Molecular Regulation of GABA(A) Receptor Function in Amygdala

描述（由申请人提供）：共病性焦虑可能使成瘾者易复发，继发于压力和焦虑对大脑的生物效应。此外，杏仁核的“过度兴奋性”，在压力或恐惧和药物戒断期间反复出现，可能会导致焦虑反应和药物渴望。基底外侧杏仁核（BLA）的兴奋性增加可能部分是由于应激或物质使用后GABAA激活减少。这些研究将通过关注恐惧条件反射和苯二氮卓类药物依赖来研究GABAA受体活性依赖调节的分子机制。这两种操作都会导致BLA中GABAA受体的下调。我们将比较恐惧应激条件下GABAA调节的分子机制与BZD给药的急性、慢性和戒断期。我们假设：1)基底外侧杏仁核内GABAA的活性依赖性调节是物质依赖和应激反应的共同机制；影响BZD反应的gaba能操作也会影响条件恐惧的习得和表达。在本研究中，我们将比较条件恐惧应激对GABAA调节的分子、行为和电生理机制，以及苯二氮卓类药物急性、慢性和戒断期的影响。我们认为这些药理学和行为事件具有类似的机制，涉及GABAA复合物的活性依赖性下调。多种小鼠分子遗传学方法将用于验证这一假设，包括：1)用原位杂交和免疫印迹法检测GABAA复合物基因和蛋白质；2)利用Cre/lox系统通过杏仁核特异性缺失GABAA α 1基因来调控GABAA受体的表达；3)利用慢病毒介导的基因沉默操作突触后GABAA α 2基因；4)通过沉默Gephyrin基因调控受体聚类。这些研究对苯二氮卓类药物依赖、戒断和复发的机制有直接的影响。鉴于杏仁核GABAA能系统在调节伏隔核内多巴胺能张力中的作用，杏仁核GABAA调节可能在成瘾性疾病中具有更普遍的作用。事实上，最近的研究表明，吗啡、酒精、可卡因和尼古丁也可能改变杏仁核中GABA的功能，并且急剧提高GABA水平会阻止药物相关线索或自我给药的恢复。这一建议代表了一种理解可能介导共病焦虑和药物滥用障碍的共享分子和细胞机制的新方法。了解应激和药物反应的共同机制对于理解病理生理学和提出治疗这些高度共病性疾病的新方法至关重要。

DESCRIPTION (provided by applicant): Comorbid anxiety may predispose addicts to relapse secondary to the biological effects of stress and anxiety on the brain. Furthermore 'hyperexcitability' of the amygdala, as repeatedly seen during stress or fear and substance withdrawal, may contribute to anxiety responses and drug craving. Such increased excitability within the basolateral amygdala (BLA) may be due in part to decreased GABAA activation following stress or substance use. These studies will examine the molecular mechanisms of activity- dependent regulation of GABAA receptors by focusing on fear conditioning and benzodiazepine (BZD) dependence. Both of these manipulations lead to downregulation of GABAA receptors in the BLA. We will compare the molecular mechanisms of GABAA regulation following the stress of fear conditioning with acute, chronic, and withdrawal phases of BZD administration. We hypothesize that: 1) activity-dependent modulation of GABAA within the basolateral amygdala is a common mechanism of both substance dependence and stress response; and 2) GABAergic manipulations that affect BZD response will also affect acquisition and expression of conditioned fear. In this proposal, we will compare the molecular, behavioral, and electrophysiological mechanisms of GABAA regulation following conditioned fear stress with acute, chronic, and withdrawal phases of benzodiazepine administration. We propose that these pharmacological and behavioral events share similar mechanisms involving activity-dependent downregulation of the GABAA complex. A variety of molecular genetic approaches in mice will be used to test this hypothesis including: 1) examination of GABAA complex genes and proteins with in situ hybridization and immunoblotting; 2) manipulation of GABAA receptor expression via amygdala-specific deletion of the GABAA alpha1 gene using the Cre/lox system; 3) manipulation of post-synaptic GABAA alpha2 gene using Lentiviral-mediated gene silencing; 4) manipulation of receptor clustering through silencing of the Gephyrin gene. These studies have direct implications for mechanisms of benzodiazepine dependence, withdrawal, and relapse. Given the role of amygdala GABAergic systems in modulating dopaminergic tone within the nucleus accumbens, amygdala GABAA regulation may have a more general role in addictive disorders. In fact, recent studies have suggested that morphine, alcohol, cocaine and nicotine may also alter GABAA functioning in amygdala, and acutely enhancing GABA levels blocks reinstatement by drug-associated cues or self-administration. This proposal represents a novel approach to understanding shared molecular and cellular mechanisms that may mediate comorbid anxiety and substance abuse disorders. Understanding the mechanisms shared by stress and drug response is critical for understanding pathophysiology and proposing new treatments for these highly comorbid disorders.