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: 8471683
• 负责人: Anjali M RAJADHYAKSHA
• 金额: $ 36.5万
• 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/8471683
• 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）：在常见滥用的非法药物中，可卡因的使用率最高，故态复燃是一个严重的公共问题，也是治疗可卡因成瘾者面临的主要挑战。尽管广泛的研究，分子底物可以作为潜在的治疗靶点，以防止复发是有限的。因此，了解复发机制和确定新的分子靶点以开发药物治疗将极大地帮助成瘾研究领域。最近的临床前啮齿动物研究表明，渴望和药物诱导的复发是由伏隔核（NAc）通过缺乏glua2，含有glua1 - ca2 +渗透性AMPA受体（CP-AMPARs）增强的突触AMPAR传递介导的。我们实验室的工作已经确定了Cav1.2 l型Ca2+通道（LTCC）作为介导可卡因诱导的长期行为反应和调节细胞表面ampar的有希望的候选通道。利用精神运动性致敏，我们发现NAc中的Cav1.2通道介导长时间戒断后可卡因诱导的致敏表达。通过可卡因CPP恢复模型，我们发现直接进入NAc的LTCC拮抗剂地尔硫卓可阻断可卡因诱导的可卡因CPP恢复。此外，我们发现cav1.2激活的激酶通路（CaMKII和ERK）调节可卡因诱导的细胞表面GluA1的增加，而不是NAc中GluA2的增加。因此，在这个RO1应用程序中，我们的目标是利用我们已经获得的知识，进一步探索介导可卡因复发的分子靶点。我们将验证NAc中Cav1.2激活的激酶途径通过增加NAc突触cp - ampar介导可卡因诱导的可卡因寻求恢复的中心假设。为了规避缺乏Cav1.2特异性阻滞剂的挑战，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Aim 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到Cav1.2固定小鼠的NAc中。小鼠将在可卡因诱导的可卡因CPP恢复中进行行为测试。在Aim 1.2中，将进行分子研究以检查cav1.2诱导的AMPAR转运。在Aim 1.3中，我们将利用电子显微镜检测NAc壳中多巴胺D1神经元中GluA1的转运。在Aim 2.1中，表达激酶抑制剂的病毒载体将立体定向递送到C57BL/6小鼠的NAc中。小鼠将在可卡因诱导的可卡因CPP恢复中进行行为测试。在目标2.2中，将审查激酶在AMPAR贩运中的作用。在Aim 3中，我们将通过药理学和基因突变小鼠检测NAc CP-AMPARs、GluA1转运和GluA1磷酸化在可卡因诱导恢复中的功能意义。本研究的结果可以通过确定离散的分子靶点来开发可卡因成瘾者的药物治疗方法，从而极大地推进可卡因成瘾领域。