Stress is an important risk factor for the development of clinical depression, yet little is known about the neurobiological mechanisms by which stress might promote depressive symptomatology. The brain is particularly susceptible to the negative effects of stress, as high levels of stress hormones result in decreased hippocampal neurogenesis, slowed cell maturation, and decreased cell complexity. Although we already know that these neurobiological changes are associated with significant impairments in important psychological functions such as learning, memory and motivation, we know little about the molecular details of this stress-induced remodeling and how it contributes to the development of depression. Currently, one candidate molecule of particular interest is reelin, an extracellular matrix protein responsible for regulating neuronal maturation and synaptic plasticity in the adult brain. Interestingly, recent post- mortem analyses indicate that reelin expression is decreased in depressed patients. Similarly, preclinical research has shown that repeated glucocorticoid administration significantly reduces reelin expression in the adult hippocampus. Combined, these results suggest that reelin may be an important protein to examine in regards to the pathogenesis of depression as well as a potential therapeutic target for the treatment of this disorder. The goal of this dissertation is to provide a comprehensive examination of the influence repeated glucocorticoid administration has on reelin expression in the rat hippocampus, and how this relates to the pathogenesis of depression. In chapter 2 we examined how co-treatment with the stress hormone corticosterone (CORT), and the antidepressant imipramine, influence reelin expression in the proliferative region of the hippocampus. In addition we determined whether changes in reelin expression are associated with alterations in neurogenesis and behavioral measures of depression. Results revealed that imipramine prevents CORT-induced downregulation of reelin in the hippocampus, and that these changes parallel improvements in FST behavior, increased neurogenesis and enhanced maturation of immature granule cells. Importantly, these data provide further evidence of reelin’s role in depression and establish this protein as a target of antidepressant treatment. In chapter 3 we examined the effect of CORT on a number of interneuron markers that co-localize with reelin throughout the hippocampus to determine whether the populations of neurons that express reelin are lost or are no longer expressing this protein. Results of this study indicate that CORT influences a number of interneuron markers in a region-specific manner in the hippocampus, but does not cause these cell populations to die, suggesting that CORT exploits an intracellular mechanism to regulate reelin expression in the hippocampus. Finally, in chapter 4, the influence of CORT on MeCP2 and DNMT1, two markers associated with DNA methylation, was examined in the hippocampus to elucidate a potential intracellular mechanism for CORT-induced reelin deficits. Results of this study indicate that CORT has no influence on global protein levels of these markers, but significantly increases the number of MeCP2-expressing cells in the proliferative subgranular zone of the hippocampus, suggesting that there is an increase in the number of methylated cells in this region. While it cannot be conclude from this study that increased methylation causes reelin deficits, the fact that an increase in MeCP2 is seen in the exact region where reelin deficits are most pronounced suggest it is possible. Moreover, these findings are novel, and suggest a role for MeCP2, and more generally, DNA methylation, in the neurobiology of depression. Collectively, the results of this dissertation enhance our understanding of the functional consequences of altered hippocampal neuroplasticity on the development of depressive symptomatology, and the role that reelin may play in this process. They also provide further support for reelin as a novel therapeutic target for the treatment of major depression.

Normal brain function is dependent on the correct positioning and connectivity of neurons established during development. The Reelin signaling pathway plays a crucial role in cortical lamination. Reelin is a secreted glycoprotein that exerts its function by binding to lipoprotein receptors and inducing tyrosine phosphorylation of the intracellular adaptor protein Dab1. Mutations in genes of the Reelin signaling pathway lead to profound defects in neuronal positioning during brain development in both mice and humans. However, the molecular mechanisms by which Reelin controls neuronal morphology and migration are unknown. We have used a systems analysis approach to identify genes perturbed in the Reelin signaling pathway and identified microtubule stabilizing CLIP-associated protein 2 (CLASP2) as a key cytoskeletal modifier of Reelin mutant phenotypes. Currently, little is known about the role of CLASP2 in the developing brain. We propose that CLASP2 is a key effector in the Reelin signaling pathway controlling basic aspects of cortical layering, neuronal morphology, and function. CLASP2 is a plus-end tracking protein and this localization places CLASP2 in a strategic position to control neurite outgrowth, directionality, and responsiveness to extracellular cues. Our results demonstrate that CLASP2 expression correlates with neurite length and synaptic activity in primary neuron cultures; however, the role of CLASP2 during brain development was unknown. In this dissertation, we have characterized the role of CLASP2 during cortical development by in utero electroporation of shRNA plasmids and found that silencing CLASP2 in migrating neurons leads to mislocalized cells at deeper cortical layers, abnormal positioning of the centrosome-Golgi complex, and aberrant length/orientation of the leading process. In addition, we found that GSK3β-mediated phosphorylation of CLASP2 controls Dab1 binding and is required for regulating CLASP2 effects on neuron morphology and migration. This dissertation provides the first steps in gaining insight into how Reelin signaling affects cytoskeletal reorganization to regulate fundamental features of neuronal migration, positioning and morphogenesis.

