Ethanol-Cannabinoid Interaction in the Regulation of Neurogenesis

慢性乙醇治疗可抑制成年大鼠脑、海马和前脑神经发生，并减少脑CB1大麻受体(CB1R)的表达。大脑中有两种不同类型的大麻素受体，CB1R和CB2大麻受体(CB2R)。在大脑中，CB1R主要是神经元，而CB2大麻素受体(CB2R)主要存在于神经胶质细胞，特别是小胶质细胞，这两种受体都参与了神经前体干细胞(Npc)的调节。 增殖、分化和神经发生。慢性酒精还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，CB2R增加，促炎细胞因子表达增加，神经再生丧失。这些研究和其他研究支持CB1R和CB2R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，我们制定了以下三个具体目标： 目的1.明确CB1受体在乙醇抑制神经发生中的作用。本研究旨在探讨CB1R在乙醇抑制神经发生中的作用(S)。通过比较CB1R和CB1R激动剂和拮抗剂以及CB1R基因敲除(KO)转基因小鼠(CB1[-/-])在乙醇抑制神经发生中的时间历程和剂量反应变化。共聚焦显微镜可以确定鼻咽癌的神经发生和鼻咽癌受体的表达。 目的2.探讨CB2R在乙醇和内毒素抑制神经发生中的作用。这一目标将检验乙醇诱导小胶质细胞激活、CB2受体和促炎细胞因子的表达减少神经发生的假设。初步研究和其他研究表明，内毒素诱导促炎细胞因子增加CB2R并抑制神经发生。乙醇可增强内毒素对致炎细胞因子的诱导，但对CB2R和神经发生的影响尚不清楚。神经发生、小胶质细胞和CB2R表达变化的时间进程和剂量反应比较 在神经遗传区确定的。单独使用乙醇、单独使用内毒素以及联合使用乙醇-内毒素可提供逐渐增强的促炎反应，以便与神经再生进行比较。CB2R激动剂和拮抗剂以及CB2R-K0转基因小鼠(CB2R[-/-])将被用来表征CB2R受体在乙醇和脂多糖抑制神经发生中的作用(S)。 瞄准。3.明确内源性大麻素在乙醇抑制神经发生中的作用。这一目的将确定内源性大麻素激活通过阻断内源性大麻素以及已知可阻断内毒素和/或乙醇效应的抗炎药物和抗氧化剂的降解，在乙醇调节神经发生中的作用。研究表明，乙醇增加了作用于和下调CBR1的内源性CB，但CBR1的激活增加了神经发生。乙醇、内源性CB和CBR1如何影响乙醇对神经发生的抑制将被确定。预计鼻咽癌对CBR1的下调不依赖于内源性CB刺激，而与促炎基因诱导的改变和神经发生的丧失有关。 总之，这些研究将为调节乙醇和大麻类药物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发生疗法。

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.