HIPPOCAMPAL BENZODIAZEPINE TOLERANCE

苯二氮卓类药物（BZs）通过GABA/A受体（GABAR）起作用，是一种有效的抗惊厥药。它们的临床用途受到功能性耐受性的限制，功能性耐受性通常被认为是由体外海马体内CA1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和蛋白下调）和功能（mIPSC振幅和c1通道电导降低）的变化外，gaba能神经元间活性的变化被认为有助于BZ耐受性。BZ拮抗剂诱导的GABAR亚基蛋白水平和mIPSC振幅的快速恢复表明，翻译和翻译后机制可能相互依赖地促进BZ耐受性。在bz治疗的大鼠中，pka介导的GABAR电流调节和cAMP类似物部分恢复mIPSC振幅的能力的初步研究表明，pka介导的事件的调节在一定程度上有助于GABAR系统功能障碍。进一步的研究发现，兴奋性氨基酸部分的改变，有助于GABAR系统功能障碍。另外的研究已经发现了兴奋性氨基酸受体（EAAR）亚基mrna和蛋白质的变化，这表明慢性BZ治疗也可以调节兴奋系统，以响应GABA抑制的减少。从这些在体外和原位海马中的发现中产生了三个中心假设，这些假设将通过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取样可视化CA1中间神经元亚群，研究内在和外在中间神经元功能在降低GABA张力中的作用。特异性目的2：利用荧光共定位和定量EM免疫金技术，探索慢性激动剂和急性拮抗剂给药后2A)翻译（即GABAR α 1亚基蛋白重分布）的作用，以及2B)翻译后机制，即外源性和内源性刺激物介导的paca介导的蛋白磷酸化对CA1锥体细胞中mIPSC振幅的调节作用。特异性目的3：检查CA1锥体细胞中兴奋性氨基酸受体（EAAR）结构和功能的代偿性变化。结构测量包括：定量免疫组织化学技术和显微解剖海马的Western blot分析，以及使用放射自显像技术检测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功能也将评估使用微荧光测量Ca2+摄取到急性解离CA1锥体细胞。了解抑制性和兴奋性突触变化的本质可能有助于设计绕过耐受性的药物和方法，并使我们更好地了解癫痫患者在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.