Neuroimmune mechanisms of alcohol reward

项目摘要/摘要 酒精领域盛行的教条是，乙醇(Etoh)的有益特性源于 大鼠腹侧被盖区(VTA)多巴胺(DA)神经活动增强及其伴随的DA释放 中级边缘奖励系统。在初步研究中，我们将证明Etoh对 中脑神经元和NAc DA释放受包括DA D2-亚型2在内的外周底物的介导 表达单核细胞来源的巨噬细胞的受体(D2R)。这些发现表明神经免疫 急性使用乙醇的相互作用和挑战乙醇唯一对DA有中枢影响的教条 神经元的活动、释放和奖励。我们建议的研究构成了对以下方面的重点调查 乙醇中的神经免疫相互作用对VTA神经元、DA传递以及乙醇奖励和消耗的影响。 核心论题是急性乙醇增强中脑边缘DA传递和乙醇奖赏 通过乙醇增强血DA，随后激活表达D2R的MDM，以及 VTA神经元随后的细胞因子调节，这是VTA慢性适应的原因 GABA神经元和DA的释放。支持我们假设的先前和初步证据包括：1) 外周DA能增加DA神经元的活性和NAc DA的释放，减少运动活动，并 通过外周D2Rs促进奖励；2)乙醇增强血液DA，抑制VTA GABA神经元，增强 脑DA，并通过外周D2Rs减少中毒；3)乙醇诱导小胶质细胞激活，增强D2 单核细胞、神经元和小胶质细胞上受体的表达；4)MDM的耗竭降低了Etoh对VTA的影响 GABA神经元和DA释放；5)选择细胞因子增强VTA神经元的兴奋性和DA释放；6)最后， 我们展示了DA和ATP从DA终末共同释放的初步证据，以及乙醇的运动效应。 小胶质细胞，这表明在体内潜在的NAC乙醇免疫相互作用的进一步研究。 这些数据将为乙醇奖赏和神经生物学提供新的基础知识。 依赖和外周底物的作用可能有助于改善药物开发工作。要测试 假设，我们提出了两个具体目标：1)明确外周神经免疫相互作用在无痛性脑出血中的作用 对VTA、GABA神经元和NAc DA释放及相关行为的影响；2)描述乙醇对NAc的影响 DA终末和小胶质细胞，DA和ATP的共同释放。我们将使用野生型和转基因小鼠模型 (GAD67-GFP敲入；VGAT-ChR2、VGAT-CRE/GAD67-GFP；和黑手党小鼠)和MDM耗竭研究 多巴胺释放的神经化学和电化学记录。将使用细胞学技术来确定 参与中边缘改变的细胞因子。研究小胶质细胞的多光子显微镜方法 用快速扫描循环伏安法测定DA和ATP的趋化作用。多光子显微镜 将在体内通过内窥镜中继梯度折射率透镜研究GFP标记的卫星小胶质细胞 使用红移光学传感器检测DA释放的同时监测多巴胺释放，该传感器 将在接受慢性间歇性无水乙醇(CIE)诱导的小鼠身上进行，从而描述神经免疫 活动，从第一次接触乙醇，到依赖。科学严谨性很高，考虑到使用 常规的行为学、药理学、电化学、显微镜和分子工具。

PROJECT SUMMARY/ABSTRACT The prevailing dogma in the alcohol field is that the rewarding properties of ethanol (EtOH) result from enhancement of ventral tegmental area (VTA) dopamine (DA) neural activity and accompanying DA release in the mesolimbic reward system. In preliminary studies, we will demonstrate that some of EtOH’s effects on midbrain neurons and NAc DA release are mediated by peripheral substrates including DA D2-subtype 2 receptor (D2R) expressing monocyte-derived macrophages (MDMs). These findings suggest a neuroimmune interaction for acute EtOH use and challenge the dogma that EtOH has exclusively central effects on DA neuronal activity, release, and reward. Our proposed studies constitute a focused investigation into the role of neuroimmune interactions in EtOH effects on VTA neurons, DA transmission, and EtOH reward and consumption. The core thesis is that acute EtOH enhancement of mesolimbic DA transmission and EtOH reward is mediated by EtOH enhancement of blood DA, subsequent activation of D2R-expressing MDMs, and subsequent cytokine modulation of VTA neurons, that are responsible for chronic adaptations in VTA GABA neurons and DA release. Prior and preliminary evidence supporting our hypothesis include: 1) Peripheral DA increases the activity of DA neurons and NAc DA release, reduces locomotor activity, and promotes reward via peripheral D2Rs; 2) EtOH enhances blood DA, inhibits VTA GABA neurons, enhances brain DA, and reduces intoxication via peripheral D2Rs; 3) EtOH induces microglia activation and enhances D2 receptor expression on monocytes, neurons, and microglia; 4) Depletion of MDMs reduces EtOH effects on VTA GABA neurons and DA release; 5) Select cytokines enhance VTA neuron excitability and DA release; 6) Last, we show preliminary evidence of DA and ATP co-release from DA terminals, and motility effects of EtOH on microglia, which indicate further study for potential NAc EtOH immune interactions in vivo. These data will provide new, fundamental knowledge on the neurobiology of EtOH reward and dependence and the role of peripheral substrates that may help improve drug development efforts. To test the hypotheses, we propose two Specific Aims: 1) Define the role of peripheral neuroimmune interactions in EtOH effects on VTA GABA neurons and NAc DA release, and related behaviors; 2) Describe effects of EtOH on NAc DA terminals and microglia, co-release of DA and ATP. We will use wild-type and transgenic mouse models (GAD67-GFP knock-in; VGAT-Chr2, VGAT-Cre/GAD67-GFP; and MaFIA mice) and MDM depletion to study neurochemical and electrochemical recordings of DA release. Cytometry techniques will be used to determine cytokine factors involved in mesolimbic alterations. Multiphoton microscopy approaches to study microglia chemotaxis in the context of DA and ATP as measured by fast scan cyclic voltammetry. Multiphoton microscopy will be used in vivo through endoscopic relay gradient index lenses to study GFP labeled satellite microglia surveillance while measured dopamine release using a red shifted optical sensor for detecting DA release, which will be performed on mice undergoing chronic intermittent EtOH (CIE) induction, thus describing neuroimmune activity from first exposure to EtOH, through to dependence. Scientific rigor is high considering the use of conventional behavioral, pharmacological, electrochemical, microscopy and molecular tools.