Role of Reelin protein in the cellular and behavioral response to drugs of abuse - PROJECT SUMMARY Substance use disorder (SUD) is a national health crisis, with over 93,000 deaths due to overdose in 2020. Currently, no FDA-approved therapeutics exist for SUD, with targeted therapeutics out of reach due to incomplete understanding of the neurobiology of addiction. Our lab has identified expression of Reln mRNA as necessary for cocaine-responsiveness in NAc Drd1+ medium spiny neurons (MSNs). Reelin is a secreted glycoprotein that plays a critical role in cortical cell migration during brain development, with mutations leading to lissencephaly, defined by a smooth-brain phenotype. In the adult brain, Reln expression remains high in the cerebellum, hippocampus and striatum. Brain-wide Reelin signaling blockade attenuates response to methamphetamines, indicating a critical role for Reelin in the striatum. Despite high striatal Reln expression and evidence suggesting Reelin modulates behavioral output of the striatum, Reelin’s role in the striatum remains under-studied. Using RNA single-molecule fluorescent in-situ hybridization (smFISH), I have validated our single-cell data showing Reln marks cocaine-sensitive Drd1+ cells. I have also found Drd1+ cells are enriched in Reln compared to Drd2+ cells in both rat and human brain tissue. My smFISH data show Reln expression is restricted to the dorsal striatum and nucleus accumbens (NAc) core and absent in the NAc shell. As these regions have distinct functions, this pattern may reflect the functional relevance of Reelin signaling. I have assessed the role of Reelin signaling using a CRISPR-interference (CRISPRi) strategy to knockdown Reln. NAc whole-cell patch clamp demonstrates that CRISPRi at Reln significantly impairs MSN excitability. Critically, in vivo CRISPRi-knockdown of striatal Reln attenuates cocaine preference in a rat model. Given these findings, I hypothesize that Reelin facilitates the dopamine-induced neuronal adaptations that follow drug exposure and promote drug-seeking behavior. The proposed study will take a multidisciplinary approach to rigorously investigate the following aims: (1) Reelin is required for cocaine-related behavioral adaptations and (2) Reelin regulates membrane excitability to facilitate dopamine signaling. The proposed studies will deepen our understanding of the molecular underpinnings of SUD, providing avenues for therapeutic exploration for a disease completely lacking targeted treatments. Under this award, I will master intravenous self-administration and locomotor sensitization assays to assess the relevant consequences of genetic manipulations. I will hone patch clamp electrophysiology skills to assess mechanisms behind behavioral adaptations. These techniques will aid my success as a physician-scientist studying mechanisms of and engineering therapeutics for brain disorders.
