Estrogen receptor (ER) involvement through genomic mechanisms have been well established in different aspects of cognition and neuronal plasticity. However, much less is known about the rapid, non-genomic effects on these processes. G-protein coupled estrogen receptor (GPER) is a relatively recently discovered estrogen receptor that has previously been shown to affect learning and memory on a long term genomic timescale. This thesis investigated the rapid effects of the GPER selective agonist G-1 on four estrogen-sensitive learning paradigms: social recognition, object recognition, object placement and social transmission of food preferences (STFP) paradigm. The rapid effects of G-1 on neuronal plasticity were also examined through changes in hippocampal dendritic spine length and density. Results show that GPER activation may rapidly enhance social recognition, object recognition and object placement learning while may impair social learning in the STFP paradigm. G-1 treatment also resulted in increases of dendritic spine density in the stratum radiatum of the CA1 hippocampus. This suggests that GPER, along with the classical ERs, mediates the rapid effects of estrogen on learning and neuronal plasticity. Thus, GPER may be a viable target for enhancing cognitive function in estrogen related therapies.

Most stroke survivors recover some degree of lost function after an ischemic event. Recovery however, is negatively affected by comorbid conditions such as diabetes. Successful recovery is dependent on the ability of adjacent surviving cortical tissue and functionally related areas to take over functions lost by the stroke. Recently our lab has shown that diabetes interferes with the remapping of sensory function to peri-infarct areas after photothrombotic stroke. Given this result, it is crucial to understand how diabetes affects the structure of neurons following stroke, particularly at the level of dendritic spines, which receive the vast majority of excitatory synaptic inputs. Type I diabetes was pharmacologically induced in transgenic mice expressing yellow fluorescent protein (YFP) in a subset of cortical neurons 4 weeks prior to receiving unilateral photothrombotic stroke in the forelimb area of the primary somatosensory cortex (FLS1). Spine density measurements were made on the apical and basilar dendrites of layer-5 pyramidal neurons at 1 and 6 weeks after stroke. Our analysis indicated that diabetes was associated with fewer apical and basilar dendritic spines in the peri-infarct region 1 week after stroke. At 6 weeks of recovery, peri-infarct dendritic spine density in both control and diabetic animals returned to baseline levels. These changes were specific to the peri-infarct cortex, as spine density in distant cortical areas such as the forelimb sensorimotor region of the contralateral hemisphere, were not affected by stroke. In order to relate changes in spine density to the recovery of forepaw function, we re-analyzed data from a previous study that employed the forepaw adhesive-tape-removal test (Sweetnam et al 2012). This analysis revealed that diabetes significantly increased the latency of tape removal from the impaired forepaw (when normalized to the unaffected paw) at 1 but not 6 weeks of recovery. Collectively, these findings indicate that diabetes exacerbates forepaw impairments and basilar spine loss initially after stroke, but does not affect the ability of the brain to replace lost spines over weeks of recovery. / Graduate / 0317

Thesis (Ph.D.)--Virginia Commonwealth University, 2006. / Title from title-page of electronic thesis. Prepared for: Dept. of Pharmacology & Toxicology. Bibliography: leaves [190]-207.

The current study aimed to determine the selective advantage of lateral amygdala (LA) neurons overexpressing the transcription factor CREB that enables their preferential incorporation into the fear memory trace. I hypothesized that overexpression of CREB drives the formation of dendritic spines, potentially providing these neurons with greater connectivity to sensory inputs at the time of learning. Using viral-mediated gene transfer, CREB tagged with GFP, or GFP as a control, was overexpressed in the LA of wild-type mice. Spine number and morphology were compared in homecage mice at the time when mice are normally trained in fear conditioning. Spine density was increased in neurons with CREB vector compared to neurons with GFP vector whereas spine head diameter and length was not different. Therefore, LA neurons overexpressing CREB have increased spine number at the time of learning, potentially providing these neurons with a selective advantage for incorporation into the fear memory trace.

The current study aimed to determine the selective advantage of lateral amygdala (LA) neurons overexpressing the transcription factor CREB that enables their preferential incorporation into the fear memory trace. I hypothesized that overexpression of CREB drives the formation of dendritic spines, potentially providing these neurons with greater connectivity to sensory inputs at the time of learning. Using viral-mediated gene transfer, CREB tagged with GFP, or GFP as a control, was overexpressed in the LA of wild-type mice. Spine number and morphology were compared in homecage mice at the time when mice are normally trained in fear conditioning. Spine density was increased in neurons with CREB vector compared to neurons with GFP vector whereas spine head diameter and length was not different. Therefore, LA neurons overexpressing CREB have increased spine number at the time of learning, potentially providing these neurons with a selective advantage for incorporation into the fear memory trace.

