Anjali Rajadhyaksha - US grants

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.

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.

Project Summary In the cerebral cortex, gamma-aminobutyric acid (GABA)ergic interneurons are the major source of inhibition. Interneuron dysfunction is strongly associated with autism and childhood epilepsy. We demonstrated that environmental influences such as electrical activity are fundamental for the maturation of GABAergic circuits. However, the identity of the activity patterns controlling interneuron development remains poorly understood. The long-term goal of this research is to uncover how early interneuron dysfunction leads to lasting neuropathologies. The objective of this proposal is to reveal the signaling pathways underlying activity- dependent development and to assess how perturbations in this process lead to aberrant brain function. To this end, we will use the murine barrel cortex as a well-established model for the study of activity-dependent circuit maturation. We will focus our studies in cortical interneurons since our previous work indicates that these neurons are exquisitely sensitive to environmental perturbations in the neonate. In the near term, this proposal is aimed at investigating the role of specific interneuron subtypes in regulating the emergence of early activity patterns (Aim 1). In addition, this project will determine the calcium-dependent signaling pathways for the functional maturation of interneuron networks. We will study the role of Cacna1c, a gene encoding for the Cav1.2 subunit of L-type calcium channels. Mutations in this gene are strongly associated with Timothy syndrome and other neurodevelopmental disorders (Aim 2). Finally, we will assess how developmental defects in interneuron number lead to abnormal brain activity during development and impaired behavior in the adult (Aim 3). With respect to the outcomes, our work is expected to identify basic mechanisms fundamental for the emergency of a healthy balance in the number of excitatory and inhibitory neurons. In addition, these results are expected to have a significant translational impact because they will expand our mechanistic knowledge on how mutations in the CACNA1C gene, strongly associated with autism, bipolar disorder, schizophrenia and Timothy syndrome, may lead to behavioral abnormalities frequently observed in these patients.

The National Science Foundation (NSF) Graduate Research Fellowship Program (GRFP) is a highly competitive, federal fellowship program. GRFP helps ensure the vitality and diversity of the scientific and engineering workforce of the United States. The program recognizes and supports outstanding graduate students who are pursuing research-based master's and doctoral degrees in science, technology, engineering, and mathematics (STEM) and in STEM education. The GRFP provides three years of financial support for the graduate education of individuals who have demonstrated their potential for significant research achievements in STEM and STEM education. This award supports the NSF Graduate Fellows pursuing graduate education at this GRFP institution.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.