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

退伍军人群体的酗酒率远远高于一般人群的频率。 尽管如此，酒精中毒的治疗选择非常少，并且非常需要新的方法来治疗。 医疗干预。这个项目源于对接受肝脏移植的酗酒者的观察， 肝脏移植手术后持续禁欲的比例非常高，我们无法解释， 心理社会或选择因素。我们问，这是否可能是由于一个共同的因素， 这些患者：移植后长时间使用免疫抑制剂，以保持移植物 器官处于健康状态。在两个独立的啮齿动物实验中，我们发现1）环孢素（CSA）具有 在乙醇（或者酒精，或乙醇）饮用的开放选择模型中的止渴作用，和2） CSA和他克莫司（TRL，或者FK 506）在黑暗中饮酒（DID）乙醇中具有相似的作用 而西罗莫司（SRL，交替雷帕霉素）没有影响，但在饮酒模型中的浓度较低， 个脑袋这些发现表明，免疫抑制剂阻断脑钙调神经磷酸酶（CLN）的活性， 通过1）大脑中的直接CLN调节或2） 通过全身免疫抑制和神经免疫途径。这项研究问是否进一步 证据支持这些可能的作用机制中的一种或另一种。 本计画的总体目标为：阐明减少酒精的机制 该研究的主要假设是， 大脑中CLN活性的抑制会减少啮齿动物对乙醇的选择。一个替代 这一假设指出，免疫系统中CLN抑制的外周效应将导致免疫功能的降低。 乙醇偏好因此，该项目的总体目标是确定这两种可能性中的哪一种 免疫抑制剂对啮齿动物选择不饮用乙醇的影响。 我们的实验方法使用基因敲除方法来解决主要假设。在 具体目标1，我们将在脑特异性CLN敲除小鼠中比较免疫抑制剂，包括a） 全脑敲除（CamKIIα Cre x floxed CLN）啮齿动物，B）CRF神经元特异性钙调磷酸酶敲除 c）在扩展的奖励区域（VTA、NAc、CeA）中的局灶性CLN敲除， 在floxed CLN小鼠中进行AAV-Cre显微注射。在具体目标2中，我们将描述可能的机制 CLN抑制对酒精消耗的中枢作用，通过评估1）大脑的代谢保护， CLN抑制剂，和2）成瘾相关的下游信号分子在扩展的大脑奖励网络。在 具体目标3，我们测试饮酒是否会导致神经炎症，从而驱动随后的饮酒。我们将 使用方法（流式细胞术、免疫组织化学、qRT-PCR、ELISA）的组合来确定 CLN抑制对乙醇诱导的神经炎症的影响。在《特定目标4》中，我们将非侵入性地 使用多参数磁共振成像技术评估小鼠大脑的解剖、代谢和功能变化 磁共振成像（MRI）。我们将在我们的小鼠上纵向使用非侵入性9.4特斯拉MRI协议 CsA、TRL和SRL治疗的饮酒偏好模型。 我们希望这些程序的数据能够以明确的方式回答研究问题， 为酒精选择的神经化学研究指出了新的答案。这反过来又可以导致更好的 了解参与停止或限制退伍军人饮酒和酒精成瘾的机制 人口。总的目标是找到新的药物，可以治疗酒精中毒的条件，影响 高达每两名患者中就有一名接受VA住院服务，估计有一半的患者积极参与 喝酒

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 (CamKIIα 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.