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

在美国，每年有超过200万人在经历了一场 创伤性脑损伤(TBI)。创伤后癫痫(PTE)发生在高达39%的中度至 严重的非穿透性颅脑损伤。与其他获得性癫痫一样，自发性反复发作与 PTE在最初受伤后有一段潜伏期(&gt；1周，最长可达多年)。这段无癫痫发作的时期 脑损伤后代表癫痫的发生时期，在此期间，大脑经历了生理，解剖， 细胞和分子的变化导致癫痫易感性的慢性增加。这一延迟 在TBI和PTE的发展之间也代表了一个时期，在这段时期内可能会采用战略 抑制导致PTE的大脑的反应性可塑性，但背后的分子机制 导致获得性癫痫的致痫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地开发出来了。创伤后癫痫(PTEgensis)点动物模型 以兴奋/抑制平衡改变为驱动因素的海马网反应性可塑性 永久性的大脑变化和癫痫状态。然而，主要的分子和电生理学 转型仍然不明朗。 有待检验的假设：损伤后海马区的活动和网络变化，诱导在 部分由于囊泡神经递质释放机制的改变，是PTE发生的主要驱动因素。这个 具体目标是：1)使用通道视紫红质-2(ChR2)来光遗传地驱动神经活动和 齿状回(DG)中特定初级神经元群去极化的PTE发生过程。2)使用 卤视紫质(NpHR)通过光遗传抑制来延缓用标准方法诱导的PTE的发生 DG的神经活动。成熟的技术将集成到一种新的独特模型中，以检测网络和 PTE和PTE发生的分子驱动因素。使用脑外伤的受控皮质撞击(CCI)模型，我们的 建议的研究结合1)独特的微电极阵列电化学(MEA)来实时监控 谷氨酸释放和氧变化作为癫痫样活动的量度；2)免疫组织化学 特定细胞表型的变化；以及3)切片电生理学与定制的Western印迹定量 一种新型的光基因修饰的海马体平台上的神经递质释放机制。 目的1：通过AAV2/5病毒转染法将ChR2启动子载体转入 利用CCI后自由漫游大鼠DG神经元的光遗传激活作用 TBI促进PTE的发生。细胞外谷氨酸、电生理、免疫组织化学和 将对处于不同行为阶段的动物进行囊泡释放生化测量 癫痫发生的研究进展。目的2：通过AAV2/5病毒转染法将NpHR- 大鼠CCI损伤前DG区启动子的构建及DG环路的光遗传抑制 CCI后抑制PTE的发生。MEA、电生理、免疫组织化学和生化指标 都会被制造出来。作为这些研究的一个组成部分，DG中的瞬时谷氨酸激增和氧气变化 MEAS的检测将触发实时光遗传抑制，作为一种潜在的终止PTE发生的手段。 我们的方法应该允许网络、神经元和突触前释放的变化明显地与 与癫痫发生有关的生化、解剖、电生理和行为结果。这个 这项提案中的研究将为开发全面的、新颖的进程分析铺平道路 并将提供初步数据，以支持确定活动依赖的因果影响的研究 PTE发育过程中的突触发生。此外，这些研究还将测试一种潜在的新疗法 PTEgensis。因此，这项研究直接关系到很大一部分军人的护理和 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.