Fragile X Syndrome (FXS) is a prevalent monogenic disease, often presenting with cognitive and neurological disorders including autism and epilepsy. The Fmr1 gene - transcriptionally silenced in FXS - normally encodes the Fragile X Mental Retardation Protein (FMRP), which acts as an activity dependent translational regulator at the base of dendritic spines. In an attempt to understand its role, dendritic spines in the dentate gyrus (DG) and cornu ammonis 1 (CA1) hippocampal regions of three-week old Fmr1- mice were analyzed and compared to wildtype (WT) littermate controls using electron microscopy. Dendritic spines with a continuous profile of the parent dendrite, spine neck, and spine head complete with synaptic components (presynaptic vesicles and postsynaptic densities) were included in our morphological analyses. We observed no changes in postsynaptic density length (DG: 5.69±0.30/6.18±0.85; SR: 7.55±0.87/6,96±0.33 µm/100 µm2; p=0.627/0.620), synapse density (DG: 32.3±3.8/30.3±1.9; SR: 34.4±1.8/30.7±0.5 synapses/100 µm2; p=0.655/0.270), spine head diameters (DG: 0.524±0.016/0.529±0.014; SR: 0.524±0.014/0.515±0.014 µm; p=0.098/0.20) or spine neck lengths (DG: 0.457±0.016/0.485±0.019; SR: 0.421 ± 0.015/0.425±0.017 µm; p=0.14/0.26), but found that in the DG spine necks were significantly narrower in the Fmr1- mice (0.193±0.0062/0.167±0.0064 µm; p=0.0002), whereas there were no changes in CA1 spine neck widths (0.162±0.0049/0.161±0.0061 µm; p=0.073). Estimated resistance calculated from spine necks morphologies revealed a ~1.7 fold increase in the Fmr1- DG compared to WT DG. These findings support that FMRP plays a role in granule cell spine neck structure and may influence synaptic signal compartmentalization and propagation in a regionally dependent manner. / Graduate

Social isolation during adolescence can have long lasting negative effects in both humans and animal models. In mice, post-weaning social isolation leads to increased addiction-like behaviors in adulthood. However, little is known about how post-weaning social isolation alters the brain. Stress during development can lead to persistent restructuring of neurons. Changes in dendritic spines can be long-lasting and have been theorized to play an important role in the maintenance of cocaine craving. We found that post-weaning isolation led to a persistent increase in spine density in adulthood within both the core and shell regions of the nucleus accumbens in male mice, but not female mice. In contrast, in the infralimbic cortex, post-weaning social isolation led to an increase in spine density only in female mice. This study highlights the long-lasting, sex-specific effects of post-weaning isolation. Microglia have been shown to assist in both the formation and elimination of dendritic spines, and are activated following exposure to stress and cocaine. Therefore, we hypothesized that microglia may be involved in the restructuring of dendritic spines during post-weaning isolation, and contribute to addiction-like behavior in adulthood. We examined whether inhibiting microglia with minocycline during the first three weeks of post-weaning isolation altered the impact of isolation in cocaine seeking. Isolated animals that received minocycline showed increased cocaine seeking in adulthood compared to group housed mice and isolated mice that received saline. Minocycline and isolation also caused sex-specific alterations in spine density. The findings of these studies provide insight into the mechanisms by which social isolation during adolescence increases vulnerability to addiction later in life. / Psychology

