D-Serine is regarded as an obligatory co-agonist required for the activation of NMDA-type glutamate receptors (NMDARs). In the retina D-serine and a second NMDAR coagonist, glycine, are present at similar concentration and the cells that produce and release them are in close apposition. This arrangement allows for an abundant supply of coagonists and under certain conditions the NMDAR coagonist binding site could be saturated. There is also evidence suggesting that D-serine can act in an inhibitory manner at AMPA/kainate-type glutamate receptors (GluRs). Glutamate receptor activation can lead to direct and indirect elevation of intracellular calcium (Ca2+) concentration ([Ca2+]i). Therefore, in this thesis, I predominantly used Ca2+ imaging techniques to study the effect of D-serine on GluR activation in the mammalian retina. I first describe a novel method I developed to load retinal cells with Ca2+ indicator dye using electroporation and show that retinas remain viable and responsive following electroporation. This technique was used to explore the excitatory role of D-serine at NMDARs and its potential inhibition of AMPA/kainate receptors using cultured retinal ganglion cells (RGCs) and isolated retina preparations. Using cultured RGCs I demonstrated that D-serine and glycine enhance NMDAR-mediated Ca2+ responses in a concentration-dependent manner and are equally effective as coagonists. In isolated retinas I showed that D-serine application enhanced NMDA-induced responses consistent with sub-saturating endogenous coagonist concentration. Degradation of endogenous D-serine reduced NMDAR-mediated Ca2+ responses supporting the contribution of this coagonist to NMDAR activation in the retina. Using imaging and two different electrophysiological approaches, I found that D-serine reduced AMPA/kainate receptor-mediated responses in cultured RGCs and isolated retinas at concentrations that are saturating at NMDARs. Antagonist experiments suggest that the majority of inhibition is due to D-serine acting on AMPA receptor activity. Degradation of endogenous D-serine enhanced AMPA/kainate-induced responses of some cells in isolated retina suggesting that, under these conditions, D-serine concentration may be sufficient to inhibit AMPA receptor activity. Overall, the work in this thesis illustrates the utility of electroporation as a method to load Ca2+-sensitive fluorescent dyes into retinal cells and highlights the potential role for D-serine as a modulator of ionotropic GluRs in the CNS.

N-methyl D-aspartate (NMDA) receptors represent integral signal transducers for excitatory glutamate neurotransmission. While NMDA receptors are critical for synaptic plasticity, the molecular events underlying this process are not fully elucidated. The potential role of NP22, a novel neuronal protein, as a downstream mediator of NMDA receptor function is explored. NP22 protein expression in genetic and pharmacological models of NMDA receptor hypofunction is examined and no significant changes are reported. Characterization of the NP22 protein complex via tandem-affinity and FLAG-purification coupled with mass spectrometry was used and no novel protein interactions are reported. GFP-tagged NP22 colocalization with F-actin decreases in cell processes of transiently transfected HEK293 cells in response to elevated intracellular calcium, while similar colocalization reductions are not seen in stably transfected HEK293 under a comparable treatment regiment. Changes in intracellular calcium affecting NP22 biology can be useful in the ongoing characterization of this novel protein.

