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 限制了它们的临床价值，并可能与慢性虐待的模式有关。 补肾益气汤增强GABA对GABAA受体(GABAR)的抑制作用 电导。慢性BZ治疗后GABAR调节良好 被确立为BZ耐受的一种机制，但 导致BZ耐受的大脑GABA突触事件不是很好 明白了。体外电生理学研究进展 海马片，正在进行的伽巴尔放射自显影研究，以及我们最初的 GABAR亚单位mRNA的原位杂交和免疫组织化学研究 和蛋白质已经证实，BZ耐受大鼠的海马区提供了一种 研究BZ耐受和HAD突触机制的实用模型 为拟议的研究提供了基础。旨在评估 慢性BZ相关功能改变的时间关系 GABARs的治疗和调节将在 停药1周后多个时间点的海马区 氟西潘(FZP)治疗。分子生物学、免疫组织化学和 将使用电生理方法来解决三个假设：1) 通过慢性BZ治疗改变Gabar亚基组成；2)BZ和 作为变化的函数，慢性BZ治疗可减弱GABA的作用 在突触前和突触后，GABA传递；以及3) 由于mRNA表达的变化导致GABAR组成的变化，因此 亚单位蛋白表达，定位于海马层相关 有GABA介导的抑制作用。地震的规模和时间进程 慢性BZ所致GABA能改变的发生和逆转 抑制系统与BZ和GABA激动剂的敏感性有关 GABAR亚基变化的程度和时间过程。这是一种变化 编码GABAR亚基的基因的表达，从而改变亚基 建议将组合作为GABAR调节的一种机制，因此 GABAR亚基(α(1-5)、β(1-3)和γ)的mRNAs表达 (1-2))将进行系统的原位定量研究 海马层的杂交方法及其时间相关性 定量免疫组织化学方法检测亚基蛋白质的变化 这是我们实验室研发的。将间接测量突触前GABA的释放 通过改变迷你ipscs的频率。的功能后果 慢性FZP治疗对GABA和BZ药理学的影响将使用 全细胞膜片钳技术测量CAL细胞GABA诱导电流 海马片和急性分离的CA1区锥体细胞 神经元。安定和唑吡坦增强GABA作用的变化 在这两个模型中也将评估海流。一个功能齐全的 GABAR突触的分子重组可能为BZ提供基础 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.