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: 8373332
• 负责人: Anjali M RAJADHYAKSHA
• 金额: $ 36.78万
• 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/8373332
• 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. PUBLIC HEALTH RELEVANCE: The results of this study have the potential to identify the Cav1.2 L-type Ca2+ channel and its downstream molecular targets as substrates that mediate relapse to cocaine-seeking behavior. These findings will greatly advance our understanding of the molecular basis of cocaine's addictive properties, which has the potential to form the basis for developing novel treatment approaches to benefit cocaine addicts.

描述（由申请人提供）：可卡因复吸是常见滥用非法药物中最严重的一种，是一个严重的公共问题，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，但可作为潜在治疗靶点以防止复发的分子底物是有限的。因此，了解复发的机制和确定新的分子靶点，以开发药物治疗将大大有助于成瘾研究领域。最近的临床前啮齿动物研究表明，渴求和药物诱导的复发是由突触增强的AMPAR传递介导的，该传递通过GluA 2-缺乏-，GluA 1-含有-Ca 2 +-可渗透的AMPA受体（CP-AMPAR）在延髓核（NAc）中进行。我们实验室的工作已经确定Cav1.2 L型Ca 2+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。使用精神敏化，我们发现，Cav1.2通道在NAc，介导可卡因诱导的表达敏化后，延长时间的撤退。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GluA 1的增加，但不调节NAc中的GluA 2。因此，在这个RO 1应用中，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。我们将测试的核心假设，Cav1.2激活的激酶途径在NAc介导可卡因诱导恢复可卡因寻求通过增加NAc突触CP-AMPAR。为了规避缺乏Cav1.2特异性阻断剂的挑战，我们建议使用最先进的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到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，将进行分子研究以检查Cav1.2诱导的AMPAR运输。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GluA 1运输。在目的2.1中，表达激酶抑制剂的病毒载体将立体定位地递送到C57 BL/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，将使用药理学和遗传突变小鼠来检查NAc CP-AMPAR、GluA 1运输和GluA 1磷酸化在可卡因诱导的恢复中的功能意义。从这项研究中获得的结果可以通过确定用于开发可卡因成瘾者的药物治疗的离散分子靶点来大大推进可卡因成瘾领域。 公共卫生关系：这项研究的结果有可能确定Cav1.2 L型Ca 2+通道及其下游分子靶点作为介导可卡因寻求行为复发的底物。这些发现将极大地推进我们对可卡因成瘾特性的分子基础的理解，这有可能为开发新的治疗方法以造福可卡因成瘾者奠定基础。