STEROIDS AND GABA:PHYSIOLOGY OF RECEPTOR SUBUNIT CHANGES

描述：（申请人摘要） 本研究的目的是确定的功能意义， GABAA受体（GABA-R）亚单位的变化产生的波动，内源性 荷尔蒙水平为此，我们将评估mRNA和蛋白质水平， α 4和δ亚单位伴随着突触电流的评估， 以及急性分离的CA 1中GABA门控电流的特征 海马神经元从GABA调节的3 α，5 α-THP中撤出后 （3 α-OH-5 α-葡聚糖-20-酮）。这种激素模式模仿激素 内源性激素戒断期的基础条件，如 经前综合症上一个供资期支持的调查结果 确定3 α，5 α-THP戒断导致焦虑， 促惊厥作用类似于从其他药物戒断后观察到的作用， GABA调节化合物，如苯二氮卓类（BDZ）。这些行为 效果与衰减时间的显着减少相关， GABA门控电流，导致总整合GABA减少 电流，一种效应，特别是由于α 4亚基的上调， GABA-R。这种α 4亚基的增加也导致了几乎全部的 BDZ不敏感。本研究的目的是确定 记录的突触电流上的α 4亚基上调 海马切片制备; mIPSC以及诱发的IPSC将被 在激素状态下测定，并与α 4亚基水平相关， Western印迹程序。我们还将评估GABA门控的 在非平衡条件下使用切除的外向贴片的电流 受体饱和和超快GABA暴露（<1毫秒）。这些条件 更接近真实的突触活动。衰减时间常数 GABA门控电流以及脱敏速率将在 含有α 4-GABA-R的细胞（经酶诱导或转染的HEK-293 细胞）并与对照含α 2受体进行比较。另外我们 将检验我们观察到的3 α，5 α-THP不敏感性是 由于GABA-R的δ亚基的上调，使用整体 细胞膜片钳技术。在这两种情况下，反义诱导的抑制 亚基表达将被用来进一步验证这些作用。 亚基在调节突触电流和3 α，5 α-THP变化中的作用 宽容这些研究将检验这一假设，即内在的 GABA-R的特性是由于在 激素暴露和戒断。

DESCRIPTION: (Applicant's Abstract) The goal of the present study is to determine the functional significance of GABAA receptor (GABA-R) subunit changes produced by fluctuations in endogenous hormonal levels. Towards this end, we will assess mRNA and protein levels of alpha4 and delta subunits concomitant with evaluation of synaptic current as well as characterization of GABA-gated current in acutely dissociated CA1 hippocampal neurons upon withdrawal from the GABA-modulatory 3alpha,5alpha-THP (3alpha-OH-5alpha-pregnan-20-one). This hormone paradigm mimics hormonal conditions underling periods of endogenous hormone withdrawal, such as pre-menstrual syndrome. Findings supported by the previous funding period established that 3alpha,5alpha-THP withdrawal results in anxiogenic, pro-convulsant effects similar to those seen after withdrawal from other GABA-modulatory compounds, such as the benzodiazepines (BDZs). These behavioral effects were well-correlated with a marked decrease in the decay time for GABA-gated current, which resulted in a decrease in total integrated GABA current, an effect due specifically to upregulation of the alpha4 subunit of the GABA-R. This increase in the alpha4 subunit also resulted in a near total BDZ insensitivity. The goal of the present studies is to determine the effect of alpha4 subunit upregulation on synaptic currents recorded from the hippocampal slice preparation; mIPSCs as well as evoked IPSCs will be determined across hormone state and correlated with alpha4 subunit levels using Western blot procedures. We will also evaluate the kinetics of GABA-gated current using excised outside-out patches under non-equilibrium conditions of receptor saturation and ultra-fast GABA exposure (<1 msec). These conditions more closely mimic those of actual synaptic events. Decay time constants for GABA-gated current, as well as rate of desensitization, will be determined in cells with alpha4-containing GABA-R (hormone-induced or transfected HEK-293 cells) and compared with control alpha2-containing receptors. In addition, we will test the hypothesis that the 3alpha,5alpha-THP insensitivity we observe is due to upregulation of the delta subunit of the GABA-R, assessed using whole cell patch clamp techniques. In both cases, antisense-induced suppression of subunit expression will be employed to further verify the role of these subunits in modifying changes in synaptic current and 3alpha,5alpha-THP tolerance. These studies will test the hypothesis that alterations in intrinsic properties of GABA-R occur as a result of altered subunit composition after hormone exposure and withdrawal.