Central Amygdala to Lateral Hypothalamus Neuropeptide Y projections as a Target in Sex-Specific Binge-Like Ethanol Consumption in Mice

项目摘要 严重的酒精使用和依赖已经成为美国的一个主要公共卫生问题， 在地球仪上，寻求治疗的人目前可获得的治疗有限。酗酒是 一个重要的危险因素，依赖的发展，这种形式的摄入导致重大健康问题， 后果虽然男性更常被诊断为酒精使用障碍，但 近年来，女性的诊断率有所上升。此外，饮酒行为的性别差异和 潜在的神经机制仍然在很大程度上未知。我们的实验室和其他实验室已经暗示了 中央杏仁核（CeA）在动物模型中的乙醇消耗。促肾上腺皮质激素释放因子 调节CeA中酗酒样的乙醇饮用，我们实验室的初步数据表明，沉默CRF+ 从CeA到外侧下丘脑（LH）的投射导致性特异性的暴食样减少， 雄性小鼠饮用乙醇，而雌性小鼠则没有。类似地，LH中的CRF 1 R拮抗和CRF 2 R激动 导致男性特定的酗酒钝化，表明这种受体系统参与调节 酗酒CRF、GABA和神经肽Y（NPY）Y1受体（Y1 R）都密切表达于 我们实验室的初步工作表明，CeA中的Y1 R激活会减弱酒精摄入量，类似于 在CeA中CRF神经元沉默的雄性中观察到的影响。然而，性别依赖的乙醇消费量 神经肽Y系统的差异仍然没有研究。因此，这项建议的重点是审查潜在的 CeA NPY和Y1 R信号传导的性别差异，以及Y1 R + CeA LH通路在 酒精摄入量的调节。拟议的实验将使用以下组合： 转基因、化学遗传学、组织学和药理学方法。经过充分验证的啮齿动物模型， 在整个过程中，将使用被称为“黑暗中饮酒”（DID）的酒精消费 实验来识别神经变化。对于目标1，我将使用设计受体技术（病毒注射 Cre依赖性Y1 R启动子DREADDs在CeA中的表达）和转基因NPY 1 R-cre中LH的插管 以确定沉默从CeA到LH的Y1 R+投射神经元是否会减弱DID狂欢样 乙醇摄入量和相关的血液乙醇浓度（BEC）在雄性而不是雌性小鼠。在目标2中，我将 关注Y1 R信号在CeA中的作用，以及Y1 R激动是否会影响酒精饮酒狂欢样 以性别依赖的方式，使用定点药理学和NPY过表达病毒载体。最后， 在目标3中，我将检测多个周期的治疗后NPY和Y1 R水平的变化，以及与CRF的共表达。 雄性和雌性小鼠饮酒过量。在神经肽Y系统中， 依赖性，并将使用免疫组织化学，Western印迹和实时PCR进行评估 来定量CeA中的蛋白质和mRNA。总之，这些结果将为我们提供新的见解， 神经回路潜在的性别差异在酗酒一样的酒精摄入量。

PROJECT SUMMARY Heavy alcohol use and dependence has become a major public health concern within the United States and across the globe, with limited treatments currently available to those who seek them. Binge intake of alcohol is a significant risk factor to the development of dependence, and this form of intake leads to major health consequences. While men are more commonly diagnosed with alcohol use disorders, the frequency for diagnoses in females has risen in recent years. Furthermore, sex differences in drinking behavior and the underlying neural mechanism remain largely unknown. Our lab, and others, have implicated the role of the central amygdala (CeA) in ethanol consumption in animal models. Corticotrophin releasing factor (CRF) modulates binge-like ethanol drinking in the CeA, and pilot data from our lab shows that silencing CRF+ projections from the CeA to the lateral hypothalamus (LH) leads to a sex-specific reduction of binge-like ethanol drinking in male, but not female, mice. Similarly, CRF1R antagonism and CRF2R agonism in the LH lead to male specific blunting of binge-drinking, suggesting this receptor system’s involvement in modulating binge drinking. CRF, GABA, and Neuropeptide Y (NPY) Y1 receptor (Y1R) are all closely expressed in the CeA, and pilot work from our lab shows Y1R activation in the CeA blunts binge-like ethanol intake, similar to effects seen in males with silenced CRF neurons in the CeA. However, sex-dependent ethanol consumption differences in the NPY system remain uninvestigated. Thus, this proposal focuses on examining potential sex-differences in CeA NPY and Y1R signaling, as well as the role of the Y1R+ CeA  LH pathway, in the modulation of binge-like ethanol consumption. Proposed experiments will use a combination of transgenic, chemogenetic, histological, and pharmacological approaches. The well-validated rodent model of binge-like ethanol consumption known as “Drinking in the Dark” (DID) will be used throughout these experiments to identify neural changes. For Aim 1, I will use the designer receptor technology (viral injections of Cre-dependent Y1R-promoter DREADDs in the CeA) and cannulation of the LH in transgenic NPY1R-cre mice to determine if silencing Y1R+ projection neurons from the CeA to the LH will attenuate DID binge-like ethanol intake and associated blood ethanol concentrations (BECs) in male but not female mice. In Aim 2, I will focus on the role of Y1R signaling in the CeA, and whether Y1R agonism can affect binge-like ethanol drinking in a sex-dependent manner, using site-directed pharmacology and an NPY over-expressing viral vector. Lastly, in Aim 3, I will examine changes in NPY and Y1R levels, and co-expression with CRF, after multiple cycles of binge-like ethanol drinking in male and female mice. Binge-induced plasticity in the NPY system that are sex- dependent are predicted, and will be assessed using immunohistochemistry, Western Blots, and real-time PCR to quantify protein and mRNA in the CeA. Taken together, these results will provide new insights into the neurocircuitry underlying sex-differences in binge-like ethanol intake.