Mechanisms of regulation of ethanol intake by lateral habenula

本应用的目的是探索外侧缰核(LHb)中的神经元在调节酒精摄入中所起的作用。厌恶的、消极的感觉输入由缰核复合体处理，缰核复合体是一种上皮性结构，涉及恐惧、焦虑、抑郁、压力和奖励。LHb主要从基底节接受输入，并将输出主要发送到多巴胺(DA)和5-羟色胺能神经元。LHb为中脑DA细胞提供了负性强化信号的重要来源。这种深刻而一致的抑制作用涉及LHb内的谷氨酸神经元与桥小脑头内侧被盖核(RMTg)中的GABA细胞的双突触联系，进而支配DA神经元。大量研究表明，缰核在尼古丁成瘾和戒断以及对吗啡自我给药和寻找可卡因行为的调节中起着关键作用。然而，LHb在乙醇成瘾中的作用还没有得到很好的研究。众所周知，DA系统，包括腹侧被盖区(VTA)，与酒精寻求和复发有关。尽管乙醇能显著激活中脑边缘多巴胺传递，但慢性酒精暴露可导致伏隔核内VTA DA神经元活动和细胞外多巴胺水平显著降低。据信，这种多巴胺功能低下会导致烦躁不安的状态，促使药物试图将多巴胺恢复到正常的药物水平。然而，导致多巴胺功能低下的机制还不是很清楚。因此，我们提议的实验将专门测试这一中心假设，即酒精依赖动物中LHb神经元的过度活动驱动抑制的RMTg神经元，从而减少VTA DA神经元的放电。这些抑制作用可能是导致过量饮酒的多巴胺功能低下和厌恶状态的基础。我们将通过使用慢性间歇性乙醇自我给药(CIESA)的大鼠模型和多学科方法来验证这一假设，包括以下两个独立但集成的特定目标的最新光遗传学技术。具体目标1将通过评估自愿饮酒的变化来检验这一假设，而LHb神经元的功能是通过电/光刺激或药理学来操纵的。我们还将通过Fos免疫反应来测量CIESA对LHb和RMTg中神经元活性的影响，使用Western blotting来测量CIESA对LHb和RMTg中谷氨酸受体蛋白水平的影响，并使用微透析技术来测量CIESA对LHb和RMTg中细胞外谷氨酸水平的影响。具体目标2将通过在酒精依赖动物脑片上进行电生理记录来研究LHb调节酒精饮酒行为的细胞机制。我们将描述CIESA在LHb和RMTg神经元的活动和谷氨酸能突触传递方面引起的变化。这些研究将提供重要的新信息，大大促进我们对LHb和RMTg在酒精使用障碍中的作用的理解。这些研究还可以提供对人类酗酒者负性强化相关饮酒的细胞机制的洞察。

DESCRIPTION The goal of this application is to explore the role that neurons in the Lateral Habenula (LHb) play in regulating ethanol intake. Aversive, negative sensory input is processed by the habenular complex, an epithalamic structure involved in fear, anxiety, depression, stress and reward. The LHb receives inputs primarily from the basal ganglia and sends outputs mainly to dopaminergic (DA) and serotonergic neurons. The LHb provides an important source of negative reinforcing signals to midbrain DA cells. This profound and consistent inhibitory influence involves a disynaptic connection from glutamate neurons in the LHb to the GABA cells in the Rostromedial Mesopontine Tegmental Nucleus (RMTg) that, in turn, innervates DA neurons. Much work has shown that the habenula plays a key role in nicotine addiction and withdrawal and in the regulation of morphine self-administration, as well as cocaine seeking behavior. However, the role of LHb in ethanol addiction has not been well explored. It is well accepted that the DA system, including the ventral tegmental area (VTA), is involved in ethanol seeking and relapse. Although ethanol acutely activates mesolimbic DA transmission, withdrawal from chronic ethanol exposure leads to substantial decrements in VTA DA neuronal activities and extracellular levels of dopamine in the nucleus accumbens. It is believed that this dopamine hypofunction leads to a dysphoric state that drives drug seeking to restore dopamine to normal, drug-na¿ve levels. However, the mechanisms causing dopamine hypofunction are not well understood. Our proposed experiments will therefore specifically test the central hypothesis that over-activity of LHb neurons in alcohol dependent animals drives inhibitory RMTg neurons which reduce firing of VTA DA neurons. These inhibitory effects may underlie the dopamine hypofunction and aversive state that may substantially contribute to excessive drinking. We will test this hypothesis by the use of a rat model of chronic intermittent ethanol self-administration (CIESA) and a multidisciplinary approach, including state-of-the-art optogenetic techniques in the following two separate but integrated Specific Aims. Specific Aim 1 will test the hypothesis by assessing changes in voluntary ethanol drinking while the function of the LHb neurons is manipulated by electrical/light stimulation or pharmacologically. We will also measure changes caused by CIESA on the activity of neurons in the LHb and RMTg by means of Fos immunoreactivity, on protein levels of glutamate receptors using Western blotting, and extracellular levels of glutamate in the LHb and RMTg using microdialysis techniques. Specific Aim 2 will examine the cellular mechanisms underlying LHb regulation of ethanol drinking behaviors by electrophysiological recording in brain slices of alcohol dependent animals. We will characterize changes caused by CIESA in the activity of, and glutamatergic synaptic transmissions to, neurons in the LHb and RMTg. These studies will provide important new information that will significantly advance our understanding of the role of LHb and RMTg in alcohol use disorders. These studies could also provide insight into the cellular mechanisms governing negative reinforcement-associated drinking in human alcoholics.