基底外侧杏仁核组蛋白乙酰化对海洛因成瘾记忆调控机制研究

The basolateral amygdala (BLA) is a crucial nucleus that regulating the heroin addiction and cue-associated memories. However, the regulatory effect of histone acetylation on heroin addiction memory has not yet been elucidated. The present study using a mouse model of heroin-induced condition place preference and conditioned rewarding to simulate the environmental and event cues, respectively. Obtain the correlative changes of synaptic plasticity in BLA neurons by using the transmission electron microscope. Screening the key histone acetylation/deacetylation enzymes, which play crucial roles in heroin addiction memory, by using RT-PCR, Western blot and immunofluorescence techniques. Then, use the gene over expression or shRNA methods to manipulate the activities of key enzymes during different stages of heroin addiction, and observe the correlative behavior changes, identify the regulatory effects of the key enzyme on addiction memory. Reveal the downstream pathway of the key enzyme by using the chromatin immunoprecipitation to detect the expression of memory related target genes. Use ACPC and MK-801 to activate or inhibit the NMDA receptor function, then detect the activation of NR1-CaMKII-CREB pathway and the key enzyme expression, which shall reveal the upstream regulatory mechanism of the key enzyme. The present study could not only clarify the molecular mechanisms of histone acetylation in BLA underlying heroin addiction memory, but also provide valuable experimental evidence and theoretical basis for the development of new treatment target in heroin addiction.

基底外侧杏仁核（BLA）是调控海洛因成瘾与线索记忆的关键核团，然而其神经元内组蛋白乙酰化修饰对成瘾记忆的调控作用却鲜有报道。本课题利用小鼠海洛因位置偏爱和条件奖赏模型分别模拟成瘾环境及事件线索，透射电镜获得BLA神经元突触可塑性证据；RT-PCR、Western blot、免疫荧光等技术筛选在海洛因成瘾记忆中发挥关键作用的组蛋白乙酰化修饰酶；进而利用过表达或shRNA干预关键酶活性，观察成瘾不同阶段行为改变，明确关键酶对海洛因成瘾记忆的调控作用；利用染色质免疫沉淀等技术检测关键酶调控的记忆相关靶基因，揭示关键酶的下游调控通路；利用ACPC、MK-801干预NMDA受体功能，检测NR1-CaMKII-CREB通路激活及关键酶表达，揭示关键组蛋白乙酰化修饰酶的上游调控机制。本课题不仅有助于阐明BLA中组蛋白乙酰化调控海洛因成瘾记忆的分子机制，也将为海洛因成瘾治疗新靶点提供实验证据与理论依据。

项目背景：基底外侧杏仁核（BLA）是调控海洛因成瘾与线索记忆的关键核团，然而其神经元内组蛋白乙酰化修饰对成瘾记忆的调控作用却鲜有报道。.研究内容：本课题利用小鼠海洛因位置偏爱和条件奖赏模型分别模拟成瘾环境及事件线索，透射电镜获得BLA神经元突触可塑性证据；RT-PCR、Western blot、免疫荧光等技术筛选在海洛因成瘾记忆中发挥关键作用的组蛋白乙酰化修饰酶；进而利用过表达或shRNA干预关键酶活性，观察成瘾不同阶段行为改变，明确关键酶对海洛因成瘾记忆的调控作用；利用染色质免疫沉淀等技术检测关键酶调控的记忆相关靶基因，揭示关键酶的下游调控通路。.结果及关键数据：BLA在关联性成瘾记忆的形成和巩固中起到关键作用，吗啡和通过加强BLA内新生突出的形成来强化成瘾记忆的强度；BLA不影响活动能力、工作记忆、识别记忆；吗啡加强了困难条件下行为应对和活动能力，但本身不引起BLA内神经元突触的形成；减少BLA内组蛋白去乙酰化酶SIRT1表达使吗啡诱导的小鼠关联性CS记忆的形成和消退均减慢；减少小鼠BLA内SIRT1表达使吗啡诱导的环境线索CPP关联记忆的形成与消退均减慢；减少小鼠BLA内SIRT1表达对小鼠的自主活动能力没有影响，可以增加小鼠的空间探索行为，改善吗啡长期戒断导致的工作记忆的损害；敲减BLA内SIRT1对吗啡学习记忆的影响可能是通过改善小鼠的焦虑样症状产生。此外，吗啡CPP的习得可增加D1R–ERK–CREB通路在vHip和mPFC的活性。而吗啡CPP的复发与D2R-Akt-GSK3在vHip和mPFC的活性有关。此外vHip–mPFC神经环路中的D1R–ERK–CREB和D2R–Akt–GSK3信号通路分别调控了吗啡CPP记忆的获得与再提取。阻断D1受体或D2受体信号可以缓解海马以来的空间参考记忆能力。这些结果表明，D1和D2信号通路分别参与了吗啡成瘾记忆的调控。.科学意义：本课题不仅有助于阐明BLA中组蛋白乙酰化调控海洛因成瘾记忆的分子机制，也将为阿片类成瘾治疗新靶点提供实验证据与理论依据。