Impulse control, an executive process that restrains inappropriate actions, is impaired in numerous psychiatric conditions. This thesis reports three experiments that utilized a novel animal model of impulse control, the response inhibition (RI) task, to examine the substrates that underlie learning this task. In the first experiment, rats were trained to withhold responding on the RI task, and then euthanized for electrophysiological testing. Training in the RI task increased the AMPA/NMDA ratio at the synapses of pyramidal neurons in the prelimbic, but not infralimbic, region of the medial prefrontal cortex. This enhancement paralleled performance as subjects underwent acquisition and extinction of the inhibitory response. AMPA/NMDA was elevated only in neurons that project to the ventral striatum. Thus, this experiment identified a synaptic correlate of impulse control. In the second experiment, a separate group of rats were trained in the RI task prior to electrophysiological testing. Training in the RI task produced a decrease in membrane excitability in prelimbic, but not infralimbic, neurons as measured by maximal spiking evoked in response to increasing current injection. Importantly, this decrease was strongly correlated with successful inhibition in the task. Fortuitously, subjects trained in an operant control condition showed elevated infralimbic, but not prelimbic, excitability, which was produced by learning an anticipatory signal that predicted imminent reward availability. These experiments revealed two cellular correlates of performance, corresponding to learning two different associations under distinct task conditions. In the final experiment, rats were trained on the RI task under three conditions: Short (4-s), long (60-s), or unpredictable (1-s to 60-s) premature phases. These conditions produced distinct errors on the RI task. Interestingly, amphetamine increased premature responding in the short and long conditions, but decreased premature responding in the unpredictable condition. This dissociation may arise from interactions between amphetamine and underlying cognitive processes, such as attention, timing, and conditioned avoidance. In summary, this thesis showed that learning to inhibit a response produces distinct synaptic, cellular, and pharmacological changes. It is hoped that these advances will provide a starting point for future therapeutic interventions of disorders of impulse control. / Thesis (Ph.D, Neuroscience Studies) -- Queen's University, 2011-07-11 09:44:54.815

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

N-methyl D-aspartate (NMDA) receptors represent integral signal transducers for excitatory glutamate neurotransmission. While NMDA receptors are critical for synaptic plasticity, the molecular events underlying this process are not fully elucidated. The potential role of NP22, a novel neuronal protein, as a downstream mediator of NMDA receptor function is explored. NP22 protein expression in genetic and pharmacological models of NMDA receptor hypofunction is examined and no significant changes are reported. Characterization of the NP22 protein complex via tandem-affinity and FLAG-purification coupled with mass spectrometry was used and no novel protein interactions are reported. GFP-tagged NP22 colocalization with F-actin decreases in cell processes of transiently transfected HEK293 cells in response to elevated intracellular calcium, while similar colocalization reductions are not seen in stably transfected HEK293 under a comparable treatment regiment. Changes in intracellular calcium affecting NP22 biology can be useful in the ongoing characterization of this novel protein.

Chronic ethanol (EtOH) exposure produces neuroadaptations within the NMDA receptor system and alterations in HPA axis functioning that contribute to neurodegeneration during ethanol withdrawal (EWD). Chronic EtOH exposure and EWD, as well as corticosteroids, also promote increased synthesis and release of polyamines, which allosterically potentiate NMDA receptor open-channel time at the NR2B subunit. The current studies investigated effects of 10 day EtOH and corticosterone (CORT) co-exposure on toxicity during EWD in rat organotypic hippocampal slice cultures, and alterations in function and/or density of the NR2B subunit of the NMDA receptor that may mediate CORT-potentiation of toxicity during EWD. We hypothesized that toxicity during withdrawal following EtOH and CORT co-exposure would be greatest in the CA1 region due to increased NMDA NR2B receptor abundance and/or function. Cultures were exposed to CORT (0.01–1 μM) during 10 day EtOH exposure (50 mM) and 1 day EWD. Additional EtOH-naïve cultures were exposed to CORT for 11 days. Propidium iodide (PI) was used to measure toxicity in the CA1, CA3, and DG hippocampal regions. In EtOH-naïve cultures, 11 day exposure to CORT (0.01 – 1 μM) produced modest toxicity and in all regions. Exposure to CORT during EtOH exposure/EWD potentiated CORT-toxicity at all concentrations in the CA1 region. Ifenprodil, an NR2B polyamine site antagonist, significantly reduced toxicity from EtOH and CORT (0.1 μM) co-exposure during withdrawal. Immunohistochemistry and Western blot analyses were conducted for measurement of NR2B immunoreactivity in organotypic cultures, and autoradiography studies were conducted for measurement of polyamine-sensitive NR2B subunits with [3H]ifenprodil. Consistent increases in NR2B subunit protein were not detected with use of any methodology. Additional studies exposed cultures to a membrane impermeable form of CORT (BSA-conjugated CORT; 0.1 μM) with or without EtOH exposure and withdrawal. BSA-CORT exposure did not produce toxicity in any hippocampal region, suggesting that CORT toxicity was not mediated by membrane bound substrates. These data suggest that CORT and EtOH co-exposure result in increased function of polyamine-sensitive NR2B subunits, but this toxicity does not appear dependent on the number of hippocampal NMDA NR2B subunits.

