Astrocytic Control of GABA Inhibition in Epilepsy

描述(申请人提供)：我们最近发现，大脑中与安定结合抑制蛋白(DBI)相关的自然产生的神经活性化合物内齐平可以模仿苯二氮类药物的活性，这是治疗癫痫的有效方法。苯二氮卓类药物是GABAA受体的变构调节剂，GABAA受体介导大脑中突触抑制的主要形式。这表明，通过产生内齐平，脑细胞和电路可以自我调节，根据需要动态增强突触抑制，以抑制持续的大脑活动引起的癫痫发作。虽然现在已经证实了与DBI相关的抗癫痫药endzepine的存在，但关于Endozepine的细胞来源，以及它们是如何从细胞中分泌或在细胞外空间中加工发挥作用的，人们知之甚少。拟议中的实验有三个目标。1)确定星形胶质细胞是否是内源性药物的来源，因为它们表达非常高水平的DBI，并且似乎具有很高的分泌能力；2)确定细胞(最有可能是星形胶质细胞)感知活动，然后通过DBI的分泌和处理做出反应的途径；3)确定最终的一个或多个Endozepine分子，它似乎不是DBI本身，而是DBI的一个蛋白质片段。我们将使用电生理学方法来记录所有三个目标的功能性内切西平活性。测试将包括测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构调节的有效方法，以及对高速离子导入GABA应用的反应，内齐平活性的灵敏分析，体外大规模丘脑网络的网络分析，以及体内癫痫敏感性的EEG分析。这些研究将通过可从星形胶质细胞中定向删除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，拟议的实验结果将提供关于内洛西平信号的机制信息，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信号改变的神经精神障碍的治疗干预的目标。

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.