Regulation of striatal microcircuits by endogenous opioids as a novel mechanism underlying cocaine seeking

项目摘要。最近的流行病学报告表明，可卡因的使用从2005年增加了45%， 2013年到2018年。可卡因引起的纹状体多巴胺水平的增加与其奖励作用有关。然而，在这方面， 驱动可卡因滥用的纹状体神经回路尚未明确界定。因此，迫切需要消除- 可卡因诱导的多巴胺释放的纹状体回路下游如何控制可卡因寻求和 拿。本提案的总体目标是确定可卡因影响GABA的机制 纹状体的两个主要神经元D1-MSNs和D2-MSNs之间的传递，最终 规范吸毒行为。我们的中心假设是可卡因可以提高阿片肽的水平 脑啡肽，其通过在D1-和D2-MSN的轴突侧支中表达的μ-阿片受体（莫尔）起作用， 方便寻找和服用可卡因。我们将在两个具体目标中对此进行测试： 目的1：确定可卡因如何影响纹状体内GABA反式神经递质可塑性的机制。 使命。根据我们的初步数据，我们假设可卡因史增加了脑啡肽再- 从D2-MSNs的租赁，这诱导了纹状体内GABA传递的MOR依赖性长期抑制， 锡永D1-MSN上。我们将通过在MSN选择性莫尔中进行全细胞电生理学来测试这一点， 脑啡肽敲除与可卡因或盐水暴露史，并记录GABA之间的传输 MSN和MSN兴奋性。药理学将用于推断电生理学中脑啡肽水平升高- 这将通过RNAscope，免疫组织化学，Western blot和我们的MSN- 选择性脑啡肽敲除 目的2：确定纹状体内回路的阿片调节如何驱动可卡因寻求和服用。 基于我们的初步数据，我们假设从D2-MSNs释放的脑啡肽作用于MORs， D1-和D2-MSNs的轴突侧支抑制纹状体内GABA并促进可卡因奖赏。我们将 在MSN选择性莫尔和脑啡肽敲除中使用操作性可卡因自我给药程序来测试这一点， 出老鼠。我们将确定来自MSN的纹状体内GABA传输如何有助于可卡因寻求 通过使用新的Gi-偶联视蛋白选择性抑制D1-MSN或D2-MSN纹状体轴突侧支， 可卡因寻找任务 成功完成拟议的研究将阐明内源性阿片类药物如何调节内 纹状体GABA传输，以及可卡因如何影响这种机制，影响电路活动。莫里奥- 总之，这项研究将通过建立一个联系，为可卡因滥用的机制提供新的见解- 阿片介导的纹状体内GABA传递调节与可卡因寻求和服用之间的关系。这 这项研究还将为我们确定驱动电子束的电路机制的长期目标奠定基础。 当鸦片剂和可卡因被共同滥用时的协同奖励。

PROJECT SUMMARY. Recent epidemiological reports indicate that cocaine use increased by 45% from 2013 to 2018. Cocaine-induced increase in striatal dopamine levels is linked to its rewarding effects. However, the striatal neural circuits driving cocaine abuse are not clearly defined. Thus, there is a critical need to delin- eate how the striatal circuits downstream of cocaine-induced dopamine release control cocaine seeking and taking. The overall objective of this proposal is to determine the mechanisms by which cocaine affects GABA transmission between the two principal neurons of the striatum, the D1-MSNs and D2-MSNs, to ultimately regulate drug seeking and taking. Our central hypothesis is that cocaine-enhances levels of the opioid peptide enkephalin, which acts via mu-opioid receptors (MOR) expressed in axon collaterals of D1- and D2-MSNs to facilitate cocaine seeking and taking. We will test this in two specific aims: Aim 1: Determine the mechanisms for how cocaine affects plasticity of intra-striatal GABA trans- mission. Based on our preliminary data, we hypothesize that a history of cocaine increases enkephalin re- lease from D2-MSNs, which induces MOR-dependent long-term depression of intra-striatal GABA transmis- sion onto D1-MSNs. We will test this by performing whole cell electrophysiology in MSN-selective MOR and enkephalin knockouts with a history of cocaine or saline exposure, and record GABA transmission between MSNs and MSN excitability. Pharmacology will be used to infer heightened enkephalin tone in electrophysi- ology recordings, and this will be confirmed by RNAscope, immunohistochemistry, Western blot and our MSN- selective enkephalin knockout. Aim 2: Determine how opioid regulation of intra-striatal circuits drive cocaine seeking and taking. Based on our preliminary data we hypothesize that enkephalin released from D2-MSNs acts on MORs in axon collaterals of D1- and D2-MSNs to suppress intra-striatal GABA and facilitate cocaine reward. We will test this using operant cocaine self-administration procedures in MSN-selective MOR and enkephalin knock- out mice. We will determine how intra-striatal GABA transmission from MSNs contributes to cocaine seeking by selectively inhibiting D1-MSN or D2-MSN striatal axon collaterals using a novel Gi-coupled opsin during a cued cocaine seeking task. Successful completion of the proposed research will elucidate how endogenous opioids regulate intra- striatal GABA transmission, and how cocaine impinges upon this mechanism to affect circuit activity. Moreo- ver, this research will provide novel insights on the mechanisms of cocaine abuse by establishing a link be- tween opioid-mediated regulation of intra-striatal GABA transmission and cocaine seeking and taking. This research will also lay the groundwork for our long-term goal of determining the circuit mechanisms driving synergistic reward when opiates and cocaine are co-abused.