The dorsal vagal complex (DVC), containing the nucleus of the solitary tract (NTS) and the dorsal motor nucleus of the vagus nerve (DMV), plays a pivotal role in autonomic regulation. Afferent fibers from peripheral organs and higher brain centers synapse in the NTS, which integrates these synaptic connections as well as information from systemically circulating hormones and metabolites. The integrated information is relayed to the dorsal motor nucleus of the vagus nerve (DMV), which in turn, projects motor fibers to elicit parasympathetic control of digestive and other viscera. Physiological functions mediated by the DVC are disrupted in diabetic patients and synaptic plasticity within the DVC has been linked to these complications. N-methyl-D-aspartic acid (NMDA) receptors have been extensively studied for their involvement in synaptic plasticity in a variety of central nervous system disorders; and their activation in the DVC modulates hepatic glucose production and feeding behavior. Although chronic disease can alter NMDA function, changes in DVC expression and/or sensitivity of NMDA receptors in diabetic states has not been addressed. Using whole cell electrophysiology, functional properties of the nuclei in the DVC were investigated in normoglycemic and type 1 diabetic mice. Preterminal NMDA (preNMDA) receptors were discovered to tonically modulate excitatory neurotransmission on terminals contacting DMV neurons. While these preNMDA receptors were not found to differentially modulate tonic excitatory neurotranmission, soma-dendritic NMDA receptor responses of NTS neurons were augmented in type 1 diabetic mice. Through the use single-cell PCR, increased NMDA receptor responses could be correlated to neurons that mediate excitatory neurotransmission and would argue that augmented NMDA receptor responses increase vagal output. In general, enhancing vagal output decreases activity of connected peripheral organs. Molecular approaches were employed to corroborate the observed functional NMDA receptors changes to their protein and mRNA expression levels. Overall, results argue that NMDA receptors are involved in synaptic plasticity in DVC of type 1 diabetic mice to enhance excitatory neurotransmission. This modulation may potentially serve as a physiological counter regulatory mechanism to control pathological disturbances of gastrointestinal homeostatic reflex responses.

Excitotoxicity is the overexcitation of neurons due to the excessive activation of excitatory amino acid receptors and is thought to be involved in many neurodegenerative states. The manner in which the neuron breaks down during excitotoxicity is still unclear. The current study used the organotypic hippocampal slice culture model to examine the time-dependent loss of the synaptic vesicular protein synaptophysin and the loss of N-methyl-D-aspartate (NMDA) receptor NR1 subunit availability following an excitotoxic insult (20 μM NMDA) to provide a better understanding of the topographical nature of neuronal death following NMDA receptor activation. Significant NMDA-induced cytotoxicity in the CA1 region of the hippocampus (as measured by propidium iodide uptake) was evident early (15 minutes after exposure) while significant loss of the NR1 subunit and synaptophysin was found at later timepoints (72 and 24 hours, respectively), suggesting delayed downregulation or degradation in axons and dendrites as compared to the soma. The addition of the competitive NMDA receptor antagonist 2-amino-7-phosphonovaleric acid (APV) significantly attenuated all NMDA-induced effects. These results suggest that NR1 and synaptophysin levels as measured by immunoreactivity are not reliable indicators of early cell death.

Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2008.