原位活体水平单细胞与神经环路分析的微流控芯片新方法研究

It is currently a great challenge for in vivo single cell analysis on living awake animals, which holds a promise to reveal the function of the single target cell and molecular mechanism of life in real-world micro-environment. In this project, we proposed a novel approach for analyzing single cell and neural circuit in vivo on living C. elegans based on microfluidic chip. To achieve this goal, an integrated microfluidic platform will be constructed for realizing living C. elegans mobilization, gas stimulation and fluorescent imaging. The interest will be focused on dynamic changes of Ca(II) ion signal in single sensory neuron upon the olfactory stimulation, and quantitative relationship between the stimuli and the calcium signal intensity and their tempo-spatial distributions, and the mechanism of the adaptation and the plasticity as well. In the further work, we will attempt to investigate the neural circuit structure how the olfactory signal transits from the sensory neuron via the interneuron to the action neuron, coupling with on-chip femto-second laser nano-surgery technique and on-chip micro-injection dissection technique. The kinetics of calcium signal on the whole olfactory circuit will be discussed. It will pave a new way to discover the chemical foundation of molecular mechanism of olfactory, and shows significant importance to life analytical chemistry and neuroscience, thus to promote the development of analytical chemistry and other related areas.

清醒状态下动物的原位活体水平(in vivo)单细胞分析，最有可能接近真实地揭示单个细胞的功能和生命活动的分子机制，是单细胞分析领域又一重大挑战，由于缺乏分析手段，较少见诸文献报道。本项目提出原位活体单细胞与神经环路分析的微流控芯片新方法研究。以线虫的嗅觉为对象，建立基于微流控芯片的线虫固定、气味刺激和荧光成像的新手段，研究气味分子刺激下活体线虫头部单个感知神经元的钙信号的变化规律、气味浓度与钙信号强度和时空分布上的定量关系，以及适应性和可塑性等；进一步，采用飞秒激光烧蚀技术和显微注射技术，寻找神经信号从感知神经元传递到中间神经元再传递到动作神经元的神经环路，揭示钙信号在神经环路上的实时动力学过程，为研究嗅觉的化学基础和分子机制提供创新平台支撑。本项目对于生命分析化学、神经生物学等基础研究领域具有重要科学价值，并促进分析化学及其相关交叉学科的发展和人才培养。

单细胞分析主要集中在离体水平(in vitro)，近年来单细胞活体水平(ex in vivo)分析发展至为迅猛，而清醒状态下动物原位活体水平(in vivo)或在体水平的单细胞分析，最有可能接近真实地揭示单个细胞的功能和生命活动的分子机制，是单细胞分析领域又一重大挑战。本项目提出原位活体单细胞与神经环路分析的微流控芯片新方法研究，建立在体单细胞和神经环路研究的微流控芯片平台，并对非极性气体和极性刺激下嗅觉神经元钙信号单细胞分辨的响应动力学进行系统观察与分析，最后发展基于光遗传和光流控芯片相结合的神经环路分析新技术。该方法对于在单细胞水平分析神经元功能及神经环路动力学具有重要科学意义，在神经生物学等研究领域具有广泛的应用前景。发表项目资助标注的SCI学术论文21篇，其中IF>10的3篇，IF>5的16篇，IF>3的5篇，获得国家发明专利2项，培养博士3名。