Pathological AMPA receptor adaptations governing dependence-escalated alcohol self-administration

项目摘要 酒精依赖和多次戒酒经历与酒精使用的严重性增加有关 精神障碍(AUD)、渴求和抗拒治疗。酗酒获得了对行为的控制，部分原因是 谷氨酸能AMPA受体(AMPAR)调节突触和 大脑奖赏通路中的行为可塑性。独特的辅助蛋白，跨膜AMPAR调节 蛋白质(TARP)γ-8，已被证明调节AMPAR的运输、活性和依赖CaMKII的可塑性， 这使得它对AMPAR介导的神经传递至关重要。TARPγ-8的一个重要特征是其高度 限制表达限于已知的调节谷氨酸对酒精反应的皮质边缘区域 包括内侧前额叶皮质(MPFC)、基底外侧杏仁核(BLA)和海马体，而 伏隔核(NAC)明显缺失。证据表明长期饮酒会增加谷氨酸 水平，进而促进钙离子的内流，并启动CaMKII磷酸化的级联反应 AMPAR以增加和维持AMPAR的活性。由于AMPAR活动是开发新的 行为(例如，学习)和对动作的保持(例如，记忆)，这一基本神经过程可能是基础 酒精的发展、维持，以及关键的是，依赖升级了自我给药。因此， K99/R00提案将确定TARPγ-8是否调节AMPAR在关键脑区的传递 在依赖升级的酒精自我给药期间。建议的目标1(K99阶段)将调查 TARPγ-8依赖的兴奋性钙信号在酒精自我刺激时奖赏相关脑区的作用 使用高度新颖的多光谱四通道光纤对mPFC、BLA、NAC和vHPC进行管理 测光平台。目标2(K99阶段)将研究TARPγ-8作为CIE蒸汽依赖的机制- 酒精自我给药的诱导升级以及由此导致的TARPγ-8和AMPAR的共同定位 使用共聚焦显微镜。AIM 3(R00阶段)结合了这些技术来评估KEY中的钙信号转导 在酒精依赖升级的自我给药过程中，奖励大脑区域。这些发现随后被推广。 通过采用基于电路的方法，使用选择性药物操作结合纤维- 用分光光度法评价TARPγ-8结合的AMPAR对“自下而上”和“顶端-顶端”的抑制作用 向下“(mPFC到NAC)钙信号。这项工作通过提供基本的机械原理来推动该领域的发展 对TARPγ-8依赖的洞察-升级的酒精自我给药对 认识和治疗AUD，并有可能为新药物治疗的发展提供信息 以高度选择性的大脑区域特定方式瞄准AMPAR功能的策略。

Project Summary Alcohol dependence and multiple withdrawal experiences are related to increased severity of alcohol use disorder (AUD), craving, and resistance to treatment. Alcohol abuse gains control over behavior, in part, through pathological adaptations of glutamatergic AMPA receptor (AMPAR) mechanisms that regulate synaptic and behavioral plasticity in brain reward pathways. The unique auxiliary protein, transmembrane AMPAR regulatory protein (TARP) γ-8, has been shown to regulate AMPAR trafficking, activity, and CaMKII-dependent plasticity, making it critical for AMPAR mediated neural transmission. An important feature of TARP γ-8 is its highly restricted expression limited to corticolimbic regions known to regulate glutamatergic response to alcohol including the medial prefrontal cortex (mPFC), basolateral amygdala (BLA), and the hippocampus, while noticeably absent from the nucleus accumbens (NAc). Evidence indicates chronic alcohol increases glutamate levels, which in turn promotes the influx of calcium, and initiating a cascade where CaMKII phosphorylates AMPARs to increase and sustain AMPAR activity. Since AMPAR activity is required for the development of new behavior (e.g., learning) and retention of actions (e.g., memory), this fundamental neural process may underlie the development, maintenance, and critically, dependence-escalated self-administration of alcohol. Therefore, this K99/R00 proposal will determine if TARP γ-8 regulates AMPAR mediated transmission in key brain regions during dependence-escalated alcohol self-administration. Aim 1 (K99 phase) of the proposal will investigate the role of TARP γ-8 dependent excitatory Ca2+ signaling in reward-related brain regions during alcohol self- administration in the mPFC, BLA, NAc, and vHPC using a highly novel multi-spectral, four-channel fiber photometry platform. Aim 2 (K99 phase) will examine TARP γ-8 as a mechanism of CIE vapor dependence- induced escalation of alcohol self-administration and the consequential co-localization of TARP γ-8 and AMPAR using confocal microscopy. Aim 3 (R00 phase) combines these techniques to evaluate Ca2+ signaling in key- reward brain regions during dependence-escalated alcohol self-administration. These findings are then extended by taking a circuit-based approach using a selective pharmacological manipulation in combination with fiber- photometry to evaluate site-specific TARP γ-8 bound AMPAR inhibition on “bottom-up” (BLA to NAc) and “top- down” (mPFC to NAc) Ca2+ signaling. This work moves the field forward by providing fundamental mechanistic insights into TARP γ-8 dependence-escalated alcohol self-administration which has high translational value for understanding and treating AUD and has the potential to inform development of new pharmacotherapeutic strategies that target AMPAR function in a highly-selective brain region specific manner.