Molecular Regulation of GABAalpha Function in Amygdala

描述（由申请人提供）：共病焦虑可能使成瘾者容易复发，继发于压力和焦虑对大脑的生物学影响。此外，杏仁核的“过度兴奋”，如在压力或恐惧和物质戒断期间反复观察到的，可能有助于焦虑反应和药物渴望。基底外侧杏仁核（BLA）内的这种兴奋性增加可能部分是由于压力或物质使用后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（BZD）依赖来研究GABAA受体的活动依赖性调节的分子机制。这两种操作都导致BLA中GABAA受体的下调。我们将比较急性，慢性和戒断阶段的BZD管理的恐惧条件应激后GABAA调节的分子机制。我们假设：1）基底外侧杏仁核内GABAA的活动依赖性调节是物质依赖和应激反应的共同机制; 2）影响BZD反应的GABA能操纵也会影响条件性恐惧的获得和表达。在这个建议中，我们将比较的分子，行为和电生理机制的GABAA调节后的条件性恐惧应激与急性，慢性和戒断阶段的苯二氮卓类药物管理。我们认为这些药理学和行为学事件具有相似的机制，涉及GABAA复合物的活性依赖性下调。将使用小鼠中的多种分子遗传学方法来检验这一假设，包括：1）用原位杂交和免疫印迹检查GABAA复合物基因和蛋白; 2）使用Cre/lox系统通过GABAA α 1基因的杏仁核特异性缺失来操纵GABAA受体表达; 3）使用慢病毒介导的基因沉默来操纵突触后GABAA α 2基因; 4）通过沉默Gephyrin基因操纵受体簇。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影响。鉴于杏仁核GABA能系统在调节丘脑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的GABAA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新的方法来理解共享的分子和细胞机制，可能介导共病焦虑和药物滥用障碍。了解应激和药物反应的共同机制对于理解病理生理学和为这些高度共病的疾病提出新的治疗方法至关重要。

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.