Brain endorphin targets of low dose alcohol

摘要 几十年来，酒精在大脑中作用的确切分子靶点一直难以捉摸，主要是作为一种 乙醇与特定蛋白质或其他细胞相互作用的暂时性和低亲和力的结果 组件。内源性阿片系统在奖赏、强化、激励、 以及包括乙醇在内的滥用药物的神经化学影响。我们之前已经证明了急性 乙醇可显著增加大鼠伏隔核细胞外内啡肽水平 (NAC)。我们还产生了新的初步数据，表明-内啡肽的免疫中和作用 NAC壳区，而不是核心，减弱了低剂量乙醇的自我给药。这些发现 提示NAC壳内的内啡肽能传递参与了低剂量的兴奋效应 乙醇。最后，我们利用转基因小鼠在 控制-内啡肽前体多肽原阿片黑素皮质素的启动子，以评估 低剂量乙醇对这些神经元的潜在激活作用。我们观察到，在这些POMC-EGFP小鼠中，Low 黑暗饮酒范例中的酒精摄入量导致大脑和血液中的酒精水平 MM，并激活下丘脑弓状核(ArcN)POMC表达神经元。总而言之， 这些数据表明，脑POMC/内啡肽系统是低剂量乙醇的分子靶点。然而， 还需要进一步的研究来检验我们的总体假设，即ArcN POMC表达的神经元是一种 低剂量乙醇在大脑中的靶标。为了实现这一目标，我们制定了以下相互关联的 独立的具体目标。在目标1中，我们将进一步表征低剂量乙醇激活ArcN的能力 表达POMC的神经元。POMC-EGFP小鼠将在黑暗中饮酒范式中进行测试，以确定 乙醇诱导的ArcN POMC表达激活的剂量依赖性、特异性和脱敏 神经元。在目标2中，我们将确定Low激活的ArcN POMC表达神经元的伏隔靶点 剂量乙醇。这将通过在POMC中使用依赖Cre的病毒逆行追踪方法来实现- Cre小鼠。最后，在目标3中，我们将确定ArcN POMC表达神经元的分子适应 发生在低剂量乙醇消费之后。这将通过使用POMC-CRE Ribotag鼠标来实现 低剂量诱导生物相关基因RNA表达的细胞类型特异性变化 乙醇消耗量。为了增加这些研究的翻译影响，所有拟议的实验都将利用 男性和女性受试者，以及允许自愿消费的酒精摄入程序，而不是 实验者被动地给药。总之，这些研究将最终指导 药理学或其他方法使酒精在大脑中的作用失效，这最终可能 减少酒精滥用给社会带来的医疗、社会经济和法律成本。

ABSTRACT The precise molecular targets of ethanol action in the brain have remained elusive for decades, primarily as a result of the transient and low-affinity nature of ethanol interactions with specific proteins or other cellular components. The endogenous opioid system has been implicated in the rewarding, reinforcing, motivational, and neurochemical effects of drugs of abuse, including ethanol. We have previously demonstrated that acute ethanol administration in rats significantly increases extracellular endorphin levels in the nucleus accumbens (NAc). We have also generated novel preliminary data indicating that immunoneutralization of -endorphin in the NAc shell subregion, but not the core, attenuates low dose ethanol self-administration. These findings suggest that endorphinergic transmission in the NAc shell contributes to the motivational effects of low dose ethanol. Finally, we utilized transgenic mice that express enhanced green fluorescent protein (EGFP) under the control of the promoter for the -endorphin precursor peptide pro-opiomelanocortin (POMC), to assess the potential activation of these neurons by low dose ethanol. We observed that in these POMC-EGFP mice, low dose ethanol consumption in the drinking-in-the-dark paradigm resulted in brain and blood ethanol levels <10 mM, and activated POMC-expressing neurons in the arcuate nucleus of the hypothalamus (ArcN). Collectively, these data indicate that brain POMC/endorphin systems are molecular targets of low dose ethanol. However, additional studies are needed to test our overarching hypothesis that ArcN POMC-expressing neurons are a target of low dose ethanol in the brain. To accomplish this, we have formulated the following inter-related yet independent Specific Aims. In Aim 1, we will further characterize the ability of low dose ethanol to activate ArcN POMC-expressing neurons. POMC-EGFP mice will be tested in the drinking-in-dark paradigm to determine the dose-dependency, specificity, and desensitization of ethanol-induced activation of ArcN POMC-expressing neurons. In Aim 2, we will determine the accumbal targets of ArcN POMC-expressing neurons activated by low dose ethanol. This will be accomplished by the use of Cre-dependent viral retrograde tracing methods in POMC- Cre mice. Finally, in Aim 3, we will determine the molecular adaptations in ArcN POMC-expressing neurons that occur following low dose ethanol consumption. This will be accomplished by utilizing POMC-Cre Ribotag mice to determine cell-type specific changes in RNA expression of biologically related genes induced by low dose ethanol consumption. To increase the translational impact of these studies, all proposed experiments will utilize both male and female subjects, and ethanol intake procedures that allow voluntary consumption as opposed to passive administration by an experimenter. Together, these studies will ultimately guide the development of pharmacological or other approaches for inactivating the effects of ethanol in the brain, which may ultimately reduce the medical, socioeconomic and legal costs of ethanol abuse to society.