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. ? ?
