An optogenetic investigation of CNS sensitization following alcohol withdrawal

描述（由申请人提供）：本项目的主要目的是表征在酒精戒断（WD）背景下发生的T型钙通道（T通道）介导的神经元和电路致敏，并确定特定T通道的潜在效用 阻断剂作为治疗WD的药物。WD症状，包括癫痫发作，是酗酒者康复的主要障碍，使许多人复吸。WD症状在随后的WD中变得更严重;可能是由于酒精通过GABA调节慢性CNS抑制后的反弹性过度兴奋。我们的实验室已经确定了T通道亚型Cav3.2作为WD过度兴奋的潜在介质，我们的研究结果表明，T通道可能是WD癫痫发作的产生和传播的基础。我建议调查的T-通道介导的致敏作用WD通过测试人口的敏感性，使用精确的光遗传学刺激范例的存在或不存在的T-通道阻滞剂。此外，我将使用光遗传学癫痫发作诱导方法来测试T通道特异性抗癫痫药物乙琥胺对标准WD癫痫发作预防药物劳拉西泮的疗效。最后，我将确定在拟议的WD发作网络的敏感化的具体网站和测试的影响T通道阻断网络的反应。所有以下具体目标将采用慢性多部位电生理学记录与转基因Thy 1-ChR 2小鼠的CA 1的光学刺激配对。具体目标1将通过量化WD之前和之后点燃动物中的发作间期尖峰来评价WD之后丘脑和海马神经元群体的致敏性。具体目标2提出在WD之后使用光遗传学癫痫发作诱导范例来确定癫痫发作扩散、强度和阈值被WD改变的程度。而Specific Aim 3将通过在一系列刺激水平下记录刺激下游多个部位的光遗传学诱发活动来表征WD后的网络变化。T通道阻滞剂将用于所有实验中，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方法，包括光遗传学，电生理学，行为研究和药理学干预，以确定WD后T通道介导的神经元敏感性效应，以及确定WD癫痫发作的潜在新疗法。这项研究的方法将提供光遗传学技术，电生理记录和分析，行为研究，组织学和显微镜的培训。在我的顾问和研究伙伴的指导和支持下，该项目将为追求基本和转化研究问题提供培训，并深入了解酗酒的负担。最后，包括我的基础研究培训期间的临床培训计划将确保我将在我的职业生涯中作为一名医生科学家全面发展。

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.