Astrocytic Control of GABA Inhibition in Epilepsy

描述（由申请人提供）：我们最近已经证明，天然存在的神经活性化合物，与脑中的蛋白质地西泮结合抑制剂（DBI）相关的内卓类药物可以模拟苯二氮卓类药物的活性，这是癫痫的有效治疗方法。苯二氮卓类是GABAA受体的变构调节剂，其介导脑中突触抑制的主要形式。这表明，通过产生内皮素，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，因为它们是由正在进行的大脑活动引起的。虽然存在DBI相关的抗癫痫endozepines现在已被证明，很少有人知道endozepines的细胞来源，也不是通过它们从细胞分泌或在细胞外空间加工发挥其作用的手段。这些实验有三个目的。1)确定星形胶质细胞是否是内卓的来源，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（最可能是星形胶质细胞）感知活性然后通过分泌和加工DBI响应的途径，和3）鉴定最终的内卓分子DBI是一种蛋白质（或分子），它似乎不是DBI本身，而是DBI的蛋白质片段。我们将使用电生理学测定来记录所有三个目标中的功能性内西平活性。试验将包括确定自发抑制性突触后电流的动力学，用于检测突触GABAA受体的外源性或内源性变构调节的有效试验，以及对高速离子电渗GABA应用的反应，内西平活性的灵敏试验，体外大规模丘脑网络的网络分析，以及体内癫痫易感性的EEG分析。这些研究将通过允许从星形胶质细胞靶向缺失DBI的突变小鼠的可用性以及允许检测缺失范围和程度的荧光报告小鼠来促进。总体而言，所提出的实验的结果将提供关于内西平信号传导的机制信息，以及这种天然脑活动是否最终可能被靶向用于癫痫和其他GABA信号传导改变的神经精神疾病的治疗干预。

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.