HIPPOCAMPAL BENZODIAZEPINE TOLERANCE

苯二氮卓类（BZ）通过GABA/A受体（GABAR）起作用，是多种癫痫的有效抗惊厥药。它们的临床用途受到功能耐受出现的限制，通常认为是由体外海马CA 1锥体细胞突触后的变化介导的。除了原位测量的突触后GABAR结构（α 1和β 3亚基mRNA和蛋白质下调）和功能（mIPSC振幅和C1通道电导降低）的变化外，GABA能中间神经元活性的变化被认为有助于BZ耐受性。快速BZ拮抗剂诱导的GABAR亚基蛋白水平和mIPSC振幅的恢复表明，翻译和翻译后机制可能相互依赖地有助于BZ耐受性。对PKA介导的GABAR电流调制和cAMP类似物部分恢复BZ处理大鼠mIPSC振幅的能力的初步研究表明，PKA介导的事件的调制部分地导致GABAR系统功能障碍。 另外的研究已经确定兴奋性氨基酸部分的变化，有助于GABAR系统功能障碍。其他研究已经确定了兴奋性氨基酸受体（EAAR）亚基mRNA和蛋白质的变化，表明兴奋性系统也受到慢性BZ治疗的调节，以响应减少的GABA抑制。从体外和原位海马的这些发现中产生了三个中心假设，这将通过3个特定目的来解决：特定目的1：通过使用细胞内和全细胞贴片技术对可视化的CA 1中间神经元亚群进行采样，检查内在和外在中间神经元功能在降低GABA张力中的作用。具体目标2：是探索2A）翻译，即，使用荧光共定位和定量EM免疫金技术和2B）翻译后机制，即，PKA介导的蛋白磷酸化的外源性和内源性刺激物调节CA 1锥体细胞中mIPSC振幅的作用。具体目标3：是为了检查在CA 1锥体细胞中兴奋性氨基酸受体（EAAR）结构和功能中发生的代偿性变化：3A）结构测量包括：显微解剖的海马的定量免疫组织化学技术和Western印迹分析以及使用放射自显影技术的EAAR受体结合的变化。3B）全细胞切片贴片技术将用于测量EAAR受体介导的、诱发的和微型EPSC振幅和衰减动力学。EAAR功能也将使用急性分离的CA 1锥体细胞的Ca 2+摄取的显微荧光测量来评估。了解抑制性和兴奋性突触的变化的性质可能允许设计药物和方法来规避耐受性，并使我们能够更好地了解癫痫患者抗惊厥药物治疗期间这些受体系统功能障碍的基本机制。

Benzodiazepines (BZs) which act through the GABA/A receptor (GABAR), are potent anti-convulsant for a variety of epilepsies. Their clinical usefulness is limited by the appearance of functional tolerance, commonly-held to be mediated by changes at the post-synaptic on CA1 pyramidal cells in the in vitro hippocampus. In addition to changes in post-synaptic GABAR structure (alpha1 and beta3 subunit mRNA and protein down-regulation), measured in situ, and function (decreased mIPSC amplitude and C1-channel conductance), changes in GABAergic interneuron activity are proposed to contribute to BZ tolerance. Rapid BZ antagonist-induced restoration of GABAR subunit protein levels and mIPSC amplitude suggested that both translational and post-translational mechanisms may interdependently contribute to BZ tolerance. Preliminary studies of PKA-mediated modulation of GABAR currents and the ability of a cAMP analogue to partially restore mIPSC amplitude in BZ-treated rats suggests that a modulation of PKA-mediated events, in part, contribute to GABAR system dysfunction. Additional studies have identified changes in excitatory amino acid part, contribute to GABAR system dysfunction. Additional studies have identified changes in excitatory amino acid receptor (EAAR) subunit mRNAs and protein suggesting that excitatory systems are also regulated by chronic BZ treatment in response to reduced GABA inhibition. Three central hypotheses were generated from these findings in in vitro and in situ hippocampus which will be addressed by 3 SPECIFIC AIMS: SPECIFIC AIM 1: to examine the role of intrinsic and extrinsic interneuron function in reducing GABA tone by sampling subpopulations of visualized CA1 interneurons using intracellular and whole-cell patch techniques. Specific Aim 2: is to explore the role of both 2A) translational, i.e., GABAR alpha1 subunit protein redistribution following chronic agonist and acute antagonist administration, using fluorescent co-localization and quantitative EM immunogold techniques and 2B) post-translational mechanisms, i.e., the effect of exogenous and endogenous stimulators of PKA-mediated protein phosphorylation to modulate mIPSC amplitude in CA1 pyramidal cells. SPECIFIC AIM 3: is to examine the compensatory changes which occur in excitatory amino acid receptor (EAAR) structure and function in CA1 pyramidal cells: 3A) Structural measures include: quantitative immunohistochemical techniques and Western blot analysis of microdissected hippocampus and changes in EAAR receptor binding using autoradiographic techniques. 3B) Whole-cell slice patch techniques will e used to measure EAAR receptor-mediated, evoked and miniature EPSC amplitude and decay kinetics. EAAR function will also be assessed using microfluorometric measurements of Ca2+ uptake into acutely dissociated CA1 pyramidal cells. Understanding the nature of the changes at inhibitory and excitatory synapses may allow the design of drugs and approaches to circumvent tolerance and allow us to gain a better understanding of basic mechanisms involved in the dysfunction of these receptor systems during anticonvulsant drug treatment of epileptic patients.