The “two-hit” schizophrenia hypothesis suggests genetic and environmental abnormalities interrupt early CNS function. This increases vulnerability of a “second hit” and schizophrenia onset. Chronic stress and decreased Reelin signaling are reportedly associated with schizophrenia. Heterozygous Reeler Mice (HRM) show a 50% reduction in Reelin and display major schizophrenia phenotypes. Receptor-Associated Protein (RAP) blocks ligand-association to Reelin receptor Apolipoprotein E receptor 2 (ApoER2). In this study, we sought to replicate major heterozygous reeler mouse (HRM) phenotypes using in vivo RAP studies to establish an experimental in vitro model. Using an in vitro model, we investigated the effects of chronic stress and decreased Reelin signaling on AMPAR subunit expression. Implantable Alzet osmotic pumps allowed bilateral ventricular 7nM RAP perfusion in 12-14 week-old mice. An assay revealed significant Dab-1 phosphorylation reduction in RAP-perfused animals. These results correspond with learning and memory and associative-fear conditioning abnormalities. Overall activity, sensory perception, and nociception remained unaltered. RAP-perfused mice displayed deficits in pre-pulse inhibition to acoustic startle, and therefore sensory-gating deficits. A significant decrease in Glur1 and Glur2/3 expression was observed in primary hippocampal/cortical neurons following chronic RAP and CORT exposure. Collectively, our results show postnatal Reelin signaling disruption produces physiological, biochemical, and behavioral phenotypes similar to the HRM model. The exact mechanism of Reelin-dependent AMPAR insertion remains unclear. Glur1 and Glur2/3 appear to be inserted by differing mechanisms. Glur1 is reported to be inserted with Reelin activation of phosphoinositol-3-kinase (PI3K) signaling. Glur2/3, whose mechanism of insertion is unknown, has not been shown to be inserted via PI3K. Our findings also demonstrate the usefulness of in vitro RAP use, in which apolipoprotein E receptor 2 (ApoER2) expression is predominant compared to other lipoprotein receptors that may be affected with RAP application.

