated. To explore long-term effects of THC after the tetrad, N= 15 animals were tested in a social interaction test with a female contact mouse, 10 and 20 days after THC treatment. Therefore, the tested mouse and the contact mouse were placed together into a cage and the time mice spent in social interaction (nose, body and anogential sniffing, allogrooming and body contact) was manually quantified during 6min of recorded testing time. Another group of N= 19 received a premedication of rimonabant (Cannabinoid- receptor 1 (CB1) antagonist, 3mg/kg bodyweight) 30min prior to THC treatment. Rimonabant prevents THC from binding to CB1 and therefore allows the assessment of the involvement of CB1 in mediating social behavior. Furthermore the suggestibility of context-dependent fear conditioning with THC treatment has been tested on N= 13 mice. Animals were placed into a conditioning chamber that delivered 6 short electric shocks with a 30sec pause to their paws (conditioning phase). Immediately afterwards mice received THC or placebo. 24h later contextdependent fear was evaluated by quantification of the time mice spent freezing in the conditioning-chamber (fear) without receiving foot shocks. Intraneuronal signaling of the ECS was analyzed with N= 29 animals using western blots. Quantities of phosphorylated (“activated”) protein kinases (ERK, AKT and S6) from different brain homogenates (hippocampus, striatum, cortex and cerebellum) were therefore measured after THC or placebo injection (30 minutes prior to sacrificing). Results: Cannabinoids induced hypothermia, hypolocomotion, analgesia and catalepsy in WTmice. These effects were significantly less detectable in Fmr1- KO mice. Effects of both cannabinoids, THC and WIN55,212, were comparable with a slightly greater but not significant efficiency of THC. THC treated WT- mice exhibited further reduced social interaction 10 days after treatment, an effect that was partially prevented by premedication with rimonabant. THC increased social interaction in Fmr1- KO mice comparable to the level of untreated WT- mice. THC had no effect on behavior of WT- mice in context-dependent fear conditioning. Fmr1- KO mice showed significant less contextdependent fear conditioning compared to WT- mice. THC facilitated the recognition of an anxiety-correlated context in Fmr1- KO mice comparable to untreated WT- mice. In western blots significant changes in the THC- induced signaling cascade were detectable and depending on genotype, brain-region and analyzed protein-kinase. In the hippocampus there were no changes in untreated Fmr1- KO mice compared to WT- mice. THC had no effect on activation of protein-kinases in WT- and Fmr1- KO mice. In the striatum there were no changes in untreated Fmr1- KO mice compared to WTmice. THC significantly increased activity of ERK, AKT and S6 in WT-mice and not in Fmr1- KO mice. In the cortex of untreated Fmr1- KO mice AKT showed a significantly increased activity compared to WT- mice. THC significantly increased AKT activity in WT- mice without having an effect on KO- mice. In the cerebellum there were no changes in untreated Fmr1- KO mice compared to WT- mice. THC significantly increased ERK- activity in Fmr1- KO mice but had no effect on protein kinase activity in WT- mice. Conclusion: We observed physiological cannabinoid effects in WT- mice after treatment with THC and WIN55,212. These effects are significantly attenuated in Fmr1- KO mice. This may be interpreted as a desensitization of the ECS in the Fmr1- KO mouse. At the same time it was demonstrated that THC has the potential to improve context dependent memory consolidation and to increase social interaction in the Fmr1- KO mouse. In particular the influence of THC on impaired social interaction should be a target of further investigations to find possible therapeutic options for this typical symptom of Autism. Underlying molecular mechanisms remain unclear and the analysis of THC stimulated intraneuronal signaling gave no clear indication of possible molecular alterations in the Fmr1- KO mouse.

This dissertation describes separate but related studies that explore visual spatial learning and memory in Fragile X Syndrome. Across all studies, either the performance of individuals affected by FXS and/or fmr1 KO mice was compared to comparison controls on seven H-W mazes of increasing difficulty levels. Study one employed the traditional configuration of the H-W mazes to evaluate performance variables that include latency to complete the maze and number of the errors. The results of study 1 revealed significant differences in performance for both FXS groups as compared to mental age-matched comparison individuals and wild type mice, respectively. In contrast to the FXS group, performance of the comparison group improved as indicated by significantly fewer errors across trials. A similar pattern of results was observed when latency across trials was analyzed. Taken together, the results of study one support the hypothesis that a selective deficit in spatial learning and memory characteristic of the FXS phenotype can be observed in the murine model of FXS, if equivalent tasks are employed in testing humans and mice. Study two expanded on these findings by adding landmarks to the maze environment to evaluate how these may impact spatial learning and memory in fmr1 KO mice. Contrary to our hypotheses, landmarks significantly impaired wild type control performance. In addition, results revealed that the performance of the fmr1 KO mice generally did not differ between landmark and non-landmark tasks, indicating that the presence of landmarks neither enhanced nor hindered mouse performance. Lastly, study three entailed a more in-depth behavior analysis of maze navigation performance for FXS individuals from study 1. Consistent with the hypotheses and findings from study 1, results revealed significant differences in performance variables between individuals, with FXS participants generally performing worse than the comparison group participants. Taken together, the results of study 3 generally supported the hypothesis that there was greater impairment in performance for individuals affected by FXS as compared to controls. This impairment was evident in the pattern of pathways taken to solve H-W mazes, consistent with the notion that affected individuals employed different behavioral strategies.

