<p> Rod and cone photoreceptors in the retina are polarized sensory neurons that possess uniquely modified primary cilium, called the outer segment, to capture photons. Circadian-mediated shedding and renewal of outer segment membrane discs requires extensive vesicular transport of protein cargo from the endoplasmic reticulum and Golgi to the base of the cilium. Endocytosis is vesicle transport process of capturing and/or recycling extrinsic components and is shown to occur in retina of early vertebrates, such as <i>Xenopus</i> laevis. In this thesis, I have explored the hypothesis that a critical endocytosis-associated protein Rabgef1 is critical for the genesis of photoreceptor outer segments in the mammalian retina. After demonstrating high expression of Rabgef1 concordant with photoreceptor maturation, I characterized morphology and function of retina from <i>Rabgef1</i>-loss of function (<i>Rabgef1</i>^{&ndash;/&ndash;}) mice. Though no gross defect was observed by histology and immunohistochemistry before eye opening (postnatal day 14), transmission electron microscopy demonstrated ultrastructural defects in photoreceptor outer segments by P8. Progressive, yet rapid, photoreceptor degeneration and near-complete ablation of the visual response were evident at and after P15. I show that the outer segment defect noted in <i>Rabgef1</i>^{&ndash;/&ndash;} mice was not due to defective ciliogenesis or trafficking of cargo proteins to the cilium. In concordance with other systems, Rabgef1 was enriched in purified endocytic vesicles from the retina and interacted with Rabaptin5, confirming its role in Rab5-mediated endocytosis. Curiously, <i>Rabgef1</i>^{&ndash;/&ndash;} photoreceptors accumulated enlarged vesicular/endosomal structures within the inner segment, similar to loss of function mutations in the yeast orthologue of Rabgef1, Vps9p. My studies provide the first evidence of an essential role of Rabgef1-mediated fusion and recycling of endocytic vesicles in the formation and/or renewal of outer segment membrane discs in the mammalian retina. Rabgef1 and other components of the endocytic pathway should therefore be considered as candidates for human retinopathies. </p><p>

<p> Visual information is relayed from retina to the brain at first order synapses within the lateral geniculate nucleus (dLGN). During development, activity-dependent synaptic competition drives the segregation of retinal ganglion cell terminals into eye-specific zones. It has been assumed that the gain of synaptic transmission within these eye-specific zones is equivalent, providing uniform information transfer from the periphery to the CNS. Here, we revise this understanding. First, we demonstrate that anatomical segregation of retinal axons triggers a profound (200&ndash;300%) potentiation of neurotransmitter release selectively within the projection zone of the ipsilateral eye. Second, optogenetic recruitment of genetically defined axons within the ipsilateral projection zone provides evidence that functional synaptic connectivity is sub-stratified within the ipsilateral dLGN. Thus, we define a new functional organization within the dLGN and propose that synaptic competition acts as a developmental timer that triggers respecification of set point synaptic gain within the ipsilateral dLGN.</p><p>

<p> The dissertation consists of three parts. The first part is a systematic review of the literature regarding transcranial direct current stimulation (tDCS) and its effects on lower extremity motor behaviors and corticospinal excitability of the lower extremity representation of the motor cortex in healthy subjects. The second part investigates how different electrode montages and electrode conductance mediums affect corticospinal excitability of the tibialis anterior (TA) representation of the motor cortex in healthy subjects. The third part studies how different electrode montage and electrode conductance medium combinations affect ankle tracking accuracy in healthy subjects regarding the dominant lower extremity.</p><p>