Depression is an extremely common, devastating psychiatric syndrome with profound effects on the structure of neurons and the proteins that they express. However, the pathophysiology of depression remains unclear despite decades of extensive research efforts, and this lack of understanding makes it difficult to develop effective treatments. It is extremely problematic that conventional antidepressant drugs do not work for many patients, and those that do respond require weeks to months of continuous treatment before adequate therapeutic improvement is achieved. Therefore, there is a clear unmet need to develop mechanistically novel antidepressant compounds that are well-tolerated, more effective, and faster acting. Subjecting rats to repeated-corticosterone (CORT; stress hormone analogous to cortisol for humans) injections produces a depressive-like phenotype that can be used to make inferences about the human condition and screen compounds for antidepressant properties. Our laboratory has previously found that stress downregulates hippocampal reelin in a similar manner to that seen in depression patients, and that drugs with antidepressant actions recover this deficit. This provided a rationale to administer reelin directly into the hippocampus, which rescued behavioral and neurochemical deficits, but intrahippocampal infusions are not clinically viable. Reelin is expressed in the periphery and blood as well as the brain, so the aims of the collection of studies described here are to evaluate the antidepressant-like properties of peripheral intravenous (i.v.) reelin. In the first experiment, the antidepressant-like effects of several dosages of reelin (3/5μg given every 5/10 days) were evaluated in rats that were exposed to 3-weeks of daily CORT (40mg/kg) injections. I found that all the dosages of reelin attenuated CORT-induced despair-like behavior in the forced-swim test (FST) and normalized alterations in serotonin (5-HT) transporter (SERT) membrane protein clustering (MPC) in blood lymphocytes. Reelin treatment also increased reelin-immunoreactive (IR) cell counts in the hippocampal dentate gyrus (DG) subgranular zone (SGZ), but it had less of an effect on neurogenesis as measured by the number and maturation rate of doublecortin (DCX)-IR cells. Interestingly, the lowest dosage used also rescued the number of reelin-IR cells in the hypothalamic paraventricular nucleus (PVN). This suggested that the restoration of SGZ-reelin plays a pivotal role in attenuating depressive-like behavior and that 3μg every 10 days was the most effective dosage that was tested. Using the lowest dosage that showed to be effective in the first experiment, I then evaluated if male and female rats responded similarly to i.v. reelin using a larger battery of behavioral tests. Post-mortem tissue analyses focused on reelin and receptors that bind gamma-aminobutyric acid (GABA) and glutamate in the SGZ, which have been implicated in psychiatric disorders and the mediation of fast-acting antidepressant responses. I found that reelin rescued the FST- behavioral and neurochemical alterations induced by CORT similarly in both sexes, indicating that it may have therapeutic effects by normalizing inhibitory/excitatory transmission. I also evaluated the effect of i.v. reelin on neurogenesis in females and found that, akin to males, the regulation of adult-born cells by peripheral reelin is unlikely to mediate the antidepressant-like effects. The goal of the third experiment was to examine whether the antidepressant-like effects of peripheral reelin are achieved in a rapid manner. I found that a single 3μg injection after 3 weeks of CORT significantly decreased behavioral deficits in the FST 24 hours later in both sexes. Reelin also partially rescued cognitive deficits and expression levels of reelin, GluN2B, and mitochondrial-related pro-apoptotic factors bcl-2 associated X protein (BAX) and cytochrome C (CytC) in the DG. In addition, a single injection of reelin fully recovered the number of GluA1-expressing cells and partially recovered SERT cluster size in males, whereas reelin partially recovered GluA1-IR cell counts and fully recovered SERT cluster sizes in females. Reelin had modest effects on DCX-IR cells in both sexes. The final chapter summarizes and discusses my findings, which suggest that the antidepressant-like effects of peripheral reelin are associated with the recovery of neurochemical deficits that strengthen neurotransmission, at least in the hippocampus. Therefore, developing reelin-based therapeutics with antidepressant activity would be a fruitful area of research, although additional mechanistic, pharmacokinetic, and pharmacodynamic studies are essential. / Graduate

Several lines of schizophrenia (SZ) research suggest that a functional downregulation of the prefrontal cortex GABAergic neuronal system is mediated by a promoter hypermethylation, presumably catalyzed by an increase in DNA-methyltransferase-1 (DNMT-1) expression. This promoter hypermethylation may be mediated not only by DNMT-1 but also by an entire family of de novo DNA-methyltransferases, such as DNA-methyltransferase-3a (DNMT-3a) and -3b (DNMT-3b). To verify the existence of an overexpression of DNMT-3a and DNMT-3b in the brain of schizophrenia patients (SZP), we compared their mRNA expression in Brodmann's area 10 (BA10) and in the caudate nucleus and putamen obtained from the Harvard Brain Tissue Resource Center (Belmont, MA) from both nonpsychiatric subjects (NPS) and SZP. Our results demonstrate that DNMT-3a and DNMT-1 are expressed and co-localize in distinct GABAergic neuron populations whereas DNMT-3b mRNA is virtually undetectable. We also found that unlike DNMT-1, which is frequently overexpressed in telencephalic GABAergic neurons of SZP, DNMT-3a mRNA is overexpressed only in layer I and II GABAergic interneurons of BA10. To ascertain whether these DNMT expression differences observed in brain tissue could also be detected in peripheral tissues, we studied whether DNMT-1 and DNMT-3a mRNAs were overexpressed in peripheral blood lymphocytes (PBL) of SZP. Both DNMT-1 and DNMT-3a mRNAs are expressed in the PBL and although DNMT-3a mRNA levels in the PBL are approximately 1/10 of those of DNMT-1, the comparison of the PBL content in NPS and SZP showed a highly significant 2-fold increase of both DNMT-1 and DNMT-3a mRNA in SZP. These changes were unaffected by the dose, the duration, or the type of antipsychotic treatment. The upregulation of DNMT-1 and to a lesser extent that of DNMT-3a mRNA in PBL of SZP supports the concept that this readily available peripheral cell type can express an epigenetic variation of specific biomarkers relevant to SZ morbidity. Hence, PBL studies may become useful to investigate a diagnostic epigenetic marker of SZ morbidity.

