Establishing the Molecular Mechanisms of Reduced Ethanol Drinking in Calcineurin-Mediated Immunosuppression Treated Rodents

Va Eastern Colorado Health Care System, Aurora CO

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Abstract

Veteran populations present alcoholism rates that are far higher than general population frequencies. Despite this, the treatment options for alcoholism are very few and much in need of new approaches for medical interventions. This project grows from the observation that alcoholic persons receiving liver transplant evidence very high rates of sustained abstinence after the liver grafting procedure that we could not explain by psychosocial or selection factors. We asked whether this might be due to a common factor shared by all of these patients: immunosuppressant medicines given for very long periods after transplant to keep the grafted organ in a state of health. In two separate rodent experiments we found that 1) cyclosporine (CSA) had an antidipsic effect in an open choice model of ethanol (alternatively alcohol, or ethyl alcohol) drinking, and 2) that CSA and tacrolimus (TRL, alternately FK506) had similar effects in a drinking-in-the-dark (DID) ethanol drinking model while sirolimus (SRL, alternately rapamycin) had no effect but had lower concentrations in the brain. These findings implicate the immunosuppressants blocking brain calcineurin (CLN) activity that may enhance the rodent's choice against drinking ethanol through 1) direct CLN modulation in the brain or 2) through the systemic immunosuppressive and neuroimmune pathways. This study asks whether further evidence supports one or the other of these possible mechanisms of action. Following the overall goal of this project--to elucidate the mechanism(s) of reduction of alcohol preference by CLN directly or by CLN-mediated immunosuppression, the study's primary hypothesis asserts that inhibition limiting CLN activity in the brain itself will decrease rodent ethanol choice. An alternative hypothesis states that the peripheral effects of CLN inhibition in the immune system will result in a decrease in ethanol preference. The overall aim of the project is therefore to establish which of these two possible mechanisms mediates the effect of immunosuppressant agents on rodent's choice not to drink ethanol. Our experimental approaches address the primary hypothesis using a genetic knockout approach. In Specific Aim 1, we will compare immunosuppressants in brain-specific CLN knock-out mice that include a) pan-brain knockout (CamKII? Cre x floxed CLN) rodents, b) CRF neuron specific calcineurin knockout (CRHCrex floxed CLN), and c) focal CLN knockouts in extended reward regions (VTA, NAc, CeA) utilizing AAV-Cre microinjections in floxed CLN mice. In Specific Aim 2, we will characterize the possible mechanisms of central effects of CLN inhibition on alcohol consumption by assessing 1) the brain's metabolic protection by CLN inhibitors, and 2) addiction related downstream signal molecules in extended brain reward networks. In Specific Aim 3, we test whether drinking causes neuroinflammation that drives subsequent drinking. We will use a combination of approaches (flow cytometry, immunohistochemistry, qRT-PCR, ELISA) to determine the effects of CLN inhibition on ethanol induced neuroinflammation. And in Specific Aim 4 we will non-invasively assess anatomical, metabolic and functional changes in the mouse brain using multi-parametric magnetic resonance imaging (MRI). We will use non-invasive 9.4 Tesla MRI protocols longitudinally on our mouse models of drinking preference treated with CsA, TRL and SRL. We expect the data from these procedures to answer the study questions in a definitive manner and to point the path to new answers in the neurochemistry of alcohol choice. This in turn can lead to better understanding of the mechanisms involved in stopping or limiting drinking and alcohol addiction in veteran populations. The overall goal is to find new medicinal agents that can treat alcoholism a condition that affects up to one in every two patients admitted to VA inpatient services with an estimated half of those actively drinking.