Mechanism of drug addiction

描述（由申请人提供）：目前，可卡因成瘾没有药物治疗，部分原因是可卡因成瘾的机制尚不清楚。可卡因在大脑中有3个高亲和力靶点，多巴胺转运蛋白（DAT）、血清素转运蛋白（SERT）和去甲肾上腺素转运蛋白（NET）。可卡因结合并阻断这些转运体的摄取功能，导致突触中递质水平延长和升高，这被认为是可卡因刺激和奖励作用的基础。令人惊讶的是，单独破坏DAT、NET或SERT的敲除小鼠仍然表现出可卡因奖励，这表明可卡因奖励不明确需要单一转运蛋白，防止可卡因抑制单一转运蛋白的药物可能无效。然而，基因敲除小鼠的代偿性变化可能改变了奖励途径。为了避免代偿性变化，我们产生了一个携带DAT突变体的敲入小鼠系，该突变体保留了摄取功能，但对可卡因抑制不敏感（DAT- ki小鼠）。在这些小鼠中，正常剂量的可卡因不再阻断DAT，刺激运动或产生奖励。我们的研究结果表明，在具有功能性DAT的小鼠中，可卡因奖励需要DAT阻断。最重要的是，这表明拮抗可卡因对DAT的抑制作用的药物应该能有效地阻断可卡因奖励。DAT-KI小鼠为研究复杂可卡因效应的机制提供了一种独特的新工具。我们建议继续目前的研究。我们将在其他几个行为测试中检查DAT- ki小鼠的可卡因反应，以剖析DAT在可卡因复杂效应中的作用。此外，我们将使用AAV载体将野生型DAT重新引入DAT- ki小鼠的选定脑区，仅在这些选定区域恢复可卡因诱导的DA升高，并研究恢复了哪些可卡因反应。这种独特的方法使我们能够将可卡因在特定大脑区域的作用与特定的可卡因效应联系起来。我们的初步数据表明，我们现在能够在受限的大脑区域注射AAV，注射不同AAV的小鼠组在条件位置偏好测试、运动刺激测试、两种测试或不进行测试中恢复可卡因反应，证明了我们方法的可行性。拟议项目的成功将大大加强我们对可卡因如何产生其复杂影响的理解，这对我们发展有效治疗可卡因成瘾的努力至关重要。公共卫生相关性：在拟议的研究中，我们将使用分子，遗传，生化和行为分析工具来了解药物成瘾的分子机制。拟议项目的成功将大大加强我们对可卡因如何产生其复杂影响的理解，这对我们发展有效治疗可卡因成瘾的努力至关重要。

DESCRIPTION (provided by applicant): Currently, there are no pharmacological treatments for cocaine addiction partly because the mechanism of cocaine addiction is not clear. Cocaine has 3 high affinity targets in the brain, dopamine transporter (DAT), serotonin transporter (SERT), and norepinephrine transporter (NET). Cocaine binds and blocks the uptake functions of these transporters, resulting in prolonged and elevated transmitter levels in the synapses, which are believed to underlie the stimulating and rewarding effects of cocaine. Surprisingly, knockout mice with DAT, NET, or SERT individually disrupted still exhibit cocaine reward, suggesting that no single transporter is explicitly required for cocaine reward and drugs preventing cocaine inhibition of a single transporter may not be effective. However, compensatory changes in the knockout mice may have altered the reward pathway. To avoid the compensatory changes, we have generated a knock-in mouse line carrying a DAT mutant that retains the uptake function but is insensitive to cocaine inhibition (DAT-KI mice). In these mice, normal doses of cocaine no longer block DAT, stimulate locomotion, or produce reward. Our results indicate that DAT blockade is required for cocaine reward in mice with a functional DAT. Most significantly, it suggests drugs that antagonize cocaine inhibition of DAT should be effective in blocking cocaine reward. DAT-KI mice provide a unique novel tool to investigate the mechanism of the complex cocaine effects. We propose to continue our current studies. We will examine cocaine responses by DAT-KI mice in several other behavioral tests to dissect out the contribution of DAT in the complex effects of cocaine. In addition, we will use AAV vector to re-introduce wild type DAT back into selected brain regions of DAT-KI mice to restore cocaine-induced DA elevation in those selected regions only and study what cocaine responses are restored. This unique approach allows us to correlate cocaine actions in specific brain regions to specific cocaine effects. Our preliminary data show that we are now able to inject AAV in confined brain regions and groups of mice with varying AAV injections restored cocaine responses in either conditioned place preference test only, locomotor stimulation only, both tests, or none of the tests, demonstrating the feasibility of our approach. The success of the proposed project will significantly enhance our understanding on how cocaine produces its complex effects, which are crucial for our efforts in the development of effective treatment for cocaine addiction. PUBLIC HEALTH RELEVANCE: In the proposed study, we will use molecular, genetic, biochemical and behavioral analysis tools to understand the molecular mechanisms of drug addiction. The success of the proposed project will significantly enhance our understanding on how cocaine produces its complex effects, which are crucial for our efforts in the development of effective treatment for cocaine addiction.