Steroids and GABA: Physiology of Receptor Subunit Change

描述（由申请人提供）：本提案将采用海马切片的电生理和免疫细胞化学技术来研究神经类固醇在改变强直性gaba能抑制和海马兴奋性方面的作用。众所周知，神经类固醇3α -5 α [β]-THP （THP）是GABA-A受体的正向调节剂，具有抗焦虑和镇静作用。然而，在某些临床条件下，THP可能具有矛盾的兴奋作用（即，青春期前后和经前不悦）。在这项研究中，我们建立了THP戒断（Wd）小鼠模型，该模型导致THP的兴奋作用。THP Wd增加CA1海马α -4和δ GABAR亚基的表达，与α -4 - β - δ GABAR的药理反应有关。在这项提议中，我们试图研究类固醇戒断后CA1海马GABAR亚基组成改变的电生理和解剖学相关性。GABAR亚基将定位于树突区域，我们预计这将与α -4共定位。初步结果表明，突触外δ亚基（突触周围到抑制性突触）的增加是由THP Wd引起的。由于THP可以在Cl通量向内时减弱α - 4- β - δ GABAR门控的电流，因此我们将验证假设，局部树突应用THP会降低THP Wd后CA1海马锥体细胞记录的张力GABAR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通过电流钳、细胞贴附贴片、成对脉冲抑制和EPSP-spike耦合来评估。由于delta亚基也位于假定的NMDAR突触的突触周围，我们还将测试THP Wd是否促进THP Wd后由于分流抑制减少而诱导长期增强（LTP）和长期抑制（LTD）。由于突触可塑性的改变表明在认知中起作用，我们将测试THP给药对海马体依赖性空间任务表现的影响。这些结果可能与青春期和整个月经周期中报告的情绪、认知和癫痫易感性（即双侧癫痫）的改变有关。

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.