An optogenetic investigation of CNS sensitization following alcohol withdrawal

描述(由申请人提供)：本项目的主要目标是描述在酒精戒断(WD)环境中发生的T型钙通道(T通道)介导的神经元和电路敏化的特征，并确定特定T通道的潜在用途 阻滞剂作为WD的治疗药物。WD症状，包括癫痫，是酗酒者康复的主要障碍，促使许多人复发。随着随后的WDS，WD症状变得更加严重；可能是由于酒精通过GABA调节慢性中枢神经系统抑郁后的反弹过度兴奋。我们的实验室已经确定T-通道亚型Cav3.2是WD超兴奋性的潜在中介，我们的发现表明T-通道可能是WD癫痫发作发生和传播的基础。我建议在存在或不存在T通道阻滞剂的情况下，通过使用精确的光遗传刺激范式测试群体敏感性，来研究T通道介导的WD的敏化效应。此外，我将使用光遗传癫痫诱导方法来测试T通道特异性抗癫痫药物乙琥胺与标准的WD癫痫预防药物劳拉西潘的疗效。最后，我将在一个拟议的WD癫痫网络中确定特定的敏感点，并测试T通道阻断对网络反应的影响。以下所有的特定目标都将采用慢性多部位电生理记录结合光刺激对转基因Thy1-ChR2小鼠的CA1。具体目标1将通过量化点燃动物在WD之前和之后的发作间期棘波来评估WD后丘脑和海马神经元群体的敏化。特定目的2建议使用WD之后的光遗传癫痫诱导范式来确定WD改变癫痫发作的传播、强度和阈值的程度。而特定目标3将通过记录刺激下游多个部位在一系列刺激水平下的光基因诱发活动来表征WD后的网络变化。T-通道阻滞剂将在所有实验中使用，以区分T-通道介导的效应与其他机制相关的效应。该培训计划提出了一种多学科的方法，包括光遗传学、电生理学、行为学和药物干预，以确定T通道对WD后神经元敏感性的影响，并寻找潜在的WD发作的新疗法。这项研究的方法将提供光遗传技术、电生理学记录和分析、行为研究以及组织学和显微镜方面的培训。在我的导师和研究助理的指导和支持下，这个项目将为追求基本和翻译研究问题提供培训，并对酗酒的负担产生洞察力。最后，一个包含我基础研究培训持续时间的临床培训计划将确保我在作为一名内科科学家的职业生涯中全面发展。

DESCRIPTION (provided by applicant): The primary objective of this project is to characterize the T-type calcium channel (T-channel) mediated neuron and circuit sensitization that occurs in the setting of alcohol withdrawal (WD) and to determine the potential utility of specific T-channel blockers as therapy in WD. WD symptoms, including seizure, represent a major barrier to recovery for alcoholics, driving many to relapse. WD symptoms become more severe with subsequent WDs; likely due to rebound hyperexcitability following chronic CNS depression through GABA modulation by alcohol. Our lab has identified the T-channel isoform Cav3.2 as a potential mediator of WD hyperexcitability, and our findings suggest that T-channels may underlie the generation and propagation of WD seizures. I propose to investigate the T-channel mediated sensitization effects of WD by testing population sensitivity using a precise optogenetic stimulation paradigm in the presence or absence of T-channel blockers. Additionally, I will use an optogenetic seizure induction method to test the efficacy of the T-channel specific anti-epileptic drug ethosuximide against the standard WD seizure prophylaxis, lorazepam. Finally, I will identify the specific sites of sensitization in a proposed WD seizure network and test the effect of T-channel blockade on the responsiveness of the network. All of the following specific aims will employ chronic multi-site electrophysiology recording paired with optical stimulation of CA1 of the transgenic Thy1-ChR2 mouse. Specific Aim 1 will evaluate sensitization of thalamic and hippocampal neuron populations following WD by quantifying interictal spikes in kindled animals before and after WD. Specific Aim 2 proposes to use an optogenetic seizure induction paradigm following WD to determine the extent to which seizure spread, intensity, and threshold are modified by WD. While Specific Aim 3 will characterize network changes following WD by recording optogenetically evoked activity at multiple sites downstream of stimulation at a range of stimulation levels. T-channel blockers will be used in all experiments to distinguish T-channel mediated effects from those related to other mechanisms. This training plan proposes a multidisciplinary approach that includes optogenetics, electrophysiology, behavioral studies, and pharmacologic intervention to identify the T-channel mediated effects on neuron sensitivity following WD as well as to identify potential novel therapies for WD seizure. The approach to this study will provide training in optogenetic techniques, electrophysiology recording and analysis, behavioral studies, and histology and microscopy. With guidance and support from my advisor and research associates; this project will provide training for the pursuit of basic and translational research questions and generate insight into the burdens of alcoholism. Finally, a clinical training plan that encompasses the duration of my basic research training will ensure that I will be well-rounded in my career as a physician-scientist.