La reelin est une glycoprotéine sécrétée de la matrice extracellulaire essentielle pour le développement embryonnaire des structures laminaires du système nerveux central (SNC): cortex, hippocampe et cervelet. Dans le cerveau postnatal et adulte, la reelin potentialise la plasticité synaptique, exerce une action trophique sur la croissance neuritique dans l’hippocampe et contrôle la maturation des récepteurs NMDA. Le but de ma thèse a été d’étudier les mécanismes cellulaires à l’origine des fonctions de la reelin dans la plasticité postnatale des structures stratifiées du SNC. Nous avons utilisé une stratégie intégrant des approches d’électrophysiologie, d’imagerie calcique, d’immunocytochimie, de biochimie et de pharmacologie, sur des modèles in vitro (culture primaires de neurones d’hippocampe et de cervelet) et ex vivo (tranches aigues de cortex frontal). Dans les neurones d’hippocampe in vitro, nous avons mis en évidence que la reelin est synthétisée et sécrétée par des neurones GABAergiques montrant un marquage reelin intense alors que les neurones cibles de la reelin sont caractérisés par une expression ponctiforme et de faible intensité. En revanche, dans le cervelet in vitro, les 2 fonctions, sécrétion et liaison de la reelin, sont assurées par la quasi totalité des cellules granulaires glutamatergiques. Nous avons finalement examiné les conséquences physiologiques de l’absence ou de la diminution de reelin endogène dans l’hippocampe et dans le cortex frontal. Nous avons mis en évidence que dans l’hippocampe in vitro la sécrétion continue de reelin régule l’homéostasie des récepteurs NMDA. Nous montrons également que dans le cortex frontal ex vivo, la reelin facilite la maturation des fonctions synaptiques glutamatergiques. Nos résultats démontrent donc que la reelin joue un rôle majeur dans la plasticité neuronale du SNC postnatal. / Reelin is an extracellular matrix protein essential for the correct formation of laminated structures during embryonic brain development. In the postnatal and adult brain, reelin promotes hippocampal dendrite development, enhances long term potentiation (LTP) at hippocampal synapses and favors the maturation of glutamatergic transmission. During my thesis, I studied the cellular mechanisms underlying the functions of reelin in laminated structures of the postnatal central nervous system: hippocampus, cerebellum and cortex. By combining immunocytochemical, biochemical and pharmacological approches, we first characterized the expression profile of reelin in primary cultures of hippocampal and cerebellar neurons. Our results showed that in the hippocampus reelin is synthesized and secreted by a population of GABAergic neurons expressing an intense reelin immunoreactivity (IR). We also showed that secreted reelin binds lipoprotein receptors present on a different neuronal population characterized by a punctate and light reelin IR. In contrast, in cerebellar cultures, we observed that reelin is synthesized and secreted by glutamatergic cells expressing a single type of reelin punctate and light staining. Using calcium imaging, we demonstrated that the continuous secretion of reelin is necessary to regulate glutamate receptor homeostasis and maintain the subunit composition of NMDARs in the hippocampus in vitro. We next examined the effect of decreased levels of reelin in the postnatal development of prefrontal cortex (PFC) glutamatergic synapses using electrophysiology on heterozygotes reeler mice (HRM) slices. Our data revealed that reelin facilitates the maturation of glutamatergic synaptic functions in the PFC and plays a central role in neuronal plasticity in the central nervous system.

