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

退伍军人的酗酒率远远高于普通人群。 尽管如此，酒精中毒的治疗选择很少，迫切需要新的方法来治疗 医疗干预。这个项目源于对酗酒者接受肝脏移植的观察 肝移植手术后持续禁酒率很高的证据，我们无法解释 心理社会或选择因素。我们问这是否可能是因为所有人都有一个共同的因素 这些患者：移植后长期服用免疫抑制药物以保持移植的移植物 器官处于健康状态。在两个不同的啮齿动物实验中，我们发现1)环孢素(CsA)有一个 酒精(可选择酒精或酒精)饮用的开放选择模型中的抗糖尿病作用，以及2) CsA和他克莫司(TRL，交替为FK506)在黑暗中饮酒(DID)中有类似的作用 西罗莫司(SRL，交替使用雷帕霉素)对小鼠饮酒无影响，但其体内药物浓度较低。 大脑。这些发现表明，免疫抑制剂阻断了脑钙调神经磷酸酶(CLN)的活性，这可能 通过1)大脑中CLN的直接调节或2)增强啮齿动物对饮酒的选择 通过全身免疫抑制和神经免疫途径。这项研究询问了进一步的 证据支持这些可能的行动机制中的一种。 遵循本课题的总体目标--阐明降酒机理(S) 这项研究的主要假设是，CLN直接或通过CLN介导的免疫抑制产生偏爱 这种限制大脑中CLN活性的抑制会减少啮齿动物对乙醇的选择。另一种选择 假说认为，抑制免疫系统中CLN的外周效应将导致 偏爱乙醇。因此，该项目的总体目标是确定这两种可能性中的哪一种 免疫抑制剂影响啮齿动物不饮酒的机制。 我们的实验方法使用基因敲除方法解决了主要假设。在……里面 具体目标1，我们将在大脑特异性CLN基因敲除小鼠中比较免疫抑制剂，包括) 泛脑基因敲除(CaMKIIαCre x Floted CLN)啮齿动物，b)CRF神经元特异性钙调神经磷酸酶基因敲除 (CRHCrex Floted CLN)，以及c)扩展奖赏区域(VTA、NAC、CEA)中的焦点CLN敲除，利用 AAV-Cre在CLN小鼠体内的微量注射。在具体目标2中，我们将描述可能的机制 抑制CLN对饮酒的中枢影响通过评估1)大脑的代谢保护 CLN抑制剂，以及2)扩展的大脑奖励网络中与成瘾相关的下游信号分子。在……里面 具体目标3，我们测试饮酒是否会导致神经炎症，从而推动随后的饮酒。我们会 使用多种方法(流式细胞仪、免疫组织化学、qRT-PCR、ELISA)相结合的方法来确定 CLN抑制对乙醇诱导的神经炎症的影响。在特定的目标4中，我们将非侵入性地 用多参数磁共振仪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身上纵向使用非侵入性的9.4特斯拉MRI协议 CsA、TRL和SRL处理的饮酒偏好模型。 我们希望来自这些程序的数据能够明确地回答研究问题，并 在酒精选择的神经化学中指出新的答案。这反过来又可以带来更好的 对退伍军人戒酒或限制饮酒和酒精成瘾机制的认识 人口。总体目标是找到可以治疗酒精中毒的新的药物制剂，酒精中毒是一种影响 每两名患者中就有一人住进退伍军人管理局的住院服务，估计有一半的人是活跃的 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.