Date of Award

5-1-2016

Degree Name

Doctor of Philosophy

Department

Molecular, Cellular, and Systemic Physiology

First Advisor

Rose, Gregory

Abstract

Intellectual Disability (ID) is a condition in which day-to-day cognitive, intellectual and adaptive functioning is negatively affected including poor performance in memory tests in human subjects. Patients with comorbid anxiety and depression demonstrated adverse memory when subject to a verbal learning test. Abnormal mutation in the human deaf1 gene has been previously reported to be associated with ID. Previous behavioral studies in mice with a brain specific conditional neuronal knock outs (NKO) of deaf 1 gene exhibited memory deficit and anxiety-like behavior. These deaf 1 NKO mice represent a convenient model for the study of the effects of ID on both in vivo and in vitro memory tests. Earlier studies in these deaf 1 NKOs have shown increased levels of anxiety in the Elevated Plus Maze and Open Field - Test along with contextual-memory deficits in Fear-conditioning experiments. In the intact animal, behavioral phenotyping experiments in mice such as Fear conditioning including contextual and cued fear conditioning measures the ability of the animal to learn, remember and associate an aversive experience to environmental cues. Studies in rodent brain slices involving Long-term potentiation (LTP) and long-term depression (LTD) have long been associated to reflect substrates for memory formation and memory loss respectively. While early-LTP (ELTP) typically lasts between 30-60 minutes, late-LTP (LLTP) lasts for hours; though there is much disagreement about the time courses. In vitro LTD was first reported in 1978 and since then has been studied in details. NMDA receptor (NMDAR) and metabotropic glutamate receptor (mGLUR) activation has been implicated in induction of both LTP and LTD among others. The CA1 region of the rodent hippocampus is the most widely explored area for LTP studies especially stratum radiatum (SR). In addition to the commissural fibers, SR receives Schaffer-collaterals (SC) and is an integral part of memory formation. Previous studies have reported that the CA1 region of the hippocampus expresses both NMDAR-LTD and mGluR-LTD. In the present study, we aim to establish whether these mice might show altered hippocampal Long-term Potentiation (LTP) and/or Long-term Depression (LTD) when brain slices from deaf1 NKO mice were subject to electrophysiological studies and if so, whether pharmacological interventions had any effect on it. Using electrophysiological techniques, hippocampal slices from DEAF1 KO mice were tested for possible alterations in LTP when compared to age-matched controls. Both early and late forms of LTP were examined, since these two types of LTP are medicated through different biochemical mechanisms. ELTP was unaltered in the NKO animals compared to their WT littermates. This experiment was followed by investigating LLTP. The control animals, as expected, exhibited a large LTP. The DEAF1 animals, in contrast, showed a paradoxical response to LLTP stimulation. Instead of the increase in response as observed in the control animals, slices from DEAF1 mice decreased to about 80% of baseline at 30 mins post train. This depression (LTD) became greater throughout the 3 hours of post-train recording, at the end of which the responses were approximately 25% of baseline. The mechanisms of this LTD were then explored with focus on glutamate receptors. Based upon existing knowledge in the literature, the possible roles of both NMDA receptors and mGlu receptors (mGluR1 and mGluR5) were explored. Treatment with D-AP5 - a selective NMDAR antagonist on slices from control animals showed no effect on the baseline evoked responses, but LLTP was blocked following D - AP5 treatment. In slices from DEAF1 NKO animals, AP5 did not affect the baseline evoked responses, but it reversed the expected LTD to a robust LLTP. Next, the involvement of mGlu receptors, known to play a role in LTD, was tested. In controls, there was once again no effect on baseline activity but LLTP at both 30 mins and 180 mins P.T was significantly enhanced as compared to aCSF-only treated slices. In slices from DEAF1 mice, similar to the AP5 study, LY367385 did not affect the baseline response, but reversed LTD to LLTP. Following this, the effect of 40 µM MPEP (an mGluR5 antagonist) was tested, and produced similar results. Thus, three receptor antagonists known to impair the expression of LTD in wild-type animals not only prevented its appearance, but lead to a robust enhancement of the response in DEAF1 hippocampus. Further exploration of the mechanics of altered LTP was undertaken by using Synaptic Tagging and Capture (STC) in combination with pharmacology. Results of the STC experiments suggest that there could be a differential effect of plasticity-inducing stimulation on downstream protein targets. Finally, whole-cell patch recordings were performed to examine the biophysical characteristics of individual CA1 pyramidal neurons. Taken together, the results suggest that multiple mechanisms may be involved in the generation and expression of LTD in the DEAF1 mice.

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