<p> Similar to the pattern observed in people with substance abuse disorders, laboratory animals will exhibit escalation of cocaine intake when the drug is readily available and will exhibit increased drug-seeking behaviors after long periods of abstinence. Additionally, there are long term changes in neuron structure, receptor function, and neurotransmission associated with abstinence from cocaine in humans and animals. DNA methylation is an epigenetic modification to the DNA structure that mediates mRNA expression to confer different cell types, but has recently been implicated in learning and memory mechanisms. The long-term control that DNA methylation has over gene expression in animals makes it a prime candidate for controlling gene expression over the course of abstinence in animals with previous drug experience. Therefore, here, I investigated the contribution of behavioral contingency of cocaine administration on escalation of cocaine intake and re-exposure to cocaine cues as well as DNA methylation and gene expression within the dorsal medial prefrontal cortex (dmPFC) in adult male Sprague-Dawley rats. I exposed rats to daily training for saline (1 h/ day) or cocaine (0.25 mg/kg/inf) in limited- (1 h access per day), prolonged- (6 h access per day), or limited + yoked-access (1 h contingent + 5 h non-contingent access per day) for 15 days. Rats were then put through forced abstinence for 1, 14, or 60 days, and then the dmPFC was dissected out. Saline- and prolonged-access rats were additionally separated into cue- and no cue- conditions after 60 days of abstinence, where cue rats were re-exposed to the operant chamber without cocaine delivery for 2 h. These studies led to 4 main findings. 1) cocaine contingency affects mRNA expression for glutamatergic genes, 2) DNA methylation changes dynamically throughout abstinence, 3) re-exposure to cocaine cues rapidly alters DNA methylation and mRNA expression, and 4) DNA methylation, hydroxymethylation, and transcription factor binding all contribute to altered mRNA expression.</p><p>

<p> Elevations in extracellular glutamate in the brain are implicated in the pathogenesis of several neurological conditions, including mesial temporal lobe epilepsy. The underlying mechanisms of this elevation are not completely understood, however, and there are no effective methods that reduce the elevation or limit its neurotoxic effects. Glutamate is normally cleared from the extracellular space and replenished in axon terminals by a series of compartmentalized processes collectively known as the glutamate-glutamine cycle. A critical step of the cycle is the conversion of glutamate to glutamine by the astrocyte-specific enzyme glutamine synthetase. In several neurological conditions including mesial temporal lobe epilepsy, studies have demonstrated that glutamine synthetase activity is pathologically low. Because glutamine synthetase is thought to be critical for glutamate metabolism, its deficiency has been postulated as a possible mechanism for the increased glutamate observed in the extracellular fluid of the epileptogenic areas of the brain.</p><p> The branched-chain amino acids valine, leucine, and isoleucine are thought to contribute to <i>de novo</i> synthesis of glutamate in the brain by transferring an amino nitrogen to the tricarboxylic acid intermediate alpha-ketoglutarate. The branched-chain amino acids have gained increasing attention in recent years for the important roles they play in cell signaling, immune modulation, protein metabolism, and glutamate synthesis. It was previously unknown, however, if increasing peripheral branched-chain amino acids concentrations can increase extracellular glutamate concentrations in the brain during physiological or in pathological conditions, particularly when glutamate metabolism is perturbed (i.e. in glutamine synthetase deficiency). Moreover, the effects of branched-chain amino acids on seizures and neuronal viability were unknown.</p><p> The objective of this thesis was to use state of-the-art methods in microdialysis, isotope tracing, mass spectrometry and video-intracranial electroencephalogram recordings to study the metabolism and functional effects of branched-chain amino acids and glutamate in na&iuml;ve and glutamine synthetase-inhibited, epileptic rats. The central hypothesis was that increased extracellular concentrations of branched-chain amino acids in the brain, in combination with alterations in enzymatic processes of glutamate metabolism, are key pathogenic features that result in brain glutamate excess and seizures in mesial temporal lobe epilepsy. To achieve the objective of this thesis, we pursued 3 specific aims. </p><p> In Aim 1, we determined the effects of intravenous branched-chain amino acid administration on brain extracellular fluid concentrations of glutamate and glutamine in na&iuml;ve rats. We found that the administration of a high-dose bolus of branched-chain amino acids significantly increased the concentrations of branched-chain amino acids and glutamine in the extracellular compartment of the brain. Glutamate concentrations transiently increased, but the elevation was not statistically significant. In Aim 2, we determined the effects of intravenous isotope-labeled leucine administration on brain extracellular fluid concentrations of glutamate and glutamine in glutamine synthetase-inhibited rats. We found that glutamine synthetase-inhibited rats, like normal rats, were remarkably efficient in handling glutamate. Moreover, we demonstrated that leucine influx across the blood brain barrier is highly dependent on glutamine levels in the extracellular fluid of the brain. In Aim 3, we investigated the effects of chronic oral branched-chain amino acid supplementation on spontaneous and induced seizures, and neuron loss in glutamine synthetase-inhibited epileptic rats. We found that the branched-chain amino acid supplementation was ineffective in reducing the frequency and severity of spontaneous seizures, but increased the threshold to pentylenetetrazole-induced seizures. Furthermore, chronic branched-chain amino acid supplementation resulted in increased loss of hippocampal hilar neurons. Future studies will explore the impact of glutamine and leucine dysregulation in the brain on cell signaling and immune modulation, which may play an important role in epilepsy as well as other disorders. We will also further explore the mechanisms underlying the branched-chain amino acid-induced neuron loss.</p><p>