L’autisme est un trouble envahissant du développement défini uniquement sur des critères comportementaux et l’âge d’apparition. Le X fragile est une pathologie d’origine monogénique dont 15-25% des patients présente le diagnostique complet de l’autisme et dont de nombreux symptômes chevauchent avec l’autisme. Une souris Fmr1 KO a été créée et validée comme modèle pour le X fragile. A l’instar de la variabilité des phénotypes du X fragile chez l’humain, les données préliminaires montrent que la mutation nulle Fmr1 chez la souris interagit avec l'arrière fond génétique. Les travaux présentés visaient à déterminer les caractéristiques autistiques exprimées par les souris Fmr1 KO, ainsi que l’interaction de la mutation nulle avec le fond génétique (souches C57BL/6J, FVB.129P2tm1Cgr /J et leurs hybrides). Les résultats de ces travaux montrent notamment que les souris Fmr1 KO présentent un évitement initial d’approche sociale, des altérations principalement qualitatives des vocalisations, de l’hyperactivité et une augmentation de l’activité diurne. La mutation interagit avec le fond génétique et les résultats actuels indiquent que les KO de fond FVB.129P2tm1Cgr /J ont le phénotype le plus marqué. / Autism is a pervasive developmental disorder defined by behavioural criteria and age of onset. Fragile X is a disorder due to the silencing of the Fmr1 gene. About 15-25% of Fragile X patients are diagnosed as autistic and many symptoms overlap between the two disorders. A mouse Fmr1 KO was created and validated as a model for Fragile X Syndrome. Preliminary data also show that the null mutation interacts with the genetic background. The work presented in this thesis aimed to determine the autistic features expressed in Fmr1 KO mice, as well as the influence of the genetic background (C57BL/6J and FVB.129P2tm1Cgr/J strains, and their reciprocal hybrids) on the expression of the Fmr1 mutation. Our results show an initial inhibition of social approach in Fmr1 KO mice and a qualitative alteration of ultrasonic vocalizations in isolated pups, as well as an increase in activity, especially during the diurnal period. The Fmr1 mutation interacts with the genetic background and the results indicate that KO on the FVB.129P2tm1Cgr/J background show the most marked phenotype.

Fragile X Syndrome (FXS) is the leading cause of inherited intellectual disability. The disorder is caused by a trinucleotide expansion that silences the Fragile X Mental Retardation 1 (Fmr1) gene resulting in the loss of its protein product, the Fragile X Mental Retardation Protein (FMRP). FXS patients show broad clinical phenotypes including intellectual disability, as well as a number of cognitive and behavioral problems. The lack of FMRP is believed to be the direct cause of the deficits seen in FXS patients. FMRP is an RNA-binding protein that is expressed in the brain and testes. This protein is believed to form a messenger ribonucleoprotein complex with mRNAs in the nucleus and subsequently export them to polyribosomes in the cytoplasm, therefore influencing translation of its bound mRNAs. Importantly, FMRP has long been suspected to be involved in synaptic plasticity due to its ability to bind several mRNAs that encode for proteins important in synaptic plasticity. Such proteins include the GluN1, GluN2A and GluN2B subunits of the N-methyl-D- aspartate receptor (NMDAR). FMRP is expressed in the hippocampus, a region of the brain involved in learning and memory processes. Recently, impaired NMDAR functioning in the dentate gyrus (DG) subregion of the hippocampus has been observed in Fmr1 knockout (-/y) mice. This impairment also resulted in reduction in long-term potentiation (LTP) and long-term depression (LTD) of synaptic efficacy, two biological models of learning and memory. In the present study, I focused on the levels of the NMDAR GluN1, GluN2B and Glu2B subunits in order to determine the synaptic plasticity alterations seen in the DG of Fmr1-/y mice. Using Western blotting, I found that there is a decrease in the GluN1, GluN2A and GluN2B subunits in the DG of young adult Fmr1-/y mice, indicating that these mice have significantly lower amounts of total NMDARs. These results could explain the altered LTP and LTD seen in Fmr1-/y mice at the molecular level and might contribute to the intellectual impairments seen in these KO mice. NMDARs appear to be important in the development and maturation of synapses. The GluN2A and GluN2B subunits are developmentally regulated, where GluN2B is predominantly expressed early in development and GluN2A in the adult brain. A dysregulation of GluN2A and GluN2B subunits has been proposed to affect the maturation and formation of synapses. Intriguingly, FMRP is also believed to play a functional role in early brain development. Thus, this study also focused on the developmental expression of the GluN1, GluN2A and GluN2B subunits in the DG, Cornu Ammonis, prefrontal cortex and cerebellum of Fmr1-/y mice, all of which are brain regions implicated in FXS. We found that the developmental expression of these subunits is altered in Fmr1-/y mice in specific brain regions. Together, these results demonstrate that the loss of FMRP differentially affects GluN1, GluN2A and GluN2B subunit expression both developmentally and spatially, further implicating NMDARs in the pathophysiology of FXS. / Graduate