CHRONIC BENZODIAZEPINE EFFECTS ON GABA RECEPTOR COMPLEX

描述：（申请人摘要） 苯二氮卓类抗惊厥药物耐受性的研究进展 限制了其临床价值，并可能与长期滥用的模式有关。 BZ增强GABA对GABAA受体（GABAR）的抑制作用，增加Cl 电导 慢性BZ治疗后GABAR的调节良好 作为BZ耐受性的一种潜在机制，然而， 导致BZ耐受的脑GABA突触事件并不好 明白 体外电生理研究结果 海马切片，正在进行的GABAR放射自显影研究，以及我们最初的 GABAR亚基mRNA原位杂交和免疫组织化学研究 和蛋白质已经确定，BZ耐受大鼠海马提供了一个 为研究BZ耐受的突触机制提供了有用的模型， 为拟议的研究提供了基础。 研究旨在评估 与慢性BZ相关的功能变化之间的时间关系 治疗和GABARs的监管将在 停止1周口服后几个时间点的海马 氟安定（FZP）治疗。 分子生物学、免疫组织化学和 电生理学方法将用于解决三个假设：1） GABAR亚基组成通过慢性BZ治疗而改变; 2）BZ和 GABA的作用因变化而被长期BZ治疗减弱 在突触前和突触后GABA传递中;和3） GABAR组成的变化导致mRNA表达的变化，因此 亚基蛋白表达，定位于海马层相关 GABA介导的抑制。 的幅度和时间进程， 慢性BZ诱导的GABA能变化的发展和逆转 抑制系统和BZ和GABA激动剂敏感性与 GABAR亚基变化的程度和时间过程。 的变化 编码GABAR亚基的基因的表达，因此亚基的改变， 组合物被提议作为GABAR调节的一种机制，因此， GABAR亚基（α（1-5）、β（1-3）和γ）的mRNA表达 （1-2））将系统地研究使用定量原位 海马层中的杂交方法，并与 用定量免疫组织化学方法测定亚基蛋白的变化 在我们的实验室里开发的。 将间接测量突触前GABA释放 通过改变小型IPSC的频率。 的功能性后果 将使用以下方法测量长期FZP治疗对GABA和BZ药理学的影响： 全细胞膜片钳法测定GABA诱导的海马CA 1区电流 海马脑片和急性分离的CAl中的锥体细胞 神经元 地西泮和唑吡坦增强GABA效应的变化 还将在这两个模型中评估电流。 一个功能和 GABAR突触的分子重组可能为BZ提供了基础 宽容

DESCRIPTION: (Applicant's Abstract) The development of tolerance to benzodiazepine (BZ) anticonvulsant actions limits their clinical value and may relate to patterns of chronic abuse. BZs potentiate GABA inhibition at the GABAA receptor (GABAR) increasing Cl conductance. Regulation of the GABAR following chronic BZ treatment is well established as one mechanism underlying BZ tolerance, yet the sequence of events at brain GABA synapses which result in BZ tolerance are not well understood. Findings of electrophysiological studies in in vitro hippocampal slices, ongoing GABAR autoradiographic studies, and our initial in situ hybridization and immunohistochemical studies of GABAR subunit mRNA and protein have established that the BZ tolerant rat hippocampus provides a useful model for studying the synaptic mechanisms of BZ tolerance and have provided a basis for the proposed studies. Studies designed to evaluate the temporal relation between the functional changes associated with chronic BZ treatment and the regulation of GABARs will be carried out in the hippocampus at several time-points after discontinuing 1 week oral flurazepam (FZP) treatment. Molecular biological, immunohistochemical and electrophysiological methods will be used to address three hypotheses: 1) GABAR subunit composition is modified by chronic BZ treatment; 2) BZ and GABA actions are attenuated by chronic BZ treatment as a function of changes in presynaptic, as well as postsynaptic, GABA transmission; and 3) that changes in GABAR composition resulting from changes in mRNA expression, thus subunit protein expression, are localized to hippocampal layers associated with GABA-mediated inhibition. The magnitude and time-course of the development and reversal of chronic BZ-induced changes in the GABAergic inhibitory system and BZ and GABA agonist sensitivity are related to the degree and time-course of changes in GABAR subunits. A change in the expression of the genes encoding GABAR subunits, thus a change in subunit composition is proposed as one mechanism for GABAR regulation, therefore the expression of mRNAs for GABAR subunits (alpha (1-5), beta (1-3) and gamma (1-2)) will be systematically studied using quantitative in situ hybridization methods in hippocampal layers and temporally correlated with changes in subunit proteins using quantitative immunohistochemical methods developed in our lab. Presynaptic GABA release will be indirectly measured by a change in the frequency of mini IPSCs. The functional consequences of chronic FZP treatment on GABA and BZ pharmacology will be measured using whole-cell patch-clamp methods to measure GABA-induced currents in CAl pyramidal cells in hippocampal slices and in acutely dissociated CAl neurons. Changes in diazepam and zolpidem's effects to potentiate GABA currents will also be assessed in these two models. A functional and molecular reorganization of GABAR synapses may provide a basis for BZ tolerance.