STEROIDS AND GABA:PHYSIOLOGY OF RECEPTOR SUBUNIT CHANGES

描述：(申请人摘要) 本研究的目的是确定神经递质的功能意义。 内源性激素波动引起的GABAA受体(GABA-R)亚单位变化 荷尔蒙水平。为此，我们将评估核糖核酸和蛋白质水平。 Alpha4和Delta亚基伴随着突触电流的评估 急性分离的CA1中GABA门控电流的特征 海马神经元退出GABA调节的3α，5α-THP (3α-羟基-5α-孕烷-20-酮)。这种荷尔蒙范例模仿荷尔蒙 内源性激素撤除时期的基础条件，如 经前综合症。上一个资助期支持的调查结果 证实了停用3α，5α-THP会导致焦虑， 促惊厥作用与停药后所见相似 GABA调节化合物，如苯二氮卓类(BDZ)。这些行为 影响与显著缩短的衰变时间相关。 GABA门控电流，导致总积分GABA下降 目前，这一效应是由于α4亚基上调所致 GABA-R。Alpha4亚基的这种增加也导致了几乎全部 BDZ不敏感。目前研究的目标是确定其影响 α4亚单位上调突触电流的研究 海马片制备；mIPSCs以及诱发的IPSCs将 跨激素状态确定，并与α4亚单位水平相关 蛋白质印迹程序。我们还将评估GABA门控的动力学 在非平衡条件下使用切除的外向外贴片的电流 受体饱和和超快GABA暴露(&lt；1毫秒)。这些条件 更接近于真实突触事件的模拟。的衰变时间常数 GABA门控电流以及脱敏速度将在 含有α4-GABA-R(激素诱导或转染HEK-293)的细胞 细胞)，并与对照组含有α2的受体进行比较。此外，我们 将检验我们观察到的3Alpha，5Alpha-THP不敏感性的假设 由于GABA-R的Delta亚基上调，使用整体评估 细胞膜片钳技术。在这两种情况下，反义诱导的抑制 将使用亚单位表达来进一步验证这些 调节突触电流和3α，5α-THP变化的亚基 宽容。这些研究将检验这样一种假设，即内在的变化 GABA-R的性质是亚基组成发生改变的结果 荷尔蒙暴露和停药。

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.