5/11 Microglial MyD88 in Mouse Models of Excessive Alcohol Intake

摘要 反复接触酒精可能是一种强有力的神经炎性刺激。酒精激活脑内的小胶质细胞 大脑通过Toll样受体(TLRs)；这种激活导致炎性介质的产生 依赖和滥用4，5.快速放电小白蛋白阳性(PV)GABA能中间神经元(PVI)是关键 对于正常的大脑功能来说。静脉曲张通常被特殊的细胞外基质分子包围，称为 周围神经网(PNN)，保护和缓冲他们免受炎症和氧化应激的影响。值得注意的是， 增加的PNN与酒精使用障碍(AUD)有关，这可能是由于关键的 在面对不利情况时寻求药物等习惯性行为的神经可塑性机制 结果2.我们的初步数据显示，小胶质细胞特异性消融关键的TLR接头的小鼠 分子髓系分化初级反应88[MyD88](CX3CR1-CreBT-MyD88f/f 小鼠“])表现出对内毒素注射的促炎反应(如IL-1、肿瘤坏死因子)减少 与CRE-Controls相比，不出所料。然而，令人惊讶的是，在对内毒素的反应中，男性(但不是女性)CRE 小鼠在额叶皮质表现出更多的PVI和PV/PNN相互作用。Cre雄性小鼠进一步 与对照组相比，在急性黑暗饮酒(DID)方案中，酒精摄入量显著增加， 与以前INIA神经免疫项目使用MyD88-/-(基因敲除)小鼠和慢性 酒精范例6.综上所述，这些数据表明小胶质细胞对PVI数量和/或 通过MyD88信号转导反应炎症信号的PNN被膜，并增加PVI/PNN 缺乏MyD88的相互作用可能是CRE小鼠饮酒增加的原因。的总目标是 这项提议是为了检验这一假设。目标1将确定小胶质细胞-MyD88是否调节PVI的数量 和/或PNN通过评估PVI特异性的吞噬作用直接响应慢性酒精 小胶质细胞的抑制性突触和PNN。Aim 2将确定小胶质细胞-MyD88是否调节 PVI和/或PNN通过评估小胶质细胞的“分泌组”间接反应慢性酒精 以及由星形胶质细胞和PVI产生PNN成分。目标3将确定变坡点和/或 小胶质细胞MyD88阳性和阴性小鼠过量饮酒行为中的PNN相互作用 (1)降解PNNS和(2)化学生成抑制与小胶质细胞激活对过量饮酒的影响。 这是一个创新的新项目，旨在补充其他几个INIA-神经免疫的现有优势 调查人员。如果完成，这些数据将是第一个显示小胶质细胞在PVI/PNN中的直接作用的数据 发展及其在过度饮酒中的作用，这可能导致新的治疗选择。

Abstract Repeated alcohol exposure can be a potent neuroinflammatory stimulus. Alcohol activates microglia within the brain via toll-like-receptors (TLRs); this activation leads to production of inflammatory mediators which contribute to dependence and abuse 4,5. Fast spiking parvalbumin positive (PV+) GABAergic interneurons (PVIs) are critical for normal brain function. PVIs are often surrounded by specialized extracellular matrix molecules called perineuronal nets (PNNs) which protect and buffer them from inflammation and oxidative stress. Notably, increased PNNs are implicated in alcohol use disorder (AUD), which may be due to a "locking in" of critical neuroplasticity mechanisms that underlie habitual behavior such as drug seeking in the face of adverse consequences 2. Our preliminary data show that mice with microglial-specific ablation of the critical TLR adaptor molecule myeloid differentiation primary response 88 [MyD88] (Cx3cr1-CreBT-MyD88f/f [hereafter called "Cre+ mice"]) exhibit decreased proinflammatory responses (e.g. IL-1, TNF) to a lipopolysaccharide (LPS) injection compared to Cre- controls, as expected. Surprisingly, however, in response to LPS, male (but not female) Cre+ mice exhibit increased numbers of PVIs and PV/PNN interactions within the frontal cortex. Cre+ male mice further exhibit markedly increased alcohol intake in an acute drinking-in-the dark (DID) protocol compared to controls, consistent with findings from previous INIA-Neuroimmune projects using MyD88-/- (knockout) mice and chronic alcohol paradigms 6. Taken together, these data suggest microglia critically regulate PVI number and/or PNN envelopment in response to inflammatory signals via MyD88 signaling, and that increased PVI/PNN interactions in the absence of MyD88 may underlie increased drinking in Cre+ mice. The overall goal of this proposal is to test this hypothesis. Aim 1 will determine if microglial-MyD88 regulates the number of PVIs and/or PNN interactions directly in response to chronic alcohol by assessing the phagocytosis of PVI-specific inhibitory synapses and PNNs by microglia. Aim 2 will determine if microglial-MyD88 regulates the number of PVIs and/or PNN interactions indirectly in response to chronic alcohol by assessing the "secretome" of microglia and the production of PNN components by astrocytes and PVIs. Aim 3 will determine the role of PVIs and/or PNN interactions in excessive drinking behavior in mice with and without microglial MyD88 by assessing the impact of (1) degrading PNNs and (2) chemogenetic inhibition vs. activation of microglia on excessive drinking. This is an innovative new project designed to complement existing strengths of several other INIA-Neuroimmune investigators. If completed, these data would be the first to show a direct role for microglia in PVI/PNN development and their role in excessive drinking, which could lead to novel treatment options.