<p> Parkinson&rsquo;s disease (PD) is an adult onset neurodegenerative disease that is characterized by deficiencies in movement, cognition, and Lewy body neuropathology within the brain. Motor and cognitive deficiencies progressively worsen through the course of disease concurrent with increasing neuropathology and neurodegeneration. Approximately 10&ndash;15% of PD patients have a family history of PD with a confirmed genetic cause. Presently PD pathogenesis is incompletely understood and there are no treatments capable of halting or reversing this disease. The extended disease-course and age-dependent nature of PD, especially in genetic cases where a mutation is present from birth, affirm that aging itself is the most important risk factor for disease. We hypothesize that specific cellular changes that occur during the normal process of aging confer susceptibility to disease-causing mutations which, while tolerated at younger ages, contribute to disease with age. Accurate animal models of PD and aging provide the ability to elucidate disease mechanisms and explore novel strategies targeting the aging process. To test the role of aging in PD we utilize the nematode <i>Caenorhabditis elegans</i> because this animal has been used extensively to study animal aging at a cellular level. We confirm that disease phenotypes in genetic <i>C. elegans</i> models of PD such as neurodegeneration, protein aggregation, and mitochondrial deficits are proportional to this organism&rsquo;s brief lifespan. This indicates that PD progresses according to biological age and not merely to chronological time. As a proof-of-principle we also show that delaying aging by mutation of the gene encoding the insulin-IGF receptor, <i>daf-2</i>, can rescue multiple deficits present in nematode models of PD. Overall we demonstrate that biological aging is a crucial for the development of various PD associated phenotypes and that delaying aging is sufficient to delay these phenotypes. Therefore targeting aging itself may be a sound strategy for the halting or the prevention of PD.</p><p>

<p> Through a mixed methodology of hermeneutic and heuristic inquiry, this thesis examines the influence of early childhood numinous, or profound spiritual, experience on the developing psyche and its relationship to repetitive compulsion over the lifespan. A dual lens of depth psychology and neuroscience is employed to explore the interstitial spaces between theories on the numen, early psychological development, and the repetition compulsion, comparing the literature with personal and clinical examples. Findings illustrated that numinous experiences are intimately connected with repetitive symptomology and that certain individuals are genetically predisposed to numinous experience. The hypothesis was proposed that repetition compulsion engendered by early numinous experiences, and perhaps all repetition phenomena, might reflect an unconscious <i> telos</i>, or the aim of an integral part of one&rsquo;s psyche seeking expression, integration, and relationship in the world.</p><p>

La libération de neurotransmetteur est provoquée par la dépolarisation de la terminaison présynaptique et l’entrée de calcium à travers les canaux calciques voltagedépendants (VDCCs). Les VDCCs à haut seuil de type-P/Q et de type-N sont classiquement impliqués dans la libération de neurotransmetteurs et sont localisés dans la terminaison axonale près de la zone active. Deux membres de la famille des VDCCs de type-L, Cav1.2 et Cav1.3 sont connus pour être exprimés dans le système nerveux central. Dans le cortex cérébelleux, les propriétés pharmacologiques des VDCCs présynaptiques ont été examinées aux synapses GABAergiques entre les interneurones de la couche moléculaire (MLIs) et entre les MLIs et les cellules de Purkinje. Bien qu’il n’y ait aucun doute que les VDCCs de type- P/Q et de type-N sont les principaux acteurs de l’entrée de calcium présynaptique et de la libération de GABA par les MLIs, l’absence d’effet des dihydropyrines antagonistes a exclut le potentiel rôle des VDCCs de type-L (Forti et al., 2000; Stephens et al., 2001). Il est intéressant de noter que les dihydropyrines antagonistes sont très peu efficaces sur les courants calciques de type-L activés par un potentiel d’action (Helton et al., 2005), ce qui suggère que l’implication des VDCCs de type-L dans la libération de neurotransmetteur a été largement négligée. Dans cette étude, nous avons montré que le BayK8644 (une dihydropyridine agoniste) augmente fortement la fréquence des mIPSCs enregistrés dans les MLIs et dans les cellules de Purkinje suggérant que les VDCCs de type-L peuvent être présents dans les terminaisons axonales des MLIs. Ce résultat a été confirmé par des expériences d’immunohistochimie utilisant la microscopie confocale et électronique ainsi que par des expériences d’imagerie calcique. Nos résultats démontrent que les VDCCs de type-L, souvent négligés dans les terminaisons axonales, ont un rôle crucial dans la libération de GABA par les MLIs. / Physiological involvement of presynaptic L-type voltage dependent calcium channels in GABA release of cerebellar molecular layer interneurons

