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

摘要 反复的酒精暴露可能是一种强有力的神经炎症刺激。酒精会激活大脑中的小胶质细胞， 通过Toll样受体（TLR）激活大脑;这种激活导致炎症介质的产生， 依赖和滥用4，5.快速尖峰小白蛋白阳性（PV+）GABA能中间神经元（PVIs）是关键的 正常的大脑功能。PVIs通常被称为细胞外基质分子的特殊细胞外基质分子包围。 神经元周围网（PNN）保护和缓冲它们免受炎症和氧化应激。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 神经可塑性机制是习惯性行为的基础，例如面对不良反应时寻求药物。 后果2.我们的初步数据表明，小鼠小胶质细胞特异性消融的关键TLR适配器， 分子髓样分化初级应答88 [MyD88]（Cx3cr1-CreBT-MyD88f/f [以下称为"Cre + 小鼠"]）对脂多糖（LPS）注射表现出降低的促炎反应（例如IL-1 β、TNF β 与Cre-对照相比，正如预期的那样。然而，令人惊讶的是，在对LPS的反应中，男性（而不是女性）Cre + 小鼠在额叶皮层内表现出增加的PVI和PV/PNN相互作用的数量。Cre+雄性小鼠进一步 与对照组相比，在黑暗中急性饮酒（DID）方案中显示出显著增加的酒精摄入， 这与先前使用MyD88-/-（敲除）小鼠和慢性 酒精范例6.总之，这些数据表明小胶质细胞严格调节PVI数量和/或 PNN通过MyD88信号传导对炎症信号作出反应，并增加PVI/PNN 在MyD88不存在下的相互作用可能是Cre+小鼠中饮酒增加的基础。的总目标 本提案旨在验证这一假设。目的1将确定小胶质细胞-MyD88是否调节PVI的数量 和/或PNN的相互作用直接响应慢性酒精通过评估PVI特异性的吞噬作用， 抑制性突触和PNN。目的2将确定小胶质细胞-MyD88是否调节细胞凋亡的数量。 通过评估小胶质细胞的"分泌组"间接响应慢性酒精的PVIs和/或PNN相互作用 以及星形胶质细胞和PVIs产生PNN组分。目标3将确定潜在受益者的作用和/或 PNN在有和没有小胶质细胞MyD88的小鼠过度饮酒行为中的相互作用， （1）降解PNN和（2）化学发生抑制与小胶质细胞激活对过度饮酒的影响。 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 investigators.如果完成，这些数据将首次显示小胶质细胞在PVI/PNN中的直接作用。 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.