Mechanisms of nicotinic acetylcholine receptor modulation of cocaine reward

项目摘要 大规模的人类全基因组关联研究已经确定了CHRNA 5中的等位基因，该基因编码 α5烟碱乙酰胆碱受体（nAChR）亚单位，可防止可卡因的使用 disorder.我们小组的初步数据显示，α5基因敲除小鼠对 可卡因的奖励作用，如使用静脉内自我管理和颅内自我管理测量， 刺激程序。延髓核（NAc）内胆碱能中间神经元（CINs）的形状奖赏相关 行为和可卡因对CIN活性的抑制作用被认为是其奖励性质的关键。 为支持本申请而收集的初步电生理学数据表明，抑制性GABA能 在α5基因敲除的小鼠中，NAc中的CINs的传递是缺乏的，这导致自发放电增加 CINs的活动此外，α5基因敲除小鼠中的CIN对可卡因的抑制作用不敏感。而 Chrna 5转录本在CIN或NAc中的任何其他细胞类型中不表达， 内侧前额叶皮层（mPFC）和少数其他大脑部位提供长距离输入， NAc。基于这些令人兴奋的发现，我推测，在NAc中，CIN衍生的胆碱能传递 刺激位于mPFC长程输入端的含α5（α5*）nAChR， 增强GABA能传递回CIN以抑制其活性。此外，我建议α5* nAChR- 介导的反馈抑制在可卡因奖赏中起允许作用。我建议严格检验一下 假设使用两个特定的目标。在AIM 1中，我将确定α5* nAChRs如何促进胆碱能神经元的功能。 可卡因奖励机制基于乙酰胆碱荧光报告的活体纤维光度法 信号传导将用于监测急性可卡因后NAc中的胆碱能传递，以证实 可卡因诱发的胆碱能信号传导的反馈抑制在α5基因敲除小鼠中是缺乏的。然后我将使用 化学遗传学方法来确定抑制CINs如何影响野生型和 α5基因敲除小鼠。AIM 2将直接测试表达α5 nAChR的NAc输入对可卡因奖赏的作用 和生理学。首先，切片电生理学结合靶向光遗传学操作将用于 评估来自mPFC的α5 nAChR表达输入如何调节CIN的活性以及 可卡因最后，我将使用CRISPR/Cas9方法切割表达α5* nAChR的mPFC→NAc中的CHRNA 5。 输入，然后进行静脉内自我给药模式，以评估CHRNA的后果5 在小鼠中对可卡因奖励的敲除。这项创新的研究提案将产生重要的新数据， 可卡因奖励的胆碱能机制与易受伤害的遗传机制直接相关， 人类的CUD此外，量身定制的职业发展计划将为我提供尖端的培训， 方法和立场，我提交了一个K99/R 00 Pathway极具竞争力的申请， 独立奖。

PROJECT SUMMARY Large-scale human genome-wide association studies have identified alleles in CHRNA5, the gene that encodes for the α5 nicotinic acetylcholine receptor (nAChR) subunit, that are protective against developing cocaine use disorder. Preliminary data from our group show that α5 knockout mice are less sensitive than wild-type mice to the rewarding effects of cocaine as measured using intravenous self-administration and intracranial self- stimulation procedures. Cholinergic interneurons (CINs) in the nucleus accumbens (NAc) shape reward-related behaviors, and inhibitory effects of cocaine on CIN activity are considered critical to its rewarding properties. Preliminary electrophysiology data collected in support of this application show that inhibitory GABAergic transmission onto CINs in the NAc is deficient in α5 knockout mice, which results in increased spontaneous firing activity of CINs. Further, CINs in α5 knockout mice are refractory to the inhibitory actions of cocaine. While Chrna5 transcript is not expressed in CINs or any other cell type in the NAc, Chrna5 transcripts are detected in the medial prefrontal cortex (mPFC) and a small number of other brain sites that provide long-range input to the NAc. Based on these exciting findings, I hypothesize that CIN-derived cholinergic transmission in the NAc stimulates α5-containing (α5*) nAChRs located on the terminals of long-range inputs from the mPFC, which enhances GABAergic transmission back onto CINs to inhibit their activity. Further, I propose that α5* nAChR- mediated feedback inhibition plays a permissive role in cocaine reward. I propose to rigorously test this hypothesis using two specific aims. In AIM 1, I will determine how α5* nAChRs contribute to cholinergic mechanisms of cocaine reward. In vivo fiber photometry with a fluorescence-based reporter of acetylcholine signaling will be used to monitor cholinergic transmission in the NAc following acute cocaine to confirm that feedback inhibition of cholinergic signaling evoked by cocaine is deficient in α5 knockout mice. I will then use a chemogenetic approach to determine how inhibiting CINs influences cocaine self-administration in wild-type and α5 knockout mice. AIM 2 will directly test the role of α5 nAChR- expressing inputs to the NAc on cocaine reward and physiology. First, slice electrophysiology combined with targeted optogenetic manipulation will be used to assess how α5 nAChR-expressing inputs from mPFC regulate the activity of CINs as well as the actions of cocaine. Finally, I will use a CRISPR/Cas9 approach to cleave CHRNA5 in α5* nAChR-expressing mPFC→NAc inputs followed by an intravenous self-administration paradigm to assess the consequences of CHRNA5 knockout on cocaine reward in mice. This innovative research proposal will generate important new data relevant to cholinergic mechanisms of cocaine reward with direct relevance to genetic mechanisms of vulnerability to CUD in humans. Further, the tailored career development plan will provide me with training in cutting-edge methodologies and position me to submit a highly competitive application for a K99/R00 Pathway to Independence Award.