Neurodevelopmental disorders (NDDs) are a heterogeneous set of disorders that are characterized by early disruptions to brain development and include autism spectrum disorder (ASD), attention deficit/hyperactivity disorder (ADHD), developmental delay (DD), intellectual disability (ID), epilepsy and schizophrenia (SZ). Although thousands of genetic risk variants have been identified, there is a lack of understanding of how they impact cellular and molecular mechanisms that underlie the clinical presentation and heterogeneity of NDDs. To investigate this, we used a combination of cellular, molecular, bioinformatic and omics methods to study NDD-associated molecular pathways in distinct neuronal populations. First, we studied the interaction between the high-confidence SZ risk genes DISC1 and NRG1-ErbB4 in cortical inhibitory neurons and found that NRG1-ErbB4 functions through DISC1 to regulate dendrite growth and excitatory synapses onto inhibitory neurons. Next, we studied the 15q13.3 microdeletion, a recurrent copy number variation (CNV) that is associated with multiple NDDs. Using a heterozygous mouse model [Df(h15q13)/+] and human sequencing data we identified OTUD7A (encoding a deubiquitinase) as an important gene driving neurodevelopmental phenotypes in the 15q13.3 microdeletion syndrome. Due to the paucity of literature on the function of OTUD7A in the brain, we used a proximity-labeling approach (BioID2) to elucidate the OTUD7A protein interaction network (PIN) in cortical neurons, and to examine how patient mutations affect the OTUD7A PIN. We found that the OTUD7A PIN was enriched for postsynaptic and axon initial segment proteins, and that distinct patient mutations have shared and distinct effects on the OTUD7A PIN. Further, we identified the interaction of OTUD7A with a high-confidence bipolar risk gene ANK3, which encodes AnkyrinG. We identified decreased levels of AnkyrinG in Df(h15q13)/+ neurons, and synaptic phenotypes were rescued by increasing AnkyrinG levels or targeting the Wnt pathway. Future investigation should include examination of the role of OTUD7A deubiquitinase activity in neural development. / Dissertation / Doctor of Philosophy (PhD) / Neurodevelopmental disorders result from disruptions to early brain development and include autism spectrum disorder (ASD), developmental delay (DD), epilepsy, and schizophrenia (SZ). These disorders affect more than 3% of children worldwide and can have a significant impact on an individual’s quality of life, including an increased risk of death in some cases. There is currently a lack of understanding of how these disorders develop and how to effectively treat them. Neurodevelopmental disorders are thought to arise from alterations in the connections between brain cells (neurons) and one of the major risk factors for these disorders is having certain variations in regions of the genome (DNA sequences), with more than 1000 of these risk variants having been identified so far. In this thesis, we analyzed how genetic risk factors interact in neurons to regulate neural connectivity. We discovered that risk variants found in individuals with different disorders actually work together to regulate similar processes important for neural connectivity, which suggests that distinct disorders may share a common underlying cause. Additionally, we established the importance of a new ASD risk gene and discovered that it interacts with other known risk genes to regulate neural connectivity. This thesis provides new insights into the processes in the brain that lead to neurodevelopmental disorders and has implications for future development of effective therapies for individuals affected by these disorders.

The majority of fast excitatory neurotransmission in the brain occurs at glutamatergic synapses. The extensive dendritic arborisations of pyramidal neurons in the neocortex and hippocampus harbor thousands of synaptic connections, each formed on tiny protrusions called dendritic spines. Spine synapses are rapidly established during early postnatal development – a key period in neural circuit assembly – and are subject to dynamic activity-dependent plasticity mechanisms that are believed to underlie neural information storage and processing for learning and memory. Recent decades have seen remarkable progress in identifying diverse plasticity mechanisms responsible for regulating synapse structure and function, and in understanding the processes underlying computation of synaptic inputs in the dendrites of individual neurons. These advances have strengthened our understanding of the biological mechanisms underlying brain function but, not surprisingly, they have also raised many new questions. Using a combination of whole-cell electrophysiology, 2-photon imaging and glutamate uncaging in rodent brain slice preparations, I have helped to document the subtype-specific regulation of glutamate receptors during a homeostatic form of synaptic plasticity at CA1 pyramidal neurons of the hippocampus, and have discovered novel synaptic calcium dynamics during a critical period of neural circuit formation. First, we found that during a homeostatic response to prolonged inactivity, both AMPA and NMDA subtypes of glutamate receptors undergo a switch in subunit composition at synapses, but exhibit a divergence in their subcellular localization at extrasynaptic regions of the plasma membrane (this work was published in the Journal of Neuroscience in 2013). In separate series of experiments using 2-photon calcium imaging, I discovered a functional coupling between NMDA receptor activation and intracellular calcium release at dendritic spines and dendrites that is selectively expressed during a critical period of synapse formation. This synaptic calcium signaling mechanism enabled the transformation of distinct spatiotemporal patterns of synaptic input into salient biochemical signals, and is thus apt to locally regulate synapse development along individual dendritic branches. Consistent with this hypothesis, I found evidence for non-random clustering of synapse development between neighboring dendritic spines. Together, these experimental results expand the current understanding of the dynamics of synapse function during homeostatic plasticity and early postnatal development. --- Les synapses glutamatergiques soutiennent la majorité de la neurotransmission excitatrice rapide du cerveau. Des milliers de ces synapses, localisées sur de minuscules saillies appelées épines dendritiques, décorent les vastes arborisations dendritiques des neurones pyramidaux du néocortex et de l'hippocampe. Ces synapses sont formées tôt lors du développement postnatal et sont soumises à des mécanismes dynamiques de plasticité qui sous-tendent, croit-on, les capacités d'apprentissage et de mémoire du cerveau. Les dernières décennies ont vu des progrès remarquables dans l'identification de divers mécanismes de régulation de la structure et de la fonction des synapses sur différentes échelles de temps, et dans la compréhension des processus qui régissent l’intégration des inputs synaptiques au niveau des dendrites individuelles. Ces progrès ont renforcé notre compréhension des éléments fondamentaux régissant la fonction cérébrale et ont ouvert de nouvelles voies d’investigations neurophysiologiques. En utilisant une combinaison d’électrophysiologie cellulaire, d'imagerie à deux-photons et de photolibération de glutamate sur des neurones pyramidaux de la région CA1 de l'hippocampe de rats, j’ai contribué à la découverte et à la caractérisation de nouvelles régulations des récepteurs du glutamate durant la plasticité synaptique homéostatique. J’ai également découvert un nouveau type de dynamique de calcium synaptique relié à une organisation spatiale du développement des synapses pendant une période critique de l’ontogénie des circuits neuronaux. Dans la première étude, nous avons constaté que lors d'une plasticité de type homéostatique induite par une inactivité prolongée, les récepteurs de glutamate de types AMPA et NMDA sont soumis à un changement important dans la composition de leurs sous-unités. De plus, nous avons observé un ciblage différentiel de ces récepteurs vers des compartiments subcellulaires spécifiques des neurones. Dans une série d'expériences séparée utilisant l’imagerie calcique à deux-photons, j’ai découvert un couplage fonctionnel durant le développent entre l'activation des récepteurs NMDA et une libération de calcium intracellulaire qui envahit tant les épines dendritiques que les dendrites. J’ai également trouvé que ce mécanisme de signalisation de calcium synaptique transforme des motifs spatiotemporels d’activités synaptiques spécifiques en signaux biochimiques post-synaptiques de manière à potentiellement réguler l’organisation spatiale des synapses durant le développement. Conformément à cette hypothèse, j’ai observé des manifestations fonctionnelles claires de regroupement dans l’espace de synapses de forces similaires le long de branches dendritiques individuelles. Ensemble, ces résultats expérimentaux élargissent notre compréhension actuelle de de la fonction des synapses durant la plasticité homéostatique ainsi que durant le développement postnatal du cerveau. En étudiant les mécanismes neurophysiologiques de base, il sera possible d'avoir un aperçu plus profond du fonctionnement du cerveau et de ses pathologies.

