Ethanol-Cannabinoid Interaction in the Regulation of Neurogenesis

慢性乙醇治疗已被证明抑制成年大脑海马和前脑神经发生，并减少大脑CB 1大麻素受体（CB 1 R）的表达。脑中存在两种不同类型的大麻素受体，CB 1 R和CB 2大麻素受体（CB 2 R）。在脑中，CB 1受体主要是神经元受体，而CB 2受体主要存在于神经胶质细胞，特别是小胶质细胞上，并且这两种受体都涉及调节神经祖干细胞（NPC）。 增殖、分化和神经发生。慢性乙醇也会增加脑小胶质细胞标记物和促炎细胞因子的表达。有趣的是，内毒素（LPS）导致脑小胶质细胞活化、CB 2 R增加、促炎细胞因子表达增加和神经发生丧失。这些研究和其他研究支持CB 1 R和CB 2 R有助于乙醇介导的神经发生抑制的总体假设。为了验证这一假设，我们制定了以下3个具体目标： 目标1。目的明确CB 1受体在乙醇抑制神经发生中的作用。该目的将使用神经发生和CB 1 R变化的乙醇时程和剂量反应比较以及CB 1 R激动剂和拮抗剂以及CB 1 R敲除（KO）转基因小鼠（CB 1 [-/-]）给药来表征CB 1 R在乙醇抑制神经发生中的作用。共聚焦显微镜将允许确定NPC神经发生和NPC上的受体表达。 目标二。目的探讨CB 2 R在乙醇和内毒素（LPS）抑制神经发生中的作用。这一目标将测试的假设，乙醇诱导的小胶质细胞活化，表达CB 2受体和促炎细胞因子减少神经发生。初步研究和其他研究表明，LPS诱导促炎细胞因子会增加CB 2 R并抑制神经发生。乙醇增强LPS诱导促炎细胞因子，但对CB 2 R和神经发生的影响尚不清楚。将比较神经发生、小胶质细胞和CB 2 R表达变化的时间过程和剂量反应。 在神经发生区域决定。单独的乙醇、单独的LPS和组合的乙醇-LPS提供与神经发生相比逐渐增加的促炎反应。将采用CB 2 R激动剂和拮抗剂以及CB 2 R-K 0转基因小鼠（CB 2 [-/-]）来表征CB 2 R受体在乙醇？LPS抑制神经发生中的作用。 瞄准3.确定内源性大麻素在乙醇抑制神经发生中的作用。这一目标将确定内源性大麻素激活在乙醇调节神经发生中的作用，通过阻断内源性大麻素的降解以及已知阻断内毒素和/或乙醇作用的抗炎药和抗氧化剂。研究表明，乙醇增加了作用于CBR 1并下调CBR 1的内源性CB，但CBR 1激活增加了神经发生。将确定乙醇、内源性CB和CBR 1如何影响乙醇对神经发生的抑制。预计NPC下调CBR 1将独立于内源性CB刺激，并与促炎基因诱导的变化和神经发生的丧失有关。 这些研究将为调节乙醇和大麻素对神经发生的影响的信号通路提供详细的见解，并发现潜在的体内神经源性疗法。

Chronic ethanol treatment has been shown to inhibit adult brain hippocampal and forebrain neurogenesis and to decrease brain CB1 cannabinoid receptor (CB1R) expression. There are two different types of cannabinoid receptors in brain, CB1R and CB2 cannabinoid receptor (CB2R). In brain CB1R are primarily neuronal, whereas CB2 cannabinoid receptor (CB2R) are primarily present on glia, particularly microglia and both receptors have been implicated in the regulation neuroprogenitor stem cell (NPC) proliferation, differentiation and neurogenesis. Chronic ethanol also increases brain microglial markers and proinflammatory cytokine expression. Interestingly, endotoxin (LPS), causes brain microglial activation, increased CB2R, increased proinflammatory cytokine expression and loss of neurogenesis. These studies and others support the overall hypothesis that CB1R and CB2R contribute to ethanol-mediated inhibition of neurogenesis. To test this hypothesis we have developed the following 3 specific aims: Aim 1. To define the role of CB1 receptors In ethanol inhibition of neurogenesis. This aim will characterize the role(s) of CB1R in ethanol inhibition of neurogenesis using ethanol time course and dose response comparisons of changes in neurogenesis and CB1R as well as administration of CB1R agonists and antagonists as well as CB1R knockout (KO) transgenic mice (CB1[-/-]). Confocal microscopy will allow determination of NPC neurogenesis and receptor expression on NPC. Aim 2. To determine the role of CB2R in ethanol and endotoxin (LPS) inhibition of neurogenesis. This aim will test the hypothesis that ethanol induced microglial activation, expression of CB2 receptors and proinflammatory cytokines reduce neurogenesis. Preliminary studies and others indicate that LPS induction of proinflammatory cytokines increases CB2R and inhibits neurogenesis. Ethanol potentiates LPS induction of proinflammatory cytokines, but the effect on CB2R and neurogenesis are not known. Time course and dose response comparisons of changes in neurogenesis, microglia, and CB2R expression will be determined in neurogeneic regions. Ethanol alone, LPS alone and combined ethanol-LPS provide progressively increased proinflammatory responses for comparisons with neurogenesis. CB2R agonists and antagonists as well as CB2R-K0 transgenic mice (CB2[-/-]) will be employed to characterize the role(s) of the CB2R receptors in ethanol ¿ LPS inhibition of neurogenesis. Aim. 3. To define the role of endogenous cannabinoids on ethanol inhibition of neurogenesis. This aim will determine the role of endogenous cannabinoids activation in ethanol regulation of neurogenesis by blocking degradation of endogenous cannabinoid as well as anti-inflammatory drugs and anti-oxidants known to block endotoxin and/or ethanol effects. Studies suggest ethanol increases endogenous CB that act on and down regulate CBR1, yet CBR1 activation increases neurogenesis. How ethanol, endogenous CB and CBR1 impact ethanol inhibition of neurogenesis will be determined. It is expected that NPC downregulation of CBR1 will be independent of endogenous CB stimulation and related to changes in proinflammatory gene induction and loss of neurogenesis. Together these studies will provide a detailed insight into the signaling pathway that regulates the effects of ethanol and cannabinoids on neurogenesis as well as discovering potential in vivo neurogenic therapies.