Alcohol/naltrexone, neuropeptide interaction with opioid receptor dynamics studie

描述(申请人提供)：尽管有相当多的科学和工业努力，仍然没有有效治疗酒精中毒的药物可用。这在一定程度上是因为酒精与大多数精神活性药物不同，它不是通过同源受体系统产生作用的。相反，酒精直接或间接地调节包括中枢阿片信号系统在内的各种信号系统。酒精-阿片类药物的相互作用是复杂的，反映在系统、突触、细胞和分子水平。然而，调节酒精-阿片相互作用的机制尚未完全解决。分子成像的最新进展为在阿片受体水平上研究酒精-阿片相互作用提供了新的方法，特别是影响受体动力学的相互作用，受体动力学在决定受体的可用性和功能方面发挥着核心作用。为此，我们目前正在开发与激光共聚焦扫描显微镜(CLSM)相结合的荧光相关光谱(FCS)，以便以终极的单分子灵敏度实时无损地观察活细胞中的分子相互作用。我们建议利用这一系统和并行功能分析来开发研究酒精(即乙醇)对阿片受体细胞动力学的影响以及这些相互作用的功能后果的方法。在前期研究的基础上，我们提出乙醇诱导的阿片受体动力学改变在中枢神经系统酒精-阿片相互作用中起重要作用，并假设这种相互作用导致神经生理功能的改变。我们还假设，已知对酒精中毒有治疗潜力的阿片受体拮抗剂，如纳曲酮，一个重要的作用是阻断乙醇对阿片受体动力学的作用。为了验证这些假设，我们提出了三个具体目标：(1)建立一个细胞模型，利用FCS/CLSM在阿片受体动力学水平上研究酒精与阿片信号系统之间的相互作用。这些研究将使用转标记或未标记阿片受体的PC12细胞。(2)通过神经元特性的电生理记录，确定酒精引起的阿片受体动力学变化对神经生理学的影响，以及(3)将酒精对阿片受体动力学调节的研究扩展到自然神经元和神经元回路，其中可以在更能反映体内条件的细胞和神经元回路中进行乙醇/阿片相互作用的研究。这些研究将使用从阿片受体基因敲除小鼠获得的小鼠海马体的原代培养。培养的细胞将被导入标记或未标记的阿片受体。拟议的研究代表了一种新的和最先进的方法来理解乙醇-阿片类药物相互作用的机制，包括可能对酒精依赖的发展至关重要的机制。项目叙事酒精是世界上滥用最广泛的精神活性物质之一。几条证据表明，大脑阿片信号系统介导了许多与酒精滥用有关的重要行为影响。拟议的研究将调查酒精在阿片受体水平上的作用，以了解酒精-阿片相互作用的调节机制。

DESCRIPTION (provided by applicant): Despite considerable scientific and industrial efforts, there are no drugs available for an efficient treatment of alcoholism. This is partially explained by the fact, that alcohol, unlike most psychoactive drugs, does not produce its actions via a cognate receptor system. Instead, alcohol modulates directly or indirectly a variety of signaling systems including the CNS opioid signaling system. Alcohol-opioid interactions are complex and reflected at the systems, synaptic, cellular and molecular levels. However, the mechanisms mediating alcohol-opioid interactions have yet to be fully resolved. Recent advances in molecular imaging now offer new approaches to study alcohol-opioid interactions at the level of opioid receptor, and especially interactions that affect receptor dynamics, which plays a central role in determining receptor availability and function. To this end, we are currently involved in the development of Fluorescence Correlation Spectroscopy (FCS) integrated with Confocal Laser Scanning Microscopy (CLSM) for nondestructive observation of molecular interactions in living cells in real time with ultimate single-molecule sensitivity. We propose to utilize this system and parallel functional analysis to develop approaches for the investigation of alcohol (i.e., ethanol) effects on cellular dynamics of opioid receptors and the functional consequence of these interactions. Based on our preliminary studies we propose that ethanol-induced changes in opioid receptor dynamics play an important role in alcohol-opioid interactions in the CNS and hypothesize that such interactions lead to functional changes in neuronal physiology. We also hypothesize that an important action of opioid receptor antagonists known to have therapeutic potential for alcoholism such as naltrexone is to block the action of ethanol on opioid receptor dynamics. To test these hypotheses, we propose 3 Specific Aims: (1) Develop a cellular model to study interactions between alcohol and the opioid signaling system at the level of opioid receptor dynamics using FCS/CLSM. These studies will employ PC12 cells transfected with tagged or untagged opioid receptors. (2) Identify the consequences of alcohol induced changes in opioid receptor dynamics to neurophysiology using electrophysiological recordings of neuronal properties, and (3) extend studies of alcohol regulation of opioid receptor dynamics to native neurons and neuronal circuits where investigations of ethanol/opioid interactions can be pursued in cells and neuronal circuits more reflective of the in vivo conditions. These studies will employ primary cultures of mouse hippocampus obtained from opioid receptor knockout mice. The cultured cells will be transfected with tagged or untagged opioid receptors. The proposed studies represents a novel and state of the art approach to an understanding of mechanisms involved in ethanol-opioid interactions including mechanisms that may be central to the development of alcohol dependence. Project Narrative Alcohol is one of the most widely abused psychoactive substances in the world. Several lines of evidence suggest that the brain opioid signaling system mediates many of the important behavioral effects alcohol associated with alcohol abused. The proposed studies will investigate actions of alcohol that occur at the level of the opioid receptor level to gain an understanding of mechanisms that mediate alcohol-opioid interactions.