Excitotoxic calcium influx and activation of calcium-regulated systems is a common event in several types of neuronal injury. This mechanism has been the focus of intense research, with the hope that a more complete understanding of how neuronal injury affects calcium-regulated systems will provide effective treatment options. This study examines one such calcium-stimulated enzyme, calcineurin, in the context of two common neurological pathologies, status epilepticus and traumatic brain injury.Status epilepticus was induced by pilocarpine injection. NMDA-dependent increases in calcineurin activity were observed in cortical and hippocampal homogenates. Upon closer examination, the most profound increases in activity were found to be present in crude synaptoplasmic membrane fractions isolated from cortex and hippocampus. A concurrent status epilepticus-induced increase in calcineurin concentration was observed in membrane fractions from cortex and hippocampus. Immunohistochemical analysis revealed an increase in calcineurin immunoreactivity in apical dendrites of hippocampal pyramidal neurons. We examined a cellular effect of increased dendritic calcineurin activity by characterizing a calcineurin-dependent loss of dendritic spines. Increased dendritic calcineurin led to increased dephosphorylation and activation of cofilin, an actin-depolymerizing factor. Calcineurin-activated cofilin induced an increase in actin depolymerization, a mechanism shown to cause spine loss in other models. Finally, via Golgi impregnation, we demonstrated that status epilepticus-induced spine loss is blocked by calcineurin inhibitors.To demonstrate that the increase in dendritic calcineurin activity was not model-specific, we examined a moderate fluid-percussion model of brain injury. Calcineurin activity was significantly increased in hippocampal and cortical homogenates. This increased activity persisted for several weeks post-injury, and may be involved in injury-induced neuronal pathologies. Also similar to the SE model, calcineurin immunoreactivity was dramatically increased in synaptoplasmic membrane fractions from cortex and hippocampus, and immunohistochemistry revealed increased calcineurin content in dendrites of hippocampal CA1-3 pyramidal neurons. These changes in calcineurin distribution also persisted for several weeks post-injury.These studies demonstrate a novel, cellular mechanism of calcium-mediated pathology in two models of neuronal injury. Elucidation of cellular events involved in the acute and chronic effects of brain trauma is essential for the development of more effective treatment options.