RNAi knockdown of Cav1.3 and addiction

描述（由申请人提供）：精神兴奋剂、安非他明和可卡因是被人类滥用的最具强化作用的药物，也是临床和科学界的一个主要健康问题。在人类和啮齿类动物的成瘾模型中，反复使用药物会导致大脑中持久的神经元适应，从而导致强迫性成瘾行为。然而，精神兴奋剂导致大脑持续变化的确切机制仍然难以捉摸。钙信号在精神兴奋剂介导的行为和分子变化中起关键作用。最近的研究强调了Cav1.3 l型Ca2+通道（LTCC）及其分子通路在神经元可塑性中的作用。我们实验室的研究发现，Cav1.3 ltcc介导多巴胺和谷氨酸信号的几个方面，这是参与精神兴奋剂作用的主要神经递质。我们发现，在安非他明致敏小鼠中，Cav1.3 ltcc通过激活背纹状体（dStr）中的多巴胺D2长（D2L）受体信号通路，介导安非他明诱导的谷氨酸受体GluR1亚基磷酸化的下调，该区域参与成瘾的习惯形成方面。我们进一步发现，这种适应只发生在延长的无药期之后，并且与致敏行为相关。因此，在本研究中，我们旨在进一步探讨Cav1.3 ltcc在安非他明诱导的行为致敏模型中D2L信号上调中的作用，该模型具有学习和记忆模型中明显的突触可塑性的许多特征。然而，Cav1.3 ltcc研究面临的挑战之一是缺乏亚基特异性药物。在这个应用中，我们建议使用RNA干扰（RNAi）技术，这是一种强大的机制，可以在空间和时间特异性的情况下，对大脑中的目标基因进行序列特异性敲除。在Specific Aim 1中，我们将生成重组腺相关病毒（rAAV）载体，将Cav1.3特异性的短发夹RNA （shRNA）分子传递到腹侧被盖区（VTA），这是启动精神兴奋剂诱导行为机制的主要神经部位。首先在体外测试具有高敲低效率的shrna，然后将在小鼠VTA体内用于特异性降解Cav1.3 mRNA，从而导致空间敲低。在Specific Aim 2中，将在安非他明行为致敏方案中对VTA特异性Cav1.3敲除小鼠进行测试，并研究VTA Cav1.3 ltcc在dStr中介导D2L和GluR1信号适应中的作用。在Specific Aim 3中，Cav1.3敲低的VTA细胞型特异性表型将通过检测Cav1.3靶点、CREB和ERK的磷酸化来表征。RNAi方法将允许阐明Cav1.3 ltcc在安非他明诱导的行为和分子可塑性中的区域和时间特异性。此外，这里产生的工具将允许靶向Cav1.3 LTCC途径的其他细胞内分子，以更好地理解导致精神兴奋剂暴露后行为持续改变的机制。

DESCRIPTION (provided by applicant): The psychostimulants, amphetamine and cocaine are among the most reinforcing drugs that are abused by humans and a major health issue in the clinical and scientific communities. Repeated drug use causes long-lasting neuronal adaptations in the brain that leads to compulsive addictive behavior both in humans and in rodent models of addiction. However the precise mechanisms by which psychostimulants cause persistent alterations in the brain remain elusive. Calcium signaling plays a pivotal role in psychostimulant-mediated behavioral and molecular changes. Recent studies have highlighted the role of the Cav1.3 L-type Ca2+ channel (LTCC) and its molecular pathways in neuronal plasticity. Work from our lab finds that Cav1.3 LTCCs mediate several aspects of dopamine and glutamate signaling, primary neurotransmitters involved in psychostimulant action. We find that in amphetamine sensitized mice, Cav1.3 LTCCs mediate downregulation of amphetamine- induced phosphorylation of the GluR1 subunit of glutamate receptors via activation of the dopamine D2 long (D2L) receptor-signaling pathway in the dorsal striatum (dStr), a region involved in the habit-forming aspects of addiction. We further find that this adaptation occurs only following extended drug-free period and is a correlate of sensitized behavior. Hence in this proposal we aim to further explore the role of Cav1.3 LTCCs in upregulation of D2L signaling in the model of amphetamine-induced behavioral sensitization that shares many features of synaptic plasticity evident in models of learning and memory. However one of challenges in studying Cav1.3 LTCCs is the lack of subunit specific pharmacological agents. In this application we propose to use RNA interference (RNAi) technology, a powerful mechanism that allows sequence-specific knockdown of target genes in the brain with spatial and temporal specificity. In Specific Aim 1, we will generate recombinant adenoassociated viral (rAAV) vectors to deliver short hairpin RNA (shRNA) molecules specific for Cav1.3 into the ventral tegmental area (VTA), the primary neural site that initiates mechanisms that underlie psychostimulant-induced behaviors. shRNAs with high knockdown efficiency first tested in vitro will then be used in vivo in mouse VTA to specifically degrade Cav1.3 mRNA resulting in a spatial knockdown. In Specific Aim 2, VTA-specific Cav1.3 knockdown mice will be tested in an amphetamine behavioral sensitization protocol and the role of VTA Cav1.3 LTCCs in mediating adaptation of D2L and GluR1 signaling in the dStr will be examined. In Specific Aim 3, VTA cell-type specific phenotype of Cav1.3 knockdown will be characterized by examining phosphorylation of Cav1.3 targets, CREB and ERK. The RNAi approach will allow the elucidation of the regional and temporal specificity of Cav1.3 LTCCs in amphetamine-induced behavioral and molecular plasticity. Furthermore the tools generated here will allow the targeting of other intracellular molecules of the Cav1.3 LTCC pathway towards a better understanding of the mechanisms that lead to persistent alteration in behavior following psychostimulant exposure.