基于光遗传-微透析技术的神经网络病变研究新方法

As a new technique employed in research on the brain’s neural networks, optogenetics allows precise modulation of the neural networks of specific brain areas by means of genetics or virus-mediated exogenous photosensitive ion channel. The abnormalities of concentration and dynamics of neurotransmitters in brain usually leads to neural network lesions. Therefore, it is very important for study on neural network lesions and clinical medicine to sensitively, rapidly and accurately determine neurotransmitters. The method of off-line analysis of neurotransmitters in brain by light stimulus has been developed. Based on our previous work, this project aims to construct animals of optogenetics, synthesize the novel BODIPY fluorescence probe with carbonyl chloride as reactive groups, develop the new method of on-line detection of neurotransmitters combining optogenetics controlling, microdialysis in vivo sampling and novel fluorescent probe labeling. Thus, the method could provide high spatial and temporal resolutions for accurately studying neurotransmitters in specific neural networks of freely moving animals. Then, this project will achieve information on concentration and dynamics of neurotransmitters of animals in the disease model group and the control group upon disturbing specific neural network. This project also will benefit further research on neural network lesions and investigation on the pathogenesis of some neurological diseases. It will also lay a foundation for developing novel diagnosing technique for some neurological diseases.

光遗传是近年发展的研究大脑神经网络的新手段，它通过遗传学或病毒介导引入外源性光敏离子通道，精确地调控大脑局部神经网络。而神经网络发生病变与大脑内神经递质的浓度及其动力学异常直接相关。因此，灵敏、快速、准确分析神经递质对于神经网络病变及临床医学研究具有重要意义。申请者已建立了光刺激扰动下脑内神经递质离线分析方法，本项目拟在此基础上，构建光遗传学动物，合成以羰酰氯为功能基团的新型BODIPY类荧光探针，建立集光遗传学控制、微透析活体取样技术和荧光探针标记为一体的神经递质在线检测新方法，实现高时空精准地研究清醒自由活动动物大脑内特定神经网络的神经递质。通过光刺激扰动特异神经网络，考查相应脑区中神经递质的改变，研究神经疾病模型组与健康对照组动物体内神经递质的浓度及其动力学差异，进而深入研究神经网络病变，阐明相关神经疾病的发病机制，为发展临床诊断新方法提供重要参考。

神经网络发生病变与大脑内神经递质的浓度及其动力学异常直接相关。本项目聚焦新型BODIPY类荧光探针的合成，结合微透析活体取样技术和荧光探针标记为一体的神经递质在线检测新方法的研究。合成了以羰酰氯为功能基团的性能优良的荧光探针，研究了外界因素对其荧光性质的影响。建立了微透析活体取样-在线分析五种氨基酸类神经递质的分析方法，建立了高效液相色谱-电化学检测测定大鼠下丘脑微透析液中组胺的分析方法。以邻苯二甲醛为柱前衍生试剂，建立了高效液相色谱分离-电化学检测瓜氨酸和精氨酸两种氨基酸的分析方法，应用于定性分析南瓜，丝瓜和西瓜中精氨酸和瓜氨酸；也用于定量分析西瓜中精氨酸和瓜氨酸的含量。此外，考察了小鼠肝脏中存在的棕色脂肪中全反式视黄酸对于与改善肥胖症及葡萄糖耐受性的关系，为进一步探索神经网络系统与脂肪代谢对肥胖及糖尿病的关系奠定下良好的基础。已发表论文5篇，培养本科生6名，联合培养博士生2名。