外侧缰核谷氨酸和γ-氨基丁酸递质信号介导酒精成瘾的作用及其分子机制研究

Alcohol (Ethanol) is addictive. The lateral habenula (LHb) has significant influence on other brain areas like the ventral tegmental area that are involved in rewarding and reinforcing actions of alcohol. Several studies have shown that habenula plays a critical role in abuse drugs such as cocaine and nicotine, however, the role of LHb in alcohol abuse and alcoholism remains unknown. Using whole-cell recordings on LHb neurons in acute brain slices of rats, we found that acute alcohol robustly increased the spontaneous firing rate of DA neurons and depolarized the membrane potential. We further found that alcohol increased the frequency without affecting the amplitude of spontaneous EPSCs, which was not altered in tetrodotoxin. In addition, alcohol increased the amplitude of eEPSCs evoked by the first but not the second stimulus of the paired pulse and reduced their paired-pulse ratio. Interestingly, GABAergic sIPSC frequency was increased by acute alcohol, too. Chronic alcohol administration altered the glutamatergic transmission and the long-term-potentiation (LTP) induction at the glutamatergic synapses in the LHb, which was mediated by PKA signaling pathway. These results show that both acute and chronic administration of alcohol can alter glutamatergic transmission and neuronal activity in the LHb. Based on these findings, we hypothesize that LHb is an integral part of brain circuits mediating alcohol abuse and alcoholism. Using patch-clamp, molecular biological and behavioral approaches in this project, we will investigate: (1) the influence of acute alcohol exposure on LHb glutamatergic transmissions, as well as GABAergic synaptic transmissions on neuronal activity; (2) the influence and the relating molecular mechanisms of chronic alcohol exposure on LHb neuronal activity and glutamatergic transmissions, glutamatergic synaptic plasticity, as well as GABAergic synaptic transmissions; (3) the effects of the cellular and molecular interference of LHb glutematergic and GABAergic signaling and CREB in the neural circuits and the behavioral responses to alcohol exposure. The results will provide the theoretical basis and tactics for prevention and treatment of alcohol abuse and alcoholism.

外侧缰核(LHb)为中脑腹侧被盖区(VTA)提供奖赏相关整合信号，被认为与可卡因、尼古丁等成瘾发生有关，但在酒精成瘾中的作用迄今未知。基于急、慢性酒精暴露能直接增强LHb神经元放电，影响兴奋性谷氨酸和抑制性GABA能突触传递及可塑性的前期研究结果，本课题将探讨LHb是否参与酒精成瘾过程及其调控机制。采用膜片钳、分子生物学和行为学等方法研究1）急性酒精暴露改变LHb谷氨酸能和GABA能突触传递对神经元兴奋活动的影响；2）慢性酒精暴露后谷氨酸和GABA能突触传递和可塑性的适应性变化对神经元兴奋活动的影响，是否由cAMP-PKA、ERK-CREB等活性改变介导；3）干扰LHb谷氨酸、GABA能突触传递及用病毒介导基因转染技术改变神经元CREB活性能否影响神经环路自平衡而改变下游VTA神经元的兴奋活动？纠正动物对酒精暴露的行为反应？本课题将为阐明LHb在酒精成瘾中的作用提供理论依据和临床干预靶标。

酒精在现代社会的消费急剧增加，我国酒精滥用已是非常严重的公共卫生问题，对酒精滥用和成瘾的神经机制研究亟待进行。外侧缰核(LHb)为中脑腹侧被盖区(VTA)提供奖赏相关整合信号，被认为与可卡因、尼古丁等成瘾发生有关，但在酒精成瘾中的作用迄今未知。基于本实验室发现急、慢性酒精暴露能直接增强LHb神经元放电，影响兴奋性谷氨酸能突触传递及可塑性的前期研究结果，提示：酒精可能通过影响谷氨酸等神经递质的突触传递，调节LHb神经元、和/或者进而调节下游VTA多巴胺神经元的兴奋活动，在酒精成瘾中起作用。本研究实验综合采用脑片技术、膜片钳、分子生物学和动物行为学等研究方法，主要分为急性和慢性动物实验两大部分，在基因、蛋白、细胞膜受体、和神经元突触和整体行为水平进行观察，实验明确了（1）酒精通过增强外侧缰核（LHb）谷氨酸能递质信号在突触前后神经元之间的传递，进而介导了酒精增强LHb神经元的电活动作用；并且酒精对谷氨酸能神经递质信号系统的改变发生在突触前递质释放和突触后的受体位点；（2）酒精暴露引起LHb核团谷氨酸递质信号改变的分子机制涉及神经元内的激酶系统MAPK ERK以及cAMP-PKA信号通路，其磷酸化状态的变化与酒精对LHb核团功能影响有关；但CaMKⅡα和CaMKⅡβ激酶系统改变不明显；（3）阻断多巴胺能信号通路（D1受体）来干扰LHb核团功能，能直接改变LHb核团及其下游脑区VTA神经元对酒精的反应以及动物的整体行为变化；（4）酒精在LHb核团的效应和5-HT能系统相关性，本研究主要确定了5-HT3受体信号系统，能部分参与介导酒精在LHb核团的作用；（5）膜片钳实验证据表明，酒精在LHb核团的效应，不仅发生在兴奋性突触传递位点，并且对抑制性突触传递过程亦有影响，表现为酒精灌流大鼠脑片，可见LHb脑区神经元的自发性抑制性突触后电流（sIPSCs）发生了改变,酒精对抑制性GABA能突触传递的急性影响，主要表现为对GABA递质的释放有促进作用（加快了sIPSC的频率），而对其突触后GABAA受体通道的本身可能没有直接作用（sIPSC的幅度未见改变），但这种影响表现下所涉及的机制以及如何介导酒精的系统性效应，有待进一步深入。本研究为阐明LHb在酒精成瘾中的作用提供了理论依据和可能的临床干预靶标。