2025 Scholars' Research Symposium Abstract: Effects of the Calcineurin Mediated Immunosuppressant Cyclosporine on Binge Alcohol Drinking and Stress Responsivity in Mice.

Introduction: Binge-drinking behavior is a prevalent and costly burden that many face. To expand potential treatments for alcohol use disorder and improve treatment efficacy, calcineurin (CN) inhibiting cyclosporine A (CsA) is investigated as a potential treatment for this disorder based on a neuroinflammation-driven approach to addictive behaviors and stress maladaptation. CsA has previously been shown to reduce binge-drinking behaviors, and this study aims to provide a connection between binge-drinking reduction and stress response to the expression of the neuroinflammasome.

Methods: CamKIIa and CRF neuronal CN knockout mice were characterized for knockout of CN expression through immunohistochemistry and RNA scope imaging of selected brain regions. Others from this cohort were then subjected to 6-week binge-drinking behavioral experiments in the format of "Drinking-in-the-Dark" (DID). Experimental mice were tracked for ethanol intake overtime and following injection of either vehicle only intraperitoneal injection or CsA and vehicle injection for any changes. These transgenic mice were separately subjected to 1-hour restraint stress experiments with exposure to either CsA or vehicle only. These cohorts were sacrificed with their brain tissue harvested and microdissected for rtPCR characterization of inflammatory gene products. Expression of CD45, COX- 2, CYC, Iba-1, IL-1b, IL-6, TNF-a, ACTB, CCL2, and CCR2 was quantified. The expression for CsA exposed mice was then compared to vehicle-only mice through the log2 fold change analysis for final comparison.

Results: Immunohistochemistry with RNA scope imaging for calcineurin expression revealed widespread CN knockout in the experimental lineage. CN knockout revealed no effect on ethanol consumption in DID models for both CamkIIa and CRF neuronal CN knockout compared to wild type. CsA did still induce a large reduction in ethanol intake for both lines of CN knockout mice compared to baseline. In the restraint stress study, rtPCR log2 fold analysis revealed that CsA reduced a wide-range of stress-induced neuroinflammatory markers. Specifically, the authors observed a generalized reduction in inflammatory gene expression with IL-1b seeing a nearly 6-fold decrease in both the central nucleus of the amygdala as well as the paraventricular nucleus.

Conclusions: The findings of normal baseline drinking behaviors and improved parameters following treatment with CsA in wild type and neuronal CN knockout lineages indicate that CN at the level of neurons is not responsible for CsA-induced reduction in binge-drinking behavior. These findings, in conjunction with the reduction of neuroinflammatory gene product expression, implicate glial cells as possibly responsible for these changes. Further investigation into glia cell specific CN knockout is warranted under similar conditions. Ultimately, these findings improve the characterization of the mechanism of CsA-induced reduction in binge drinking behaviors and aid in discovering new treatments for alcohol use disorder.