HIPPOCAMPAL BENZODIAZEPINE TOLERANCE

苯二氮类药物(BZS)通过GABA/A受体(GABAR)发挥作用，是一种有效的抗癫痫药。它们的临床应用受到功能性耐受的出现的限制，通常认为是由体外海马区CA1锥体细胞突触后的变化所介导的。除了原位测量的突触后GABAR结构的变化(α1和β3亚单位的mRNA和蛋白下调)和功能的变化(mIPSC幅度和C1通道电导降低)外，GABA能中间神经元活性的变化也被认为与BZ耐受有关。BZ拮抗剂诱导的GABAR亚单位蛋白水平和mIPSC幅度的快速恢复表明，翻译和翻译后机制可能相互依赖地促进了BZ的耐受。对PKA介导的GABAR电流的调制以及cAMP类似物部分恢复BZ治疗大鼠mIPSC幅度的初步研究表明，PKA介导的事件的调制在一定程度上导致了GABAR系统的功能障碍。更多的研究已经发现兴奋性氨基酸部分的变化，导致GABAR系统功能障碍。更多的研究已经发现兴奋性氨基酸受体(EAAR)亚单位mRNAs和蛋白质的变化，这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。根据这些发现，在体外和原位的海马区产生了三个中心假说，这三个假说将由三个特定的目标来解决：特定的目标1：通过使用细胞内和全细胞贴片技术采样可见的CA1中间神经元亚群，研究内在和外在神经元间功能在降低GABA张力中的作用。具体目的2：利用荧光共定位和定量EM免疫金技术，探讨翻译过程，即慢性激动剂和急性拮抗剂给药后GABARα1亚单位蛋白再分布的作用，以及翻译后机制，即外源性和内源性PKA介导的蛋白磷酸化刺激物对CA1锥体细胞mIPSC幅度的调节作用。特异性目的3：研究CA1区锥体细胞兴奋性氨基酸受体(EAAR)结构和功能的代偿性变化：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EAAR受体结合的放射自显影技术的变化。3b)全细胞切片技术将用于测量EAAR受体介导的、诱发的和微小的EPSC的幅度和衰减动力学。EAAR功能也将通过显微荧光测量急性分离的CA1锥体细胞的钙摄取来评估。了解抑制性和兴奋性突触变化的本质可能有助于设计避免耐受的药物和方法，并使我们更好地了解癫痫患者抗惊厥药物治疗过程中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.