<p> Working memory is a collection of cognitive resources that allow for the temporary maintenance and manipulation of information. This information can then be used to accomplish task goals in a variety of different contexts. To do this, the working memory system is able to process many different kinds of information using resources dedicated to the processing of those specific types of information. This processing is modulated by a control component which is responsible for guiding actions in the face of interference. Recently, the way in which working memory handles the processing of this information has been the subject of debate. Specifically, current models of working memory differ in their conceptualization of its functional architecture and the interaction between domain-specific storage structures and domain-general control processes. Here, domain-specific processing is when certain components of a model are dedicated to processing certain kinds of information, be it spatial or verbal. Domain-general processing is a when a component of a model can process multiple kinds of information. One approach conceptualizes working memory as consisting of various discrete components that are dedicated to processing specific kinds of information. These multiple component models attempt to explain how domain-specific storage structures are coordinated by a domain-general control mechanism. They also predict that capacity variations in those domain-specific storage structures can directly affect the performance of the domain-general control mechanism. Another approach focuses primarily on the contributions of a domain-general control mechanism to behavior. These controlled attention approaches collapse working memory and attention and propose that a domain-general control mechanism is the primary source of individual differences. This means that variations in domain-specific storage structures are not predicted to affect the functioning of the domain-general control mechanism. This dissertation will make the argument that conceptualizing working memory as either domain-specific or domain-general creates a false dichotomy. To do this, different ways of measuring working memory capacity will first be discussed. That discussion will serve as a basis for understanding the differences, and similarities between both models. A more detailed exposition of both the multiple component model and controlled attention account will follow. Behavioral and physiological evidence will accompany the descriptions of both models. The emphasis of the evidence presented here will be on load effects: observed changes in task performance when information is maintained in working memory. Load effects can be specific to the type of information being maintained (domain-specific), or occur regardless of information type (domain-general). This dissertation will demonstrate how the two models fail to address evidence for both domain-specific and domain-general load effects. Given these inadequacies, a new set of experiments will be proposed that will seek to demonstrate both domain-specific and domain-general effects within the same paradigm. Being able to demonstrate both these effects will go some way towards accounting for the differing evidence presented in the literature. A brief conceptualization of a possible account to explain these effects will then be discussed. Finally, future directions for research will be described.</p><p>

<p> Cannabinoid signaling is known to modulate behavioral and physiological responses associated with stress, and could be manipulated in the management of stress-aggravated neuropsychiatric disorders. However, there is still a limited understanding of how individual components of this signaling system contribute to stress responsivity. Zebrafish are a genetically tractable vertebrate model that would be particularly useful for studying this interface. Accordingly, we utilized genome editing technology to provide a platform for studying how cannabinoid signaling modifies stress responses in zebrafish. <b>Chapter 1</b> reviews how zebrafish have been leveraged in the field of cannabinoid biology. <b>Chapter 2</b> describes the deployment of transposon technology to develop the SR4G zebrafish stress response reporter line, which is used to show that cannabinoid signaling impacts the transcriptional activity of glucocorticoid receptors. <b>Chapter 3</b> details the deployment of nuclease technology to develop lines with mutations in genes encoding components of the endocannabinoid system, which are used to establish roles for these genes in modulating stress-associated behavior. <b>Chapter 4</b> concludes with a summary of this thesis, and synthesizes content from the preceding chapters in a discussion about potential future research directions. </p><p>