BDNF在戒酒后机体对酒精敏感性增强中的作用及其机制研究

The mesolimbic dopaminergic pathway consisting the ventral tegmental area (VTA) has evidentially been shown to be a key mediator in the ethanol rewarding after withdrawal from chronic intermittent ethanol exposure. Dopamine (DA) neurons are susceptible to ethanol excitation after withdrawal from repeated ethanol exposure, which might result in ethanol relapse, however the cellular and molecular mechanisms that underlie this phenomenon remain elusive. We recently found that local injection of exogenous BDNF into VTA in rats decreased sensitization of DA neurons to ethanol, and acute ethanol perfusion blocked Long-Term Potentiation of GABAergic Synapses (LTPGABA) induced by High Frequency Stimuli (HFS) in the VTA in brain slices of rats. Several lines of evidence indicate that back-propagating action potentials trigger dendritic release of BDNF, which can serve as a target-derived messenger for activity-dependent synaptic plasticity. Binding BDNF to TrkB not only contributes to a process of LTP in multiple brain areas, but also is involved in ethanol rewarding effects mediated by VTA. Therefore, we would use patch-clamp and molecularly biological techniques on rat model for voluntary ethanol intake to further test the mechanims of BDNF decreasing sensitization to ethnaol and accomplish the objective of this application by pursuing the following Specific Aims: ① to determine if BDNF is not only necessary but also sufficient for the production of LTPGABA induced by HFS in VTA, and what is the mechanism; ② to assess expression of BDNF in VTA after withdrawal from chronic ethanol; whether expression of BDNF is low enough to abolish LTPGABA, which contributes to hypersensitivity of DA neurons after withdrawal from repeated ethanol exposure; ③ to determine if application of exogenous BDNF could decrease DA neuron's sensitivity through rescuing the LTPGABA inhibited by withdrawal from chronic ethanol consumption so that BDNF would reduce animal's ethanol intake. Accomplishment of this project will provide evidence for an new effective therapeutic strategy of clinical prevention and cure for drug addiction including alcoholism.

戒酒后机体对酒精敏感性增强，机制不清。腹侧被盖（Ventral tegmental area, VTA）富含多巴胺（Dopamine，DA）神经元，介导酒精奖赏。我们最近发现，戒酒后VTA注射BDNF降低大鼠对酒精敏感性。酒精急性灌流脑片，阻断高频电刺激诱导VTA抑制性突触发生长时程增强（Long-term potentiation of GABAergic synapse, LTPGABA)。LTP是酒精依赖早期的重要神经基础。本课题通过膜片钳、分子生物学等技术，在主动饮酒大鼠模型上进一步探讨BDNF降低酒精敏感性的机制：①内源性BDNF在LTPGABA中的作用；②戒酒后，VTA BDNF低表达导致抑制LTPGABA，增强DA神经元敏感性；③外源性BDNF通过解救LTPGABA而降低DA神经元敏感性，减少再饮酒。本课题通过BDNF降低戒酒后酒精敏感性机制的发现，为防治酒精成瘾提供新线索。

酒精成瘾是一种反复饮酒造成的失去“控制酒精摄入”能力的慢性复发性脑病，但机制不清楚。药物对中枢突触可塑性及神经环路的影响，如长时程增强(Long-Term Potentiation, LTP)，被认为是与成瘾早期密切相关的重要神经生物学基础。中脑腹侧被盖区（Ventral tegmental area, VTA）富含多巴胺能（Dopamine，DA）神经元，脑VTA DA投射系统介导酒精的中枢奖赏效应。我们发现酒用精孵浴脑片，抑制高频电刺激（High Frequency Stimuli, HFS）诱导的VTA区DA神经元上GABAA受体介导LTPGABA，然而诱发LTPGABA的生理学机制及其在主动饮酒中的作用尚未知晓。.本课题从动物整体、细胞及分子水平，观察BDNF在介导VTA区DA神经元上的LTPGABA中的作用及其机制；BDNF的表达在主动饮酒抑制LTPGABA中的作用；BDNF介导的LTPGABA对主动饮酒大鼠饮酒量的影响（生物学功能）。.我们发现酒精能够长时程抑制突触前氨基丁酸类（抑制性）神经递质释放增强(LTPGABA)，从而使多巴胺能神经元的活动脱抑制，合成和分泌的多巴胺水平升高，产生成瘾的欣快感，此作用可能是酒精成瘾的重要神经生物学机制，并且本人发现酒精的作用位点是在氨基丁酸能神经纤维末梢的突出前膜上，可能通过mu阿片受体发挥作用，mu阿片受体阻断剂纳洛酮可阻断酒精的这种作用。.进一步研究发现，BDNF通过激活TrkB介导HFS引起的大鼠VTA区DA神经元上LTPGABA。BDNF介导LTPGABA的机制：BDNF激活突触后DA神经元TrkB受体，激活NOS，使NO生成增多，激活GABA能神经末梢鸟苷酸环化酶（GC），cGMP合成增多，引起突出前GABA释放增多，形成LTPGABA。主动饮酒戒断后由于VTA区BDNF低表达而抑制LTPGABA，外源性BDNF可以通过解救主动饮酒对LTPGABA的抑制并减少饮酒大鼠的饮酒量。.另外，我们还发现，在大鼠VTA区微量注射甘氨酸阻断高频刺激诱发的抑制性突触长时程增强，此作用可被士的宁阻断，并且显著抑制酒精成瘾大鼠的饮酒量。本项目资助的研究成果为临床治疗酗酒及减轻酗酒患者的饮酒量提供了新思路和实验依据。