杏仁核突触功能可塑性的CaMKIIβ调控及其抗抑郁作用研究

The amygdala has been documented as a key brain region in the depression-related fear circuitry and the alteration of its synaptic plasticity has become a critical access to diseases. Nevertheless, relatively fewer protein studies on the amygdala have been reported. Our previous quantitative proteomics of the amygdala found that the expression of postsynaptic protein CaMKIIβ in both stress-susceptible and stress-insusceptible rats was significantly changed with the opposite trend. As an important synaptic plasticity-related molecule, the regulation mechanism and role of CaMKIIβ still remain to be explored. Therefore, this project aims to address the key scientific issue on CaMKIIβ regulation over the amygdalar synaptic functional plasticity and its antidepressant role. After the establishment of animal models with chronic mild stress and antidepressant treatment, the expression level of CaMKIIβ in the basolateral and lateral nuclei and central nucleus of the amygdala that was associated with fear memory will be respectively detected. With protein targeting and broad-spectrum analysis, the molecule network of CaMKIIβ regulation over the amygdalar synaptic functional plasticity at the cellular and animal levels will be systematically constructed. Then, the patch-clamp technique will be employed to analyze the corresponding changes of LTP. Thereby, some new potential pathogenetic mechanism(s) of depression will be revealed. Further, by a combination of brain region targeted intervention strategy and chronic mild stress model, the CaMKIIβ regulation over the synaptic functional plasticity changes in the amygdala as a new antidepressant way will be consolidated from stress resistance, depression development and reversal.

杏仁核作为抑郁症相关恐惧环路的关键脑区，其突触可塑性改变已成疾病发生的重要途径，然相应蛋白质研究相对较少。前期杏仁核定量蛋白质组学发现应激敏感和应激抵抗大鼠突触后蛋白CaMKIIβ表达异常且趋势相反，作为重要的突触功能可塑性关联分子，所涉调控机制及作用尚需剖析。籍此，本项目拟围绕“CaMKIIβ如何调控杏仁核突触功能可塑性及其抗抑郁作用”这一关键科学问题，借助慢性温和应激及其药物治疗动物模型，待分检杏仁核基底外侧核、外侧核和中央核等恐惧记忆相关分区CaMKIIβ水平后，有机整合蛋白质靶向和广谱分析策略，从细胞和动物水平系统构建CaMKIIβ调节杏仁核突触功能可塑性的分子网络，同时利用膜片钳技术分析相应LTP变化，继而发现潜在的抑郁症发生新机制；进一步采用脑区靶向干预策略并结合慢性温和应激模式，从抵抗应激、引发抑郁和逆转抑郁等角度以期夯实CaMKIIβ调节杏仁核突触功能可塑性的抗抑郁新途径。

抑郁症作为一种常见的情绪障碍，可由各种原因引起，具有严重的危害性。已有研究表明，杏仁核突触可塑性改变已成疾病发生的重要途径，然相应蛋白质研究相对较少。由此，本项目首先采用慢性温和应激模式建立了抑郁大鼠模型，通过糖水偏好和强迫游泳实验得到了对照、应激敏感和不敏感三个组别；随后利用稳定同位素标记的相对和绝对定量（iTRAQ）技术结合二维液相色谱串联质谱对各组大鼠杏仁核组织的蛋白质组进行定量分析；通过生物信息学和免疫印迹分析，发现应激敏感大鼠杏仁核中异常的突触形态和功能可塑性分子机制。进一步选取了感兴趣蛋白CaMKIIβ并对其调控机制进行了解析，在体外培养神经元中稳定过表达和低表达CaMKIIβ后，使用基于iTRAQ标记的定量蛋白质组技术对CaMKIIβ所引起的蛋白质表达变化进行比较分析，并通过生物信息学和免疫印迹分析初步探讨了CaMKIIβ调节的分子机制，增进了我们对抑郁症杏仁核突触可塑性的理解。另外，我们还借助糖水偏好和高架十字迷宫实验对应激敏感大鼠进行抑郁/焦虑敏感亚组分析，并利用定量蛋白质组学和生物信息学技术发现了前额叶、下丘脑、垂体和纹状体等多个脑区中蛋白质表达变化，为疾病研究提供了潜在新靶点，该分子基础将有助于更好理解应激诱导的抑郁/焦虑和应激抵抗的病理生理机制的共性和特异性。本项目利用定量蛋白质组学技术发现的所有差异数据，将给抑郁症的诊治、抗抑郁药物的研发和临床使用提供新的理论依据。