The Role of Cav1.2 L-type Ca2+ Channels in Cocaine-Induced Reinstatement

Cav1.2 L 型 Ca2 通道在可卡因诱导恢复中的作用

• 批准号: 8657425
• 负责人: Anjali M RAJADHYAKSHA
• 金额: $ 38.03万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8657425
• 关键词: AMPA Receptors; Addictive Behavior; Behavior; Behavioral; C57BL/6 Mouse; Cell Fractionation; Cell surface; Cocaine; Cocaine Dependence; Data; Development; Diltiazem; Dopamine; Electron Microscopy; Extinction (Psychology); Face; Genetic; Illicit Drugs; Immunoblot Analysis; Immunoblotting; Investigation; Knock-out; Knowledge; Label; Laboratories; Learning; Measures; Mediating; Mediator of activation protein; Memory; Modeling; Molecular; Molecular Target; Mus; Mutant Strains Mice; Neurons; Nucleus Accumbens; Pathway interactions; Peptides; Pharmaceutical Preparations; Pharmacological Treatment; Pharmacology; Phosphorylation; Phosphorylation Site; Phosphotransferases; Property; Relapse; Research; Rodent; Rodent Model; Role; Surface; Synapses; Techniques; Technology; Testing; Therapeutic Intervention; Viral; Viral Vector; Withdrawal; Work; addiction; base; calmodulin-dependent protein kinase II; cocaine use; drug craving; drug relapse; genetic technology; inhibitor/antagonist; kinase inhibitor; mouse model; neurotoxic; novel; pre-clinical; preference; prevent; recombinase; response; therapeutic target; trafficking; transmission process; treatment strategy

DESCRIPTION (provided by applicant): Relapse to cocaine use, the highest among commonly abused illicit drugs, is a serious public problem and represents the primary challenge that exists for the treatment of cocaine addicts. Despite extensive investigation, molecular substrates that can serve as potential therapeutic targets to prevent relapse are limited. Thus, understanding the mechanisms of relapse and identifying new molecular targets for developing pharmacological treatments will greatly aid the field of addiction research. Recent preclinical rodent studies have suggested that craving and drug-induced relapse is mediated by enhanced synaptic AMPAR transmission in the nucleus accumbens (NAc) via GluA2-lacking-, GluA1-containinng-Ca2+-permeable AMPA receptors (CP-AMPARs). Work from our laboratory has identified the Cav1.2 L-type Ca2+ channel (LTCC) as a promising candidate for mediating cocaine-induced long-term behavioral responses and in regulating cell surface AMPARs. Using psychomotor sensitization, we find that Cav1.2 channels in the NAc, mediates cocaine-induced expression of sensitization following extended periods of withdrawal. Using the reinstatement of cocaine CPP model, we find that the LTCC antagonist, diltiazem delivered directly into the NAc blocks cocaine-induced reinstatement of cocaine CPP. Furthermore, we have found that Cav1.2-activated kinase pathways (CaMKII and ERK) regulate cocaine-induced increase in cell surface GluA1, but not GluA2 in the NAc. Thus, in this RO1 application we aim to capitalize on the knowledge we have gained to further explore molecular targets that mediate relapse to cocaine. We will test the central hypothesis that Cav1.2- activated kinase pathways in the NAc mediate cocaine-induced reinstatement of cocaine seeking via increase in NAc synaptic CP-AMPARs. To circumvent the challenge of the lack of Cav1.2-specific blockers, we propose to use the cutting edge Cre-lox P technology to generate local Cav1.2 knockout in the mouse NAc. Kinases will be manipulated by the use of viral vectors. In Aim 1.1, adenoassociated viral (AAV) vectors expressing Cre recombinase will be stereotaxically delivered into the NAc of Cav1.2 floxed mice. Mice will be behaviorally tested in cocaine-induced reinstatement of cocaine CPP. In Aim 1.2, molecular studies will be pursued to examine Cav1.2-induced AMPAR trafficking. In Aim 1.3, electron microscopy will be utilized to examine GluA1 trafficking in dopamine D1 neurons in the NAc shell. In Aim 2.1, viral vectors expressing kinase inhibitors will be stereotaxically delivered into the NAc of C57BL/6 mice. Mice will be behaviorally tested in cocaine-induced reinstatement of cocaine CPP. In Aim 2.2, role of kinases in AMPAR trafficking will be examined. In Aim 3, the functional significance of NAc CP-AMPARs, GluA1 trafficking and GluA1 phosphorylation in cocaine-induced reinstatement will be examined using pharmacology and genetic mutant mice. The results obtained from this study could greatly advance the field of cocaine addiction by identifying discrete molecular targets for developing pharmacological treatments for cocaine addicts.

