Adaptations in an insular cortex microcircuit following escalated alcohol drinking

项目摘要/摘要 酒精使用障碍(AUD)是一个普遍存在的公共健康问题，给个人和经济带来巨大的影响 成本。尽管AUD的性质很严重，但我们对所涉及的大脑机制缺乏透彻的了解 在过度饮酒方面。因此，未来研究的一个重要目标是更准确地绘制 伴随着酒精摄入量增加而来的分子和回路适应。虽然有几种型号 这使得对酒精饮酒如何改变大脑回路和信号的机械性研究成为可能 该项目将专注于间歇性酒精接触(IA)范例，因为它可靠地推动了高水平的 在酒精剥夺期间自愿饮酒和戒酒。虽然众多的神经化学系统 已被确定为在AUD中发挥作用，治疗干预最有希望的线索之一是 Kappa阿片受体(KOR)及其内源性配体强啡肽(Dyn)。DYN/KOR系统是 在酒精依赖者和反复接触酒精的啮齿动物中都有上调。此外，Dyn/Kor 信号已被证明有助于酒精摄入量的增加和相关的负面情绪状态 戒酒。与Dyn/KOR系统在过量饮酒中的重要作用一致， 我已经生成了初步结果，表明对KOR的药物封锁抑制了升级 IA范例中的饮酒。其中一个可能的大脑区域是岛叶皮质(IC)。 尽管在酒精研究的背景下，IC是一个高度未被研究的大脑区域，但有几项研究 显示IC参与酒精自我给药，并在高血糖后经历结构适应 酒精摄入量。在我的初步工作中，我已经确定了Dyn表达的一个离散的亚群 位于IC(ICDyn)第2/3层的锥体细胞，与长期IA对酒精的接触。此外，我还有 发现Dyn降低了表达KOR的第5层中间神经元的兴奋性，而长期IA对酒精的作用 增加IC层5锥体细胞的兴奋性驱动。这表明KOR信号在局部调制 5层神经元活动和IA对酒精的作用可诱导ICDyn板层微回路的可塑性。这个 拟议的实验将彻底表征长期对酒精的影响如何影响新发现的 集成多个融合的电生理、药理学和光遗传学的ICDyn微电路 接近了。使用这些方法，我将绘制ICDyn微电路的连接性图并评估 伴随长期IA对酒精的IC的基础兴奋性和Dyn/KOR信号的适应性 喝酒。我还将使用多路复用器将KOR定位到IC第五层中不同的神经元亚群 荧光原位杂交法。通过对这部小说如何产生更机械的理解 ICDyn微电路受到酒精摄入量增加的影响，我将通知未来旨在减轻酒精摄入量的研究 虐待和依赖。最终，这项提案将确定一个依赖于站点、特定于细胞和信号的- 可用于AUD靶向治疗的选择性机制。

Project Summary/Abstract Alcohol use disorder (AUD) is a pervasive public health problem that carries great personal and economic costs. Despite the serious nature of AUD, we lack a thorough understanding of the brain mechanisms involved in excessive alcohol consumption. Therefore, a significant goal for future research is to more precisely map the molecular and circuit adaptations that accompany escalated alcohol intake. Although there are several models that allow mechanistic investigation of how escalated alcohol drinking can alter brain circuits and signaling, this project will focus on the intermittent access (IA) to alcohol paradigm, as it reliably drives high levels of voluntary alcohol intake and withdrawal during alcohol deprivation. While numerous neurochemical systems have been identified as playing a role in AUD, one of the most promising leads for therapeutic intervention is the kappa opioid receptor (KOR) and its endogenous ligand dynorphin (Dyn). The Dyn/KOR system is upregulated in both alcohol-dependent humans and rodents repeatedly exposed to alcohol. Further, Dyn/KOR signaling has been shown to contribute to escalated alcohol intake and negative-affective states associated with alcohol withdrawal. Consistent with the important role of the Dyn/KOR system in excessive alcohol intake, I have generated preliminary results that show pharmacological blockade of KOR suppresses escalated alcohol drinking in the IA paradigm. One possible brain region underlying this effect is the insular cortex (IC). Though the IC is a highly understudied brain region in the context of alcohol research, several studies have shown the IC is involved in alcohol self-administration and undergoes structural adaptations following high levels of alcohol intake. In my preliminary work, I have identified a discrete subpopulation of Dyn-expressing pyramidal cells in layer 2/3 of the IC (ICDyn) that are engaged by long-term IA to alcohol. In addition, I have found that Dyn decreases excitability in KOR-expressing layer 5 interneurons, and that long-term IA to alcohol increases excitatory drive in IC layer 5 pyramidal cells. This indicates that KOR signaling locally modulates layer 5 neuronal activity and IA to alcohol may induce plasticity within an ICDyn laminar microcircuit. The proposed experiments will thoroughly characterize how long-term IA to alcohol impacts this newly identified ICDyn microcircuit by integrating multiple converging electrophysiological, pharmacological, and optogenetic approaches. Using these approaches, I will map the connectivity of this ICDyn microcircuit and assess adaptations in basal excitability and Dyn/KOR signaling in the IC that accompany long-term IA to alcohol drinking. I will also localize KOR to distinct subpopulations of neurons in layer 5 of the IC using a multiplexed fluorescence in situ hybridization assay. By generating a greater mechanistic understanding of how this novel ICDyn microcircuit is impacted by escalated alcohol intake, I will inform future studies aimed at mitigating alcohol abuse and dependence. Ultimately, this proposal will identify a site-dependent, cell-specific, and signaling- selective mechanism that can be used for the targeted treatment of AUD.