Molecular Component - Contet

摘要 斯克里普斯研究所酒精研究中心的总体工作模式是 内侧前额叶皮质(Mpfc)的边缘下亚区是调节失调的关键中枢。 慢性间歇性乙醇(CIE)暴露对大脑网络活动的影响，部分原因是来自 下丘脑和对杏仁中央核(CEA)自上而下的控制受损。在这个范围内 由Contet博士和Dunning博士领导的分子组件框架将测试这一假设 MPFC星形胶质细胞中代谢型谷氨酸受体3(GRM3)的丰富与行为有关 通过维持局部高谷氨酸能升高小鼠的酒精依赖表型 局部中间神经元对投射到CEA的锥体神经元的抑制性传入。这一假设得到了支持 根据上一个供资期间产生的结果，这些结果揭示了CIE引发的长期影响 过量饮酒对星形细胞标志物丰度的影响，GRM3达到蛋白质组 在预检和下检mPFC样本中均有显著意义。GRM3在星形胶质细胞中高度富含 控制谷氨酸摄取，但也可以调节突触传递和神经元的可塑性。具体目标1 因此，该项目的重点将是确定在大宗蛋白质组中检测到的GRM3丰度降低 源于星形胶质细胞对神经元的减少。要做到这一点，我们将利用最先进的量化质量 从代谢性标记的大脑样本中提取蛋白质丰度的变化 单元类型。我们预计CIE诱导的mPFC GRM3缺陷将是星形胶质细胞特有的，并将是 与谷氨酸转运体丰度降低有关。我们还将探讨CRF1的潜在作用 这一效应中的受体信号。然后我们将检验这样的假设，即GRM3丰度降低既是 产生CIE行为表型的必要条件(具体目标2)和充分(具体目标3) 利用基因表达调控技术在有慢性过量饮酒史的小鼠中戒断 靶向mPFC星形胶质细胞。分析GRM3过表达和基因敲除对细胞的影响 特定类型的蛋白质组、细胞外谷氨酸水平和突触传递将提供机制 洞察这些行为结果可能是如何产生的。此外，具体目标2将包括一项 通过抑制谷氨酸羧基肽酶II(GCPII)来挽救GRM3信号的翻译方法。 总之，该项目有望确定星形细胞GRM3作为一种重要的调节因子的相关性。 MPFC中的谷氨酸稳态可以靶向减少过度饮酒。它将从中受益 来自神经蛋白质组学和动物模型提供的专业知识和资源，并将相互作用 无论是在概念上还是在实验上，都与其他四个TSRI-ARC研究组件一起使用。

SUMMARY The overarching working model of The Scripps Research Institute Alcohol Research Center (TSRI-ARC) is that the infralimbic subdivision of the medial prefrontal cortex (mPFC) represents a critical hub for the dysregulation of brain network activity by chronic intermittent ethanol (CIE) exposure, driven in part by inputs from the hypothalamus and impairing top-down control over the central nucleus of the amygdala (CeA). Within this framework, the Molecular Component, led by Drs. Contet and Dunning, will test the hypothesis that reduced abundance of metabotropic glutamate receptor 3 (GRM3) in mPFC astrocytes contributes to behavioral phenotypes of alcohol dependence in mice by maintaining a local hyperglutamatergic tone that elevates the inhibitory input of local interneurons onto pyramidal neurons projecting to the CeA. This hypothesis is supported by results generated during the previous funding period, which revealed a long-lasting impact of CIE-induced excessive alcohol drinking on the abundance of astrocytic markers, with GRM3 reaching proteome-wide significance, in both prelimbic and infralimbic mPFC samples. GRM3 is strongly enriched in astrocytes where it controls glutamate uptake, but it can also regulate synaptic transmission and plasticity in neurons. Specific Aim 1 of this project will thus be to determine whether the GRM3 abundance reduction detected in bulk proteomes stems from a reduction in astrocytes vs. neurons. To do this, we will leverage state-of-the-art quantitative mass spectrometry from metabolically labeled brain samples to tease apart protein abundance changes in different cell types. We anticipate that CIE-induced mPFC GRM3 deficiency will be specific to astrocytes and will be associated with reduced abundance of glutamate transporters. We will also probe a potential role of CRF1 receptor signaling in this effect. We will then test the hypotheses that reduced GRM3 abundance is both necessary (Specific Aim 2) and sufficient (Specific Aim 3) to produce the behavioral phenotypes of CIE withdrawal in mice with a history of chronic excessive alcohol drinking, using gene expression manipulations targeted to mPFC astrocytes. Analyzing the consequences of GRM3 overexpression and knockdown on cell type-specific proteomes, extracellular glutamate levels, and synaptic transmission will provide mechanistic insights into how these behavioral outcomes might be generated. Furthermore, Specific Aim 2 will include a translational approach to rescue GRM3 signaling via inhibition of glutamate carboxypeptidase II (GCPII). Altogether, this project is anticipated to establish the relevance of astrocytic GRM3 as an important regulator of glutamate homeostasis in the mPFC that can be targeted to reduce excessive alcohol drinking. It will benefit from the expertise and resources provided by the Neuroproteomics and Animal Models Cores and will interact both conceptually and experimentally with the four other TSRI-ARC Research Components.