Mechanisms of Ligand-Induced GABA Receptor Plasticity

该部分的目的是分析慢性GABA能配体诱导中枢神经系统(CNS)可塑性的分子机制。不同的可塑性诱导处理导致GABA受体(GABA)亚单位α4及其相关伙伴，伽马2或8水平的变化，并伴随着对 内源性神经类固醇。在这个项目中，我们已经确定含有α4β3β亚基的GABAR是突触外定位的，参与紧张性抑制，并且是神经类固醇和麻醉剂(包括乙醇)调节中枢兴奋性的主要靶点，而对苯二氮卓类药物不敏感。特别是，我们通过几条证据表明，GABAR的Alpha6beta3Delta亚型很可能是乙醇在小脑中的作用靶点。这一重要发现需要证实，并形成了AIM I的理论基础。我们认为，α4/6β3βGABAR既对可塑性敏感，又通过神经类固醇调节的紧张性抑制来调节兴奋性。在这个项目Aim II中，我们将比较和对比慢性类固醇(含突触外三角洲亚基的靶)和慢性BZ(含突触的Gamma2亚基的靶)后GABAR的变化。在接受药物治疗的小鼠中，我们将使用抗体以生化方法测量海马和小脑中GABAR亚单位的水平和亚单位的配对，以及指示亚单位开关的GABAR水平和变构调节的结合分析。在药物处理的原代培养的海马神经元中，我们将测量细胞表面GABAR的含量 使用生物素化的亚基和亚基伙伴。慢性GABA能药物的可塑性改变不仅与临床上有用的药物如苯二氮类药物(BZ)的耐受性的形成以及戒断和依赖有关，而且与GABA系统对兴奋性的控制有关。这反过来又对正常的中枢信息处理和对常见或不寻常的经历(可塑性)做出反应的变化至关重要，包括致痫现象。

The objectives of this component are to analyze the molecular mechanisms of chronic GABAergic ligand-induced plasticity in the central nervous system (CNS). Various plasticity-inducing treatments produce changes in levels of the GABAA receptor (GABAR) subunit alpha4 and its associated partners, gamma2 or 8, accompanied by altered sensitivity to endogenous neurosteroids. In this program, we have determined that alpha4beta3delta subunit-containing GABAR are extrasynaptically localized, involved in tonic inhibition, and the major target of modulation of CNS excitability by neurosteroids and anesthetics including ethanol, while being insensitive to benzodiazepines. In particular we showed by several lines of evidence that the alpha6beta3delta subtype of GABAR is very likely the target of action of ethanol in the cerebellum. This important discovery needs substantiation and forms the rationale for Aim I. We suggest that the alpha4/6beta3delta GABAR are both susceptible to plasticity and positioned to regulate excitability via neurosteroid-modulated tonic inhibition. In this project Aim II we will compare and contrast changes in GABAR following chronic steroid (extrasynaptic delta subunit-containing target) and chronic BZ (synaptic gamma2 subunit-containing target). In drug-treated mice, we will measure biochemically the levels of GABAR subunits in hippocampus and cerebellum and partnering of subunits using antibodies, as well as binding assays for GABAR levels and allosteric modulation indicative of subunit switches. In drug-treated primary cultured hippocampal neurons, we will measure the content of cell surface GABAR subunits and subunit partners using biotinylation. Plastic changes following chronic GABAergic drugs are relevant not only to the development of tolerance to clinically useful agents like benzodiazepines (BZ) as well as withdrawal and dependence, but also to the control of excitability by the GABA system. This in turn is critically important to normal CNS information processing and changes that occur in response to usual or unusual experiences (plasticity), including epileptogenic phenomena.