Excess ethanol drinking after prenatal exposure to ethanol: chemokine signaling and orexigenic neuropeptides

 描述(由申请人提供)：临床研究表明，在生命早期，特别是在怀孕期间，接触乙醇会增加乙醇的消费和偏好，并增加酒精中毒的风险。这一现象涉及的分子和细胞机制尚不清楚。我们最近在大鼠身上进行的研究，集中在下丘脑的促食欲素神经肽、黑色素浓缩激素(MCH)和增食欲素(OX)，它们增加了乙醇的摄入量和相关的情绪行为，证明了胚胎暴露在相对较低的水平和仅在下丘脑神经发生的高峰期，对穹隆周围外侧下丘脑(Pf-Lh)产生这些多肽的神经元的密度和长期表达的刺激作用。有证据表明，像乙醇一样的炎性介质脂多糖会增加外周趋化因子水平、多肽表达和饮酒，我们在最近的研究中调查并证明了这些促食欲素多肽与局部趋化因子系统CCL2及其受体CCR2之间的密切联系。与CCL2或CCR2基因突变显著减少酒精摄入量的证据一致，我们发现，从胚胎第10天(E10)到E15的低剂量产前酒精暴露，增加了循环中的CCL2水平，增加了含有CCR2但不含CCL2的MCH神经元青春期前后代的密度，以及青春期对乙醇的摄取和偏好。这一证据，以及我们广泛的初步发现，为我们的假设提供了基础，该假设将在四个目标中进行验证，即：胚胎和DAM中CCL2/CCR2信号系统的激活有助于产前低水平和短时间的乙醇暴露对Pf Lh内促食氧肽神经元的发育和迁移的刺激作用，导致它们在出生后密度和表达增加，随后促进青春期酒精摄取和相关行为。在目标1中，我们将研究在青春期子代中，产前接触CCL2是否具有类似于乙醇的作用，并介导其对神经细胞发育和多肽诱导的行为的刺激作用。在目标2中，我们将直接比较酒精和CCL2对胚胎的影响，并确定CCL2是否介导了乙醇诱导的共表达CCR2的多肽神经元的增殖、分化和迁移。根据我们的初步发现，下丘脑神经上皮(NEP)中的放射状胶质细胞含有CCL2并对乙醇高度敏感，我们在目标3中提出在胚胎E12至E19中测试乙醇或CCL2对NEP中这些神经前体细胞和肽神经元发育的影响，以及这些CCL2细胞在介导乙醇效应中的作用。在目标4中，我们将通过直接操作胚胎进行测试，包括第三脑室注射 对于CCL2或CCL2 siRNA，神经上皮细胞CCL2是否参与了乙醇对神经元发育和青少年饮酒的影响。这项研究应该从根本上提供新的信息，说明孕期接触乙醇如何通过神经免疫系统控制导致物质滥用的多肽神经元的发育。

 DESCRIPTION (provided by applicant): Clinical studies demonstrate that exposure to ethanol early in life, particularly during gestation, increases the consumption of and preference for ethanol and the risk for alcoholism. The molecular and cellular mechanisms involved in this phenomenon have yet to be characterized. Our recent studies in rats, focusing on the hypothalamic orexigenic neuropeptides, melanin-concentrating hormone (MCH) and orexin (OX), that increase ethanol intake and related emotional behaviors, demonstrate a stimulatory effect of embryonic exposure to ethanol, at relatively low levels and only during peak hypothalamic neurogenesis, on the density and long-term expression of neurons that produce these peptides in the perifornical lateral hypothalamus (PF+LH). Building on evidence that the inflammatory agent lipopolysaccharide, like ethanol, increases peripheral chemokine levels, peptide expression, and drinking of ethanol, we investigated and demonstrated in our most recent study a close link between these orexigenic peptides and local chemokine system, CCL2, and its receptor, CCR2. Consistent with evidence that mutations of CCL2 or CCR2 genes markedly reduce ethanol intake, we found that prenatal ethanol exposure, at low doses from embryonic day 10 (E10) to E15, increases circulating CCL2 levels, density in pre-pubertal offspring of MCH neurons that contain CCR2 but not CCL2, and intake of and preference for ethanol during adolescence. This evidence, along with our extensive preliminary findings, provides the foundation for our hypothesis, to be tested in 4 aims, that: Activation of the CCL2/CCR2 signaling system in the embryo as well as dam contributes to the stimulatory effects of prenatal ethanol exposure, at low levels and for a brief period, on development and migration of orexigenic peptide neurons in the PF+LH, resulting in their increased density and expression after birth that subsequently promote ethanol intake and related behaviors during adolescence. In Aim 1, we will examine in adolescent offspring whether prenatal exposure to CCL2 acts similarly to ethanol and mediates its stimulatory effects on neuronal development and behaviors induced by the peptides. In Aim 2, we will directly compare in the embryo effects of prenatal exposure to ethanol and CCL2 and determine whether CCL2 mediates ethanol-induced proliferation, differentiation and migration of peptide neurons that co-express CCR2. Building on our preliminary finding that radial glia in the hypothalamic neuroepithelium (NEP) contain CCL2 and are highly responsive to ethanol, we propose in Aim 3 to test in the embryo, from E12 to E19, the effects of ethanol or CCL2 on development of these neuroprogenitor cells and peptide neurons in NEP and the role of these CCL2 cells in mediating ethanol's effects. In Aim 4, we will test, with direct manipulations of the embryo involving third ventricular injections of CCL2 or CCL2 siRNA, whether neuroepithelial CCL2 is involved in ethanol's effects on neuronal development and adolescent drinking. This research should provide fundamentally new information on how gestational exposure to ethanol acts through the neuroimmune system to control the development of peptide neurons that contribute to substance abuse.