BDNF-TrkB介导LTPGABA调控大鼠饮酒行为的分子机制

Alcohol abuse is a major public problem. However, the neural basis underlying control of ethanol behaviors is still unclear. The mesolimbic dopamine (DA) system has evidentially been shown to be a key mediator in the rewarding effects of ethanol and alcohol-drinking behavior, which consists of dopaminergic projections from the ventral tegmental area (VTA) of the midbrain to various areas in the limbic system. Synaptic plasticity in VTA is thought to be related with ethanol behaviors further closely. Our group found that BDNF-TrkB mediated Long-term potentiation of GABAergic synapse in VTA, which was inhibited by acute application of ethanol. Our hypothesis in this project is that LTPGABA mediated by BDNF-TrkB regulates ethanol behaviors. Exogenous agonist of TrkB-R could regulate ethanol behaviors through rescuing LTPGABA while voluntary alcohol drinking inhibits LTPGABA via decreasing BDNF/TrkB expression in VTA. We previously found that microinjection of BDNF into VTA reduced ethanol intake four week voluntary ethanol drinking in rats. In this project, we would use behavior test, optogenetics, patch-clamp, PCR, WB, confocal, electron microscope and other molecular biological techniques on rat model for voluntary ethanol intake to test our central hypothesis and accomplish the objective of this application by pursuing the following specific aims: to assess dynamically effects of LTPGABA mediated by BDNF-TrkB on ethanol intake and ethanol Conditioned Place Preference (CPP) respectively; to determine expression of LTPGABA in anterior VTA on different time points in ethanol drinking rats; to determine expression and subcellular localization of BDNF, TrkB and cGMP to elucidate molecular mechanisms underlying LTPGABA regulating ethanol behaviors; to determine ultrastructure of GABAergic synapse dynamically during alcohol drinking weeks. Accomplishment of this project will provide evidence and inventive cues for clinical prevention and cure for alcohol abuse.

酒精滥用是公众健康问题。调节饮酒行为的神经基础尚不清楚。中脑腹侧被盖（VTA）突触可塑性与饮酒行为有关。课题组发现BDNF-TrkB介导VTA抑制性突触长时程增强（LTPGABA），酒精抑制LTPGABA。课题假说LTPGABA参与调控饮酒行为。饮酒通过降低BDNF/TrkB表达抑制LTPGABA，外源性TrkB受体激动剂通过解救LTPGABA干预饮酒行为。我们前期发现，饮酒4周VTA微量注入BDNF减少酒精成瘾大鼠饮酒量。本课题拟在主动饮酒大鼠模型上，通过光遗传学等技术明确BDNF-TrkB介导LTPGABA对大鼠CPP及饮酒量的调节作用；用膜片钳技术阐明其调节饮酒行为电生理机制；通过PCR、WB、免疫荧光和Confocal等阐明BDNF、TrkB、cGMP等相关分子机制；透射电镜分析GABA能突触超微结构，阐明调控饮酒行为的形态学基础。项目完成为酒精滥用防治提供科学依据。

酒精依赖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，调控饮酒行为的机制不清楚。中脑腹侧被盖区（Ventral tegmental area, VTA）富含多巴胺能（Dopamine，DA）神经元，GABA神经元调节DA神经元活动，VTA DA投射系统与酒精奖赏效应关系密切。. 本项目在从动物整体、细胞及分子水平，集中动态研究了BDNF-TrkB诱导VTA抑制性突触长时程增强(Long-term potentiation of GABAergic synapse, LTPGABA)调控大鼠饮酒行为的分子机制。检测了VTA微量注射BDNF及腹腔注射7,8-DHF对大鼠饮酒量和酒精戒断焦虑行为的影响及相关机制；通过PCR、WB等技术检测了饮酒大鼠VTA BDNF、NO及cGMP等表达，用电镜观察了VTA GABA能突触超微结构改变，通过膜片钳技术检测BDNF诱导饮酒大鼠VTA LTPGABA电生理机制；阐明了LTPGABA调控大鼠饮酒行为的分子机制。. 我们发现BDNF和7,8-DHF均可显著减轻酒精依赖大鼠戒断症状及饮酒量，机制与VTA TrkB（BDNF受体）有关；腹腔注射7,8-DHF通过激活伏隔核TrkB减轻酒精依赖小鼠戒断后焦虑行为；大鼠VTA NO/cGMP/PKG信号通路参与BDNF(或7,8-DHF)-TrkB对饮酒行为的调控；BDNF抑制大鼠酒精偏好作用与NO/cGMP信号通路有关；主动饮酒降低了VTA GABA神经元K+-Cl-协同转运蛋白2(K+-Cl-cotransporter 2, KCC2)的表达，外源性KCC2激活剂可抑制主动饮，7,8-DHF通过增加VTA KCC2表达减轻饮酒行为，提示KCC2可能是BDNF-TrkB调节饮酒行为的分子机制；主动饮酒导致VTA GABA神经元形状缩小、超微结构改变、数量减少，学习记忆能力减退；海马α2肾上腺素能受体介导右美托咪定减轻酒精戒断后焦虑；主动饮酒4周抑制VTA LTPGABA，BDNF通过TrkB解救被抑制的LTPGABA可能是其调控饮酒行为的电生理学基础。结论：BDNF-TrkB诱导VTA抑制性突触长时程增强调节饮酒行为和戒断反应，机制与NO/cGMP及KCC2信号分子有关。研究结果为酒精依赖临床防治提供了新思路。