Electrophysiology of alcohol in extended amygdala

描述（由申请人提供）：该项目基于行为发现，即中央杏仁核（CeA）和蓝斑（LC）是参与应激反应和滥用药物强化特性的关键脑区，并且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，阿片类药物和甘丙肽）。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性动物中过量饮酒的乙醇。因此，我们提出了几组实验：1）通过比较来自对照和过度饮酒小鼠（WID模型）的脑切片中的CeA细胞和网络功能，结合CRF和CRF受体的细胞化学定位，关于乙醇增强GABA能IPSC或抑制谷氨酸能EPSP，来评估CRF受体在过度饮酒中的作用。2)为了确定κ阿片受体（KOR）在过量饮酒中的作用，通过检查脑KOR敲除（KO）的WID小鼠脑切片中的CeA细胞功能，与我们的mu和delta受体数据进行比较。3)为了确定甘丙肽及其受体在过量饮酒中的作用，通过检查来自WID小鼠和具有脑Gal 1和Gal 2受体的科斯KO和甘丙肽过表达的小鼠的切片中的CeA和LC细胞，以及通过CeA和LC神经元中的神经化学和分子生物学测量。4)在由Crabbe和Finn组选择性饲养的HDID小鼠中，根据特定目标1-3的结果，确定对最大WID诱导变化的影响，以在黑暗中大量饮酒与其对照相比，以及SHAG与SLAG品系，选择用于计划的大量和少量饮酒。电生理学研究将使用CeA和LC脑切片，并涉及标准细胞内和全细胞钳夹方法。我们将使用一系列措施来评估乙醇和肽效应的突触前和突触后作用部位。甘丙肽研究将采用RIA、实时PCR和受体结合研究。这个项目应该提供重要的新信息，在细胞水平上的乙醇中毒的可能尖叫声，并通过比较乙醇和肽的行动在控制，过量饮酒，敲除模型，也将提供线索的突触，细胞和离子通道相关的乙醇依赖。

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.