描述（由申请人提供）：可卡因吸毒复吸是最常见的非法药物滥用率最高的一种，是一个严重的公共问题，也是可卡因成瘾者治疗面临的主要挑战。尽管进行了广泛的研究，但可作为预防复发的潜在治疗靶点的分子底物仍然有限。因此，了解复发机制并确定开发药物治疗的新分子靶点将极大地有助于成瘾研究领域。最近的临床前啮齿动物研究表明，渴求和药物引起的复发是通过缺乏 GluA2、含有 GluA1 的 Ca2+ 可渗透性 AMPA 受体 (CP-AMPAR) 增强伏隔核 (NAc) 中的突触 AMPAR 传递来介导的。我们实验室的工作已确定 Cav1.2 L 型 Ca2+ 通道 (LTCC) 是介导可卡因诱导的长期行为反应和调节细胞表面 AMPAR 的有希望的候选者。利用精神运动敏化，我们发现 NAc 中的 Cav1.2 通道在长时间戒断后介导可卡因诱导的敏化表达。使用可卡因 CPP 恢复模型，我们发现 LTCC 拮抗剂地尔硫卓直接递送到 NAc 中可阻断可卡因诱导的可卡因 CPP 恢复。此外，我们发现 Cav1.2 激活的激酶途径（CaMKII 和 ERK）调节可卡因诱导的细胞表面 GluA1 的增加，但不调节 NAc 中 GluA2 的增加。因此，在这个 RO1 应用中，我们的目标是利用我们所获得的知识来进一步探索介导可卡因复发的分子靶点。我们将测试中心假设，即 NAc 中的 Cav1.2 激活激酶途径通过 NAc 突触 CP-AMPAR 的增加介导可卡因诱导的可卡因寻求恢复。为了规避缺乏 Cav1.2 特异性阻断剂的挑战，我们建议使用尖端的 Cre-lox P 技术在小鼠 NAc 中产生局部 Cav1.2 敲除。激酶将通过使用病毒载体来操纵。在目标 1.1 中，表达 Cre 重组酶的腺相关病毒 (AAV) 载体将立体定向递送至 Cav1.2 floxed 小鼠的 NAc 中。将在可卡因诱导的可卡因 CPP 恢复中对小鼠进行行为测试。在目标 1.2 中，将进行分子研究来检查 Cav1.2 诱导的 AMPAR 贩运。在目标 1.3 中，将利用电子显微镜检查 NAc 壳中多巴胺 D1 神经元中的 GluA1 运输。在目标 2.1 中，表达激酶抑制剂的病毒载体将立体定向递送至 C57BL/6 小鼠的 NAc 中。将在可卡因诱导的可卡因 CPP 恢复中对小鼠进行行为测试。在目标 2.2 中，将研究激酶在 AMPAR 运输中的作用。在目标 3 中，将使用药理学和基因突变小鼠检查 NAc CP-AMPAR、GluA1 运输和 GluA1 磷酸化在可卡因诱导的恢复中的功能意义。这项研究获得的结果可以通过确定离散的分子靶标来开发针对可卡因成瘾者的药物治疗，从而极大地推进可卡因成瘾领域的发展。