Optogenetics: A tool to probe mechanism and an agent to block TBI-induced epileptogenesis.

在美国，每年有超过200万人在遭受了 创伤性脑损伤（TBI）。创伤后癫痫（PTE）发生在高达39%的中度至重度癫痫患者中。 严重的非穿透性脑外伤与其他获得性癫痫一样， PTE患者在初始损伤后具有潜伏期（>1周和长达多年）。在这段没有烦恼的时期 TBI后代表癫痫发生期，在此期间大脑经历生理，解剖， 细胞和分子的变化，导致癫痫易感性的慢性增加。该延迟 在创伤性脑损伤和PTE发展之间也代表了一个可以采用战略的时期 抑制大脑中导致PTE的反应可塑性，但导致PTE的分子机制 导致获得性癫痫的癫痫发生过程在很大程度上是未知的，也没有抗癫痫治疗 到目前为止已经成功开发。创伤后癫痫发生点的动物模型 海马网络的反应可塑性，兴奋/抑制平衡的改变作为驱动因素 永久性大脑变化和癫痫状态的症状然而，主要的分子和电生理 转型仍然模糊不清。 待检验的假设：海马中的损伤后活动和网络变化， 部分通过囊泡神经递质释放机制的改变，是PTEgenesis的主要驱动因素。的 具体目的是：1）使用通道视紫红质-2（ChR 2）来光遗传地驱动神经活动， 通过去极化齿状回（DG）中特定的初级神经元群体来进行PTE发生的过程。2)使用 盐视紫红质（NpHR）通过光遗传学抑制来延迟使用标准方法诱导的PTE发生， DG的神经活动。网络检测技术将被集成到一个新的和独特的模型中，以检测网络， PTE和PTE发生的分子驱动因素。使用TBI的受控皮质撞击（CCI）模型，我们 所提出的研究结合联合收割机1）独特的微电极阵列电化学（MEA）以实时监测 谷氨酸释放和氧变化作为癫痫样活动的度量; 2）免疫组织化学来定义 特异性细胞表型的变化;和3）切片电生理学与定制的Western印迹定量， 在一个新的，光遗传学修饰的海马平台上的神经递质释放机制。 目的1：研究将通过将ChR 2启动子构建体用AAV 2/5病毒转染入 大鼠海马DG，利用CCI诱导后自由漫游大鼠DG神经元的光遗传学激活 TBI以增强PTEgenesis。细胞外谷氨酸，电生理，免疫组织化学， 囊泡释放生物化学测量将在动物的离散行为阶段进行， 癫痫发生的进展目的2：研究将通过NpHR-1的AAV 2/5病毒转染来完成。 CCI损伤前大鼠海马DG启动子构建及DG通路的光遗传学抑制 CCI后抑制PTEgenesis。MEA、电生理学、免疫组织化学和生化测量 将被制造。作为这些研究的一个组成部分，短暂的谷氨酸激增和DG中氧的变化， 通过MEA检测到的光致突变将触发实时光遗传学抑制，作为中止PTEgenesis的潜在手段。 我们的方法应该允许网络，神经元和突触前释放的变化，以明确地联系到 与癫痫发生相关的生物化学、解剖学、电生理学和行为结果。的 这项建议中的研究将为全面、新颖地分析这一进程铺平道路。 并将提供初步数据，以支持确定活动依赖性肺栓塞的病因效应的研究。 突触发生在PTE的发展。此外，这些研究将测试一种潜在的新疗法， PTEgenesis.因此，这项研究直接关系到很大一部分军人的护理， 妇女、退伍军人和普通民众。

Over two million people are treated medically each year in the United States after sustaining a traumatic brain injury (TBI). Posttraumatic epilepsy (PTE) develops in up to 39% of patients with moderate to severe, non-penetrating TBI. As with other acquired epilepsies, spontaneous recurrent seizures associated with PTE develop with a latency (>1 week and up to many years) after the initial injury. This seizure-free period after TBI represents the period of epileptogenesis, during which the brain undergoes physiological, anatomical, cellular, and molecular changes leading to a state of chronically increased seizure susceptibility. This delay between the TBI and development of PTE also represents a period during which strategies might be employed to inhibit the reactive plasticity in the brain that leads to PTE, but the molecular mechanisms underlying the epileptogenic process leading to acquired epilepsy are largely unknown and no anti-epileptogenic therapies have been successfully developed to date. Animal models of posttraumatic epileptogenesis (PTEgenesis) point to reactive plasticity of hippocampal networks, with alteration in the balance of excitation/inhibition as a driver of permanent brain changes and the epileptic state. However, the prime molecular and electrophysiological transformations remain murky. The hypothesis to be tested: Post-injury activity and network changes in the hippocampus, induced in part by alterations in the vesicular neurotransmitter release machinery, are primary drivers of PTEgenesis. The Specific Aims are to: 1) Use channelrhodopsin-2 (ChR2) to optogenetically drive neural activity and the process of PTEgenesis by depolarizing specific primary neuronal populations in dentate gyrus (DG). 2) Use halorhodopsin (NpHR) to retard PTEgenesis, induced using a standard method, by optogenetically inhibiting neural activity in DG. Proven techniques will be integrated into a new and unique model to detect network and molecular drivers of PTE and PTEgenesis. Using the controlled cortical impact (CCI) model of TBI, our proposed studies combine 1) unique microelectrode array electrochemistry (MEA) to monitor real-time glutamate release and oxygen change as a metric of epileptiform activity; 2) immunohistochemistry to define changes in specific cell phenotypes; and 3) slice electrophysiology with custom Western blot quantitation of neurotransmitter release machinery on a novel, optogenetically-modified hippocampal platform. Aim 1: Studies will be accomplished by AAV2/5 viral transfection of a ChR2-promotor construct into hippocampal DG of rats, utilizing optogenetic activation of DG neurons of free-roaming rats after CCI-induced TBI to enhance PTEgenesis. Extra-cellular glutamate, electrophysiological, immunohistochemical, and vesicular release biochemical measures will be made on animals at discrete behavioral stages during the progression of epileptogenesis. Aim 2: Studies will be accomplished by AAV2/5 viral transfection of an NpHR- promoter construct into hippocampal DG of rats prior to CCI injury and optogenetic inhibition of DG circuits after CCI to inhibit PTEgenesis. MEA, electrophysiological, immunohistochemical, and biochemical measures will be made. As one component of these studies, transient glutamate surges and changes in oxygen in DG detected by MEAs will trigger real-time optogenetic inhibition as a potential means to abort PTEgenesis. Our approach should allow network, neuronal, and presynaptic release changes to be clearly tied to biochemical, anatomical, electrophysiological, and behavioral outcomes associated with epileptogenesis. The studies in this proposal will pave the way to development of comprehensive, novel analyses of the progression of PTE and will provide preliminary data to support studies identifying causative effects of activity-dependent synaptogenesis in the development of PTE. In addition, these studies will test a potentially novel therapy for PTEgenesis. Thus, this research is directly relevant to the care of a large proportion of service men and women, veterans, and the general population.