Electrophysiology of alcohol in extended amygdala

描述(申请人提供)：该项目基于行为学研究结果，即杏仁中央核(CEA)和蓝斑(LC)是参与应激反应和滥用药物增强特性的关键大脑区域，这些行为可能涉及几种递质(GABA、谷氨酸、去甲肾上腺素)和神经肽(CRF、类阿片和甘丙肽)。这两个区域都与动机行为和焦虑状态有关，我们假设CEA和LC中的这些相同的神经化学系统参与了依赖动物中看到的过量饮酒。因此，我们提出了几组实验：1)通过比较正常小鼠和过度饮酒小鼠(WID模型)脑片CEA的细胞和网络功能，结合CRF和CRF受体的细胞化学定位，评估CRF受体在过度饮酒中的作用。2)为了确定kappa阿片受体(Kors)在过量饮酒中的作用，为了与我们的Mu和Delta受体数据进行比较，通过检测大脑Kors基因敲除(KO)的WID小鼠脑片的CEA细胞功能。3)通过检测WID小鼠和KOS小鼠脑片CEA和LC细胞的GALL和Gal2受体及Galanin过度表达，以及CEA和LC神经元的神经化学和分子生物学方法，确定甘丙素及其受体在过量饮酒中的作用。4)根据特定目标1-3的结果，确定在由Crabbe和Finn组选择性饲养的HDID小鼠中，与其对照组相比，在黑暗中高饮酒的HDID小鼠，以及为计划的高和低酒精摄入量而选择的SHIG和SLAG系小鼠中，WID1-3诱导的最大变化的影响。电生理学研究将使用CEA和LC脑片，并涉及标准的细胞内和全细胞钳制方法。我们将使用一系列方法来评估乙醇和多肽效应在突触前和突触后的作用部位。放射免疫分析、实时聚合酶链式反应和受体结合研究将用于甘丙素研究。该项目将在细胞水平上为乙醇中毒可能的尖叫提供重要的新信息，并通过比较乙醇和肽在控制、过度饮酒和基因敲除模型中的作用，也将为酒精依赖的突触、细胞和离子通道相关提供线索。

DESCRIPTION (provided by applicant): This project is based on behavioral findings that the central amygdala nucleus (CeA) and locus coeruleus (LC) are key brain areas involved in stress reactions and the reinforcing properties of abused drugs, and that these behaviors may involve several transmitters (GABA, glutamate, norepinephrine) and neuropeptides (CRF, opioids and galanin). Both regions are implicated in motivated behaviors and anxiety states, and we hypothesize that these same neurochemical systems within the CeA and LC are involved in the excessive ethanol drinking seen in dependent animals. Therefore, we propose several sets of experiments: 1) T assess the role of CRF receptors in excessive drinking, by comparing the CeA cellular and network function in brain slices from control and excessively drinking mice (WID model) mice, with respect to the ethanol augmentation of GABAergic IPSCs or inhibition of glutamatergic EPSPs, combined with cytochemical localization of CRF and CRF receptors. 2) To determine the role of kappa opiate receptors (KORs) in excessive drinking, for comparison to our mu and delta receptor data, by examining CeA cellular function in brain slices from WID mice with a knockout (KO) for brain KORs. 3) To determine the role of galanin and its receptors in excessive drinking, by examining CeA and LC cellular in slices from WID mice and those with KOs for brain Gall and Gal2 receptors and with galanin over-expression, and by neurochemical and molecular biological measures in CeA and LC neurons. 4) To determine the effects on the largest WIDinduced changes from the results of Specific Aims 1-3, in the HDID mice selectively bred by the Crabbe and Finn groups for high drinking in the dark versus their controls, and for SHAG vs.SLAG lines, selected for scheduled high and low alcohol consumption. The electrophysiological studies will use CeA and LC brain slices and involve standard intracellular and whole-cell clamp methods. We will use a battery of measures to assess the pre- versus postsynaptic sites of action of ethanol and peptide effects. RIA, real-time PCR and receptor binding studies will be used in the galanin studies. This project should provide important new information on the possible squeal of ethanol intoxication at the cellular level, and, by comparisons of ethanol and peptide actions in control, excessively drinking, and knockout models, will also provide clues as to the synaptic, cellular and ion channel correlates of ethanol dependence.