Dans de nombreuses espèces doiseaux chanteurs (ou Passériformes), dont fait partie le canari domestique (Serinus canaria), le comportement de chant est produit à la fois pour défendre un territoire ou attirer un partenaire. Le Système de Conntrôle du Chant est un réseau nerveux central spécialisé, principalement localisé au niveau du télencéphale et associé au contrôle de lapprentissage, la perception et la production du chant. Ce comportement a été décrit subir la modulation de facteurs externes, tels que la testostérone, la photopériode et les interactions sociales. En parallèle avec le comportement de chant, certains des noyaux appartenant au Système de Contrôle du Chant (HVC, RA et Area X) présentent un phénomène de plasticité saisonnière nerveuse fascinante. Le volume de ces noyaux augmente notamment par espacement des cellules, agrandissement de la taille du neuropile et de larborisation dendritique et, dans le cas particulier dHVC, par incorporation de neurones nouveaux-nés. Nous proposons ici une synthèse de la littérature concernant ce phénomène tout à fait particulier ; en effet, la régénération des neurones du système nerveux central est considérée comme inexistante -ou uniquement limitée à la production de quelques interneurones- chez les mammifères. Létude de la neuroplasticité chez loiseau chanteur constitue dès lors un modèle tout à fait remarquable et offrant des perspectives nouvelles dans létude du cerveau des vertébrés.

<p>Autism is a pervasive developmental disorder (PDD) that&rsquo / s incidence is approximately 1 in 158. It is four times more prevalent in males than females and is believed to be caused by both genetic and environmental factors. Research indicates that several genes are involved in autism and it is believed that these genes act together to produce autism. Many genes implicated in this disorder are involved with brain structure formation and brain functioning. Studies have identified the reelin (RELN) gene as necessary for proper formation of brain, which indicates that RELN abnormalities could contribute to the aetiology of several neurogenetic diseases such as schizophrenia, bipolar and autism. The aims of the study were (i) to genotype two SNPs (exonic rs3622691 and intronic rs736707) in the RELN gene using Taqman&reg / SNP Genotyping assays to detect association with autism in three distinct South African (SA) ethnic groups (Black, Caucasian and Mixed), and (ii) to detect candidate genes that are over and under-expressed in the samples taken from a SA Caucasian autistic group and compare those with samples taken from a healthy Caucasian group using cDNA microarray. The Taqman&reg / study indicated significant association for the intronic SNP, rs736707, with a p-value of 0.0009 in the total SA group. More so, the Mixed group displayed the highest significance amongst the ethnic groups, with a p-value of 0.00014. The microarray study yielded 21 genes with 95% significance in the Caucasian sample group. Most genes were hypothetical proteins and formed part of the FAM90A family. The LOC83459 showed the highest level of expression in the autistic samples, while the BTNL8 gene was shown to be highly suppressed in the control samples.</p>

Amygdala kindling is commonly used to study the neural mechanisms of temporal lobe epilepsy and its behavioral consequences. The repetitive seizure activity that occurs during kindling is thought to induce an extensive array of structural and functional modifications within the brain, particularly in the hippocampus and dentate gyrus regions. Some of these changes include the growth or sprouting of new axonal connections as well as the birth and integration of new neurons into hippocampal circuits. Previous work has shown that these changes in structural and functional plasticity are not necessarily beneficial events. For instance, the growth and reorganization of synaptic terminals in the hippocampus and other brain regions might serve as a substrate that enhances hyperexcitability and seizure generation. In addition, although seizures induce the birth of new neurons, many of these newly generated cells migrate and function improperly within the hippocampal networks. Considering the prominent role of the hippocampus in a variety of behaviours, including learning, memory, and mood regulation, it would appear that alterations involving the structural and functional properties of both mature and newly born neurons in this region could impact these hippocampal-dependent functions. However, to date, the role of kindling-induced changes in hippocampal structural plasticity and neurogenesis on behaviour is incomplete, and the molecular mechanisms that govern these pathological events are poorly understood.