Astrocytic Control of GABA Inhibition in Epilepsy

描述（由申请人提供）：我们最近证明，大脑中与蛋白质地西泮结合抑制剂（DBI）相关的天然神经活性化合物，内啡肽可以模拟苯二氮卓类药物的活性，苯二氮卓类药物是治疗癫痫的有效药物。苯二氮卓类药物是GABAA受体的变构调节剂，介导大脑突触抑制的主要形式。这表明，通过产生内啡肽，脑细胞和回路可以自我调节，动态增强突触抑制，以抑制癫痫发作，因为它们是由持续的大脑活动引起的。虽然目前已证实存在与dbi相关的抗癫痫药物，但对药物的细胞来源知之甚少，也不知道它们是如何从细胞分泌或在细胞外空间加工以发挥作用的。拟议中的实验有三个目的。1)确定星形胶质细胞是否是内啡肽的来源，因为它们表达非常高水平的DBI，并且似乎具有高分泌能力；2)确定细胞（最有可能是星形胶质细胞）通过哪些途径感知活性，然后通过分泌和加工DBI做出反应；3)确定最终的内啡肽分子（或分子），这似乎不是DBI本身，而是DBI的一个蛋白质片段。我们将使用电生理分析来记录所有三个目标的功能性内啡肽活性。实验将包括确定自发抑制性突触后电流的动力学、检测突触GABAA受体外源性或内源性变抗调节的有效实验、对高速离子吸附GABA应用的反应、对内啡肽活性的敏感实验、体外大规模丘脑网络的网络分析以及体内癫痫易感性的脑电图分析。能够从星形胶质细胞中靶向删除DBI的突变小鼠的可用性，以及能够检测删除范围和程度的荧光报告小鼠的可用性，将促进这些研究。总的来说，拟议的实验结果将提供有关内啡肽信号传导的机制信息，以及这种自然的大脑活动是否最终可能成为癫痫和其他GABA信号传导改变的神经精神疾病的治疗干预的目标。

DESCRIPTION (provided by applicant): We have recently shown that naturally occurring neuroactive compounds, endozepines related to the protein Diazepam Binding Inhibitor (DBI) in the brain can mimic the activity of benzodiazepines, which are effective treatments in epilepsy. Benzodiazepines are allosteric modulators of GABAA receptors, which mediate the primary form of synaptic inhibition in the brain. This suggests that, through production of endozepines, brain cells and circuits can-self regulate, to dynamically enhance synaptic inhibition as needed to suppress seizures as they arise from ongoing brain activity. While the existence of DBI-related antiepileptic endozepines has now been demonstrated, little is known regarding the cellular source of endozepines, nor of the means through which they are secreted from cells or processed in the extracellular space to exert their action. The proposed experiments have three aims. 1) Determine whether astrocytes are the source of endozepines, as they express very high levels of DBI, and appear to have a high capacity for secretion, 2) determine the pathways through which cells, most likely astrocytes, sense activity and then respond through secretion and processing of DBI, and 3) Identify the final endozepine molecule (or molecules), which does not appear to be DBI itself, but a protein fragment of DBI. We will use electrophysiological assays to document functional endozepine activity in all three aims. Assays will include determining the kinetics of spontaneous inhibitory post-synaptic currents, an effective assay for detection of exogenous or endogenous allosteric modulation of synaptic GABAA receptors, and responses to high-speed iontophoretic GABA application, a sensitive assay for endozepine activity, network analysis of large-scale thalamic networks in vitro, and EEG analysis of seizure susceptibility in vivo. These studies will be facilitated by the availability of mutant mice that alow for targeted deletion of DBI from astrocytes, and by fluorescent reporter mice that allow for detection of the range and extent of deletion. Overall the results of the proposed experiments will provide mechanistic information regarding endozepine signaling and whether this natural brain activity might ultimately be targeted for therapeutic intervention in epilepsy and other neuropsychiatric disorders of altered GABA signaling.