Mechanisms of nicotinic acetylcholine receptor modulation of cocaine reward

烟碱乙酰胆碱受体调节可卡因奖赏的机制

• 批准号: 10537011
• 负责人: Janna K Moen
• 金额: $ 6.72万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10537011
• 关键词: Abstinence; Acetylcholine; Acute; Affect; Alleles; Attenuated; Award; Back; Behavior; Behavioral; Brain; CRISPR/Cas technology; Cells; Chronic; Cocaine; Cocaine Dependence; Cocaine use disorder; Code; Complex; Corpus striatum structure; Data; Development; Development Plans; Dopamine; Dose; Electrophysiology (science); Feedback; Fiber; Fluorescence; Foundations; Future; Genes; Genetic; Glutamates; Goals; Health; Human; Human Genome; Hyperactivity; Individual; Infusion procedures; Interneurons; Intravenous; Knock-out; Knockout Mice; Laboratories; Light; Measures; Medial; Mediating; Mediator of activation protein; Methodology; Methods; Molecular; Monitor; Mus; Neurons; Nicotinic Receptors; Nucleus Accumbens; Opsin; Optics; Pathway interactions; Patients; Pattern; Phenotype; Photometry; Physiological; Physiology; Play; Positioning Attribute; Prefrontal Cortex; Procedures; Property; Psychological reinforcement; Receptor Signaling; Refractory; Reporter; Research Personnel; Research Proposals; Rewards; Role; Self Administration; Self Stimulation; Shapes; Signal Transduction; Site; Slice; Source; Stimulus; Testing; Training; Transcript; Transgenic Organisms; Variant; Viral; Wild Type Mouse; abuse liability; addiction; base; career development; cell type; cholinergic; cocaine self-administration; experimental study; gamma-Aminobutyric Acid; genetic approach; genome wide association study; in vivo; innovation; interest; neurotransmission; optogenetics; sensor; transmission process

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.

项目摘要 大规模的人类基因组关联研究已经确定了CHRNA5中的等位基因，该基因是编码的基因 对于α5烟碱乙酰胆碱受体（NACHR）亚基，可免受可卡因使用的保护 紊乱。来自我们组的初步数据表明，α5敲除小鼠对野生型小鼠的敏感性不太敏感 可卡因的奖励作用是使用静脉自我给药和颅内自我给出的 刺激程序。伏隔核（NAC）形状奖励相关的胆碱能中间神经元（CINS） 可卡因对CIN活性的行为和抑制作用被认为对其奖励性质至关重要。 为支持此应用的初步电生理学数据表明，抑制性GABA能 在NAC中的CIN上的传输缺乏α5基因敲除小鼠，这导致赞助增加 CIN的活性。此外，α5基因敲除小鼠中的CIN对可卡因的抑制作用难治性。尽管 CHRNA5转录本未在CINS或NAC中的任何其他细胞类型中表达，ChRNA5转录本在 中位前额叶皮层（MPFC）和其他少数其他大脑位点，这些位点为远程输入提供 NAC。根据这些令人兴奋的发现，我假设NAC中的Cin衍生的胆碱能传播 刺激位于MPFC的远程输入端子上的含α5的nACHRS（α5*） 增强GABA能传播回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励中起允许的作用。我建议严格测试这个 假设使用两个特定目标。在AIM 1中，我将确定α5* NACHR如何促进胆碱能 可卡因奖励的机制。体内纤维光度法具有乙酰胆碱的基于荧光的记者 急性可卡因后，信号传导将用于监测NAC中NAC中的胆碱能传播，以确认 可卡因引起的胆碱能信号传导的反馈抑制在α5基因敲除小鼠中缺乏。然后我将使用 化学发生方法来确定抑制CIN如何影响可卡因在野生型和 α5敲除小鼠。 AIM 2将直接测试对可卡因奖励的NAC表达输入的α5NACHR的作用 和生理学。首先，将切片电生理与靶向的光遗传操作相结合 评估MPFC的α5NACHR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方法清除表达NACHR的MPFC→NAC中的CHRNA5 输入，然后是静脉自我给药范式，以评估CHRNA5的后果 小鼠可卡因奖励的淘汰。这项创新的研究建议将产生重要的新数据 可卡因奖励的胆碱能机制，与脆弱性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 方法并定位我向K99/R00途径提交高度竞争的申请 独立奖。