Steroids and GABA: Physiology of Receptor Subunit Change

描述（由申请人提供）：本提案将在海马切片中采用电生理学和免疫细胞化学技术，以研究神经类固醇在改变紧张性GABA能抑制和海马兴奋性中的作用。神经类固醇3 α-5 α [β]-THP（THP）是众所周知的GABA-A受体的正调节剂，具有抗焦虑和镇静作用。然而，存在THP可能具有矛盾兴奋作用的临床状况（即，围青春期和PMDD）。在这项研究中，我们已经开发了一种THP戒断（Wd）的小鼠模型，该模型导致THP的兴奋作用。THP Wd增加CA 1海马中α-4和δ GABAR亚基的表达，与α 4-β-δ GABAR的药理学反应相关。在这个建议中，我们试图检查改变GABAR亚基组成的电生理和解剖学相关的CA 1海马类固醇戒断后。δ GABAR亚基将定位于树突状区域，我们预计该区域将与α-4共定位。初步结果表明，增加突触外δ亚基，突触周围抑制性突触，结果从THP Wd。由于当Cl通量向内时，THP可以减弱由α 4-β-δ GABAR门控的电流，因此我们将测试以下假设：THP Wd后，局部树枝状细胞应用THP将减少从CA 1海马锥体细胞记录的强直性GABA能电流，导致苯二氮卓类药物不敏感（用全细胞膜片钳技术记录）。THP对神经元兴奋性的影响将使用电流钳、细胞贴附膜片、成对脉冲抑制和EPSP-尖峰耦合来评估。由于δ亚基也定位于突触周围推定的NMDAR突触，我们还将测试THP是否有利于诱导长时程增强（LTP）和长时程抑制（LTD）后THP Wd作为减少分流抑制的结果。由于突触可塑性的改变表明在认知中的作用，我们将测试THP管理对性能的影响，在一个依赖于大脑的空间任务。这些结果可能与情绪、认知和癫痫易感性的改变有关（即，月经性癫痫），报告在青春期和整个月经周期。

DESCRIPTION (provided by applicant): This proposal will employ electrophysiological and immunocytochemical techniques in the hippocampal slice to investigate the effect of neurosteroids in altering tonic GABAergic inhibition and hippocampal excitability. The neurosteroid 3alpha-5alpha[beta]-THP (THP) is well-known as a positive modulator of the GABA-A receptor, with anxiolytic and sedative effects. However, there are clinical conditions where THP may possess paradoxical excitatory effects (i.e., the peri-pubertal period and PMDD). In this study, we have developed a mouse model of THP withdrawal (Wd) which results in excitatory effects of THP. THP Wd increases expression of alpha-4 and delta GABAR subunits in CA1 hippocampus, associated with the pharmacological responses of alpha4-beta-delta GABAR. In this proposal, we seek to examine the electrophysiological and anatomical correlates of altered GABAR subunit composition in CA1 hippocampus after steroid withdrawal. The delta GABAR subunit will be localized to dendritic regions which we expect will co-localize with alpha-4. Preliminary results suggest that increases in extrasynaptic delta subunit, perisynaptic to inhibitory synapses, result from THP Wd. Because THP can attenuate current gated by alpha4-beta-delta GABAR when Cl flux is inward, we will test the hypothesis that local dendritic application of THP will decrease tonic GABAergic current recorded from CA1 hippocampal pyramidal cells following THP Wd, resulting in benzodiazepine insensitivity (recorded with whole cell patch clamp techniques). THP effects on neuronal excitability will be assessed using current clamp, cell-attached patch, paired-pulse inhibition and EPSP-spike coupling. Because the delta subunit is also localized perisynaptic to putative NMDAR synapses, we will also test whether THP facilitates the induction of long-term potentiation (LTP) and long-term depression (LTD) following THP Wd as a result of reduction in shunting inhibition. Because alterations in synaptic plasticity suggest a role in cognition, we will test the effect of THP administration on performance in a hippocampal-dependent spatial task. These results may be relevant for alterations in mood, cognition and seizure susceptibility (i.e., catamenial epilepsy) reported during puberty and across the menstrual cycle.