The Role of Amygdalar Endocannabinoids in Alcohol Drinking after Traumatic Brain Injury (TBI)

项目摘要 创伤性脑损伤（TBI）和酒精使用障碍（AUD）各自对公共卫生造成重大负担： 每年约有250万美国人发生TBI，约7.2%的美国成年人（约1700万） 有一个AUD。特别重要的是这两个健康问题的相互作用：酒精中毒 是引起TBI的主要危险因素，TBI促使人类患者和动物中饮酒的增加。 模型，并且TBI神经行为后遗症在TBI时和TBI后因酒精中毒而恶化 饮酒。大约25%的TBI患者在治疗后一年报告重度或有问题的饮酒。 TBI，无论受伤前是否饮酒。不幸的是，酒精的神经机制 加重TBI的病理生理学或TBI刺激酒精升级的机制 消费是很好理解的。基底外侧杏仁核（BLA）介导应激反应和焦虑样 行为，并受到急性和慢性酒精的影响。BLA能锥体神经元有助于 饮酒量增加和酒精戒断引起的焦虑样行为。突触输入调节 BLA锥体神经元上的神经传导至少部分地由内源性大麻素（eCB）介导。在大脑中，eCB（2- 花生四烯甘油[2-AG]和花生四烯酰胺[AEA]）逆行转运以激活突触前大麻素 受体，从而抑制谷氨酸能和GABA能传递。eCB信号传导介导酒精效应 对BLA突触传递的影响：例如，急性酒精引起兴奋性 在BLA中，2-AG可增强抑制性传递，而这种作用可能是由2-AG介导的。类型-- 1大麻素受体（CB 1 Rs）在BLA神经末梢介导eCB对焦虑样行为的影响， 慢性间歇性酒精暴露通过选择性下调BLA的表达， CB 1 R在BLA的多巴胺能末梢上。因此，在CB 1 R的2-AG信号调节BLA介导的 这可能是减少TBI后饮酒升级的一个有希望的目标。这里我们 我建议使用轻度TBI的啮齿动物侧向流体冲击（LFP）模型来检查以下神经生物学基础： 合并TBI和AUD。具体来说，我们将测试BLA内源性大麻素在TBI后的 饮酒。将训练雌性和雄性Wistar大鼠在操作环境中自我给药酒精， 将在所有大鼠中的一半通过LFP接受TBI之前进行开颅术。在所有的实验中，我们将使用男性和 雌性饮酒的Wistar大鼠，经历轻度TBI或假损伤：我们将使用c-fos免疫组织化学 为了测量BLA神经元活化，用Western印迹法测量eCB系统组分（即，受体， 合成酶和降解酶），以及质谱法以测量BLA中的eCB水平。 我们还将测量主要的2-AG降解酶，单酰基甘油脂肪酶的活性。最后我们 将测试BLA内注射MAGL抑制剂JZL 184阻断TBI后酒精升高的预测 自我管理。我们的总体假设是TBI降低了BLA中的eCB信号传导和CB 1 R表达， 并且BLA中2-AG水平的增加将挽救TBI后饮酒的升级。拟议 本实验将（1）研究TBI对饮酒大鼠BLA-eCB系统的影响，（2）评价 药理学eCB调节在阻断TBI后酒精自我给药升级中的功效。 此外，本提案中描述的实验和活动将提供一个有前途的医学博士/博士学生 与研究培训和专业发展，将形成一个成功的职业生涯的基础， 从事酒精转化研究的医生兼科学家

PROJECT ABSTRACT Traumatic brain injury (TBI) and alcohol-use disorder (AUD) each pose significant burdens to public health: approximately 2.5 million Americans incur a TBI annually, and roughly 7.2% of American adults (~17 million) have an AUD. Of particular importance is the reciprocal interaction of these two health issues: alcohol intoxication is a major risk factor for incurring a TBI, TBI drives escalation of alcohol drinking in human patients and animal models, and TBI neurobehavioral sequelae are worsened by alcohol intoxication at time of TBI and by post-TBI alcohol drinking. Approximately 25% of TBI patients report heavy or problematic alcohol drinking one year post- TBI, regardless of pre-injury alcohol intake. Unfortunately, neither the neurological mechanisms by which alcohol exacerbates TBI pathophysiology nor the mechanisms by which TBI stimulates escalation of alcohol consumption are well-understood. The basolateral amygdala (BLA) mediates stress responses and anxiety-like behavior, and is impacted by acute and chronic alcohol. BLA glutamatergic pyramidal neurons contribute to escalated alcohol drinking and alcohol withdrawal-induced anxiety-like behavior. Regulation of synaptic input onto BLA pyramidal neurons is mediated, at least in part, by endocannabinoids (eCBs). In the brain, eCBs (2- arachidonylglycerol [2-AG] and anandamide [AEA]) transport retrogradely to activate pre-synaptic cannabinoid receptors, thereby inhibiting glutamatergic and GABAergic transmission. eCB signaling mediates alcohol effects on BLA synaptic transmission: for example, acute alcohol produces eCB-dependent decreases in excitatory transmission and increases in inhibitory transmission in BLA, and these effect may be mediated by 2-AG. Type- 1 cannabinoid receptors (CB1Rs) on BLA glutamatergic terminals mediate eCB effects on anxiety-like behavior, and chronic intermittent alcohol exposure augments excitatory BLA transmission by selectively down-regulating CB1R on glutamatergic terminals in BLA. Therefore, 2-AG signaling at CB1R modulates BLA glutamatergic transmission, and may be a promising target for reducing post-TBI escalation of alcohol drinking. Here, we propose to use a rodent lateral fluid percussion (LFP) model of mild TBI to examine the neurobiological basis for co-morbid TBI and AUD. Specifically, we will test the role of BLA endocannabinoids in post-TBI escalation of alcohol drinking. Female and male Wistar rats will be trained to self-administer alcohol in an operant setting, and will undergo craniotomy before half of all rats receive TBI via LFP. In all experiments, we will use male and female alcohol-drinking Wistar rats that undergo mild TBI or sham injury: we will use c-fos immunohistochemistry to measure BLA neuronal activation, Western blots to measure eCB system components (i.e., receptors, synthetic enzymes, and degradative enzymes) in BLA, and mass spectroscopy to measure eCB levels in BLA. We will also measure the activity of the major 2-AG degradative enzyme, monoacylglycerol lipase. Finally, we will test the prediction that intra-BLA injection of MAGL inhibitor, JZL184, blocks post-TBI escalation of alcohol self-administration. Our overall hypotheses are that TBI reduces eCB signaling and CB1R expression in BLA, and that increasing 2-AG levels in BLA will rescue post-TBI escalation of alcohol drinking. The proposed experiments will (1) investigate the effects of TBI on BLA eCB system of alcohol-drinking rats and (2) evaluate the efficacy of pharmacologic eCB modulation in blocking post-TBI escalation of alcohol self-administration. Additionally, the experiments and activities described in this proposal will provide a promising M.D./Ph.D. student with research training and professional development that will form the foundation for a successful career as a physician-scientist conducting translational alcohol research.