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.

项目总结 大规模的人类全基因组关联研究已经确定了CHRNA5的等位基因，该基因编码 对于α5烟碱型乙酰胆碱受体(NAChR)亚单位，它可以防止吸食可卡因 无序。我们小组的初步数据显示，α5基因敲除小鼠比野生型小鼠对 用静脉给药和颅内给药测定可卡因的奖赏效应 刺激程序。伏核胆碱能中间神经元(CINs)的形状奖赏相关 行为，以及可卡因对CIN活性的抑制作用被认为是其奖励特性的关键。 为支持这一应用而收集的初步电生理学数据显示，抑制性GABA能 在α5基因敲除小鼠中，NAC向CIN的传递存在缺陷，这导致自发放电增加 CIN的活跃度。此外，α5基因敲除小鼠的CIN对可卡因的抑制作用是无效的。而当 CHRNA5转录本在NAC的CIN或任何其他细胞类型中不表达，CHRNA5转录本在 内侧前额叶皮质(MPFC)和少数其他为大脑提供远程信息输入的脑区 南汽。基于这些令人兴奋的发现，我假设NAC中CIN来源的胆碱能传递 刺激位于来自mPFC的远程输入端子上的含有α5的(α5*)nAChR，这 增强GABA能传递回CIN以抑制其活性。此外，我建议α5*nAChR- 中介反馈抑制在可卡因奖赏中起允许作用。我提议对此进行严格的测试 假设使用两个特定的目标。在目标1中，我将确定α5*nAChRs如何参与胆碱能 可卡因奖赏机制。乙酰胆碱荧光报告体内纤维光度法 信号将被用来监测急性可卡因后NAC的胆碱能传递，以证实 在α5基因敲除小鼠中，可卡因引起的胆碱能信号的反馈抑制是缺乏的。然后，我将使用 化学遗传学方法确定抑制CINs如何影响野生型和非野生型可卡因自我给药 α5基因敲除小鼠。Aim 2将直接测试表达α5nAChR的输入对可卡因奖赏的作用 和生理学。首先，将使用切片电生理和靶向光遗传操作相结合的方法来 评估来自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方法在表达α5*nAChR的mPFC→NAC中切割chrn5 输入后静脉注射自我给药范例以评估CHRNA5的后果 在小鼠身上敲除可卡因奖励。这一创新的研究提案将产生重要的新数据 可卡因奖赏的胆碱能机制与易感性的遗传机制直接相关 人类体内的CUD。此外，量身定做的职业发展计划将为我提供尖端技术培训 方法论和定位我提交一份竞争激烈的K99/R00途径申请 独立奖。