<p/> The aim of this dissertation is to gain a better understanding of the changes in synaptic plasticity and neurogenesis within the hippocampus that occur after amygdala kindling. In chapter 2, we will examine if kindling alters the expression of synapsin I, a molecular marker of synaptic growth and activity, in both the hippocampus and other brain regions. In addition, we will also set out to determine if changes in synapsin I are related to the development of behavioural impairments associated with kindling. In chapter 3, the effect of kindling on hippocampal neurogenesis will be examined. In addition, we will also evaluate the effect of kindling on the expression of Reelin and Disrupted-in-Schizophrenia 1 (DISC1), two proteins instrumental for mediating proper neuronal migrational and maturation during development. In chapter 4, the effect of altered DISC1 expression in the dentate gyrus after kindling will be examined more extensively. We will examine whether altered DISC1 expression in the dentate contributes to some of the pathological features associated with seizure-induced hippocampal neurogenesis, such as ectopic cell migration and dentate granule cell layer dispersion. Finally, in chapter 5, the impact of aberrant seizure-induced neurogenesis on behaviour will be examined by determining if seizure-generated neurons functionally integrate and participate in hippocampal circuits related to memory processing. The results of this dissertation enhances our understanding of the functional consequences that altered hippocampal synaptic plasticity and neurogenesis may have on the development of epilepsy and emergence of cognitive impairments associated with chronic seizures.<p/>

Amygdala kindling is commonly used to study the neural mechanisms of temporal lobe epilepsy and its behavioral consequences. The repetitive seizure activity that occurs during kindling is thought to induce an extensive array of structural and functional modifications within the brain, particularly in the hippocampus and dentate gyrus regions. Some of these changes include the growth or sprouting of new axonal connections as well as the birth and integration of new neurons into hippocampal circuits. Previous work has shown that these changes in structural and functional plasticity are not necessarily beneficial events. For instance, the growth and reorganization of synaptic terminals in the hippocampus and other brain regions might serve as a substrate that enhances hyperexcitability and seizure generation. In addition, although seizures induce the birth of new neurons, many of these newly generated cells migrate and function improperly within the hippocampal networks. Considering the prominent role of the hippocampus in a variety of behaviours, including learning, memory, and mood regulation, it would appear that alterations involving the structural and functional properties of both mature and newly born neurons in this region could impact these hippocampal-dependent functions. However, to date, the role of kindling-induced changes in hippocampal structural plasticity and neurogenesis on behaviour is incomplete, and the molecular mechanisms that govern these pathological events are poorly understood.<p/> The aim of this dissertation is to gain a better understanding of the changes in synaptic plasticity and neurogenesis within the hippocampus that occur after amygdala kindling. In chapter 2, we will examine if kindling alters the expression of synapsin I, a molecular marker of synaptic growth and activity, in both the hippocampus and other brain regions. In addition, we will also set out to determine if changes in synapsin I are related to the development of behavioural impairments associated with kindling. In chapter 3, the effect of kindling on hippocampal neurogenesis will be examined. In addition, we will also evaluate the effect of kindling on the expression of Reelin and Disrupted-in-Schizophrenia 1 (DISC1), two proteins instrumental for mediating proper neuronal migrational and maturation during development. In chapter 4, the effect of altered DISC1 expression in the dentate gyrus after kindling will be examined more extensively. We will examine whether altered DISC1 expression in the dentate contributes to some of the pathological features associated with seizure-induced hippocampal neurogenesis, such as ectopic cell migration and dentate granule cell layer dispersion. Finally, in chapter 5, the impact of aberrant seizure-induced neurogenesis on behaviour will be examined by determining if seizure-generated neurons functionally integrate and participate in hippocampal circuits related to memory processing. The results of this dissertation enhances our understanding of the functional consequences that altered hippocampal synaptic plasticity and neurogenesis may have on the development of epilepsy and emergence of cognitive impairments associated with chronic seizures.<p/>