应用在位活体实时共聚焦成像研究斑马鱼毛特纳运动神经与少突胶质细胞发育的相互作用

The myelination of axon is critical to the morphological structure and function of central nervous system. However, many questions such as the dynamic process of myelin development and related mechanisms remain unclear. Especially the interaction of cell behavior during myelination in vivo between neurons and oligodendrocytes is poorly understood. In order to examine the effect of axonal electrical activities on myelination in central nervous system, we propose to use zebrafish larva as a model, which is myelin and neuronal axon co-labeled, to observe dynamic changes of oligodendrocytes and axons in the development of myelination. Meanwhile, we are prepared to modulate axonal electrical activity, by means of pharmacology or optogenetics tools, and explore how axonal electrical activity may affect early development of myelination in zebrafish larva. As labeling specific cells by single-cell electroporation, we aim to observe the distribution, motility and dynamics of mitochondria in neuronal axon and/or oligodendrocytes in myelination under normal development condition or altering axonal electrical activity. In this proposal, we aim to observe development of myelination from the perspectives of oligodendrocytes, neuronal axons and mitochondria. Thus, it will provide us a new insight for in vivo imaging study of myelination in central nervous system, the dynamic processes and its underlying mechanisms.

髓鞘对于中枢神经系统的形态结构形成和功能完备至关重要。但目前对髓鞘发育的动态过程及具体机制不甚了解，尤其是对神经元与少突胶质细胞之间的交互作用在髓鞘化行为中的作用更是知之甚少。为了在位活体验证神经元活动性对于中枢神经系统髓鞘化行为的影响，申请人拟以斑马鱼为模型，构建髓鞘和神经元轴突双重标记的髓鞘化模型，以观察髓鞘化过程中少突胶质细胞和轴突的动态变化。并且通过药理学或光遗传的手段对神经元的活动性进行调控，探究神经元活动性对斑马鱼髓鞘形成的影响。进一步利用单细胞电转技术标记并观察神经元或少突胶质细胞中的线粒体的活动，探究其在髓鞘发育过程及神经元活动性影响髓鞘化过程中发挥的作用。该项目从少突胶质细胞、神经元轴突和亚细胞多角度观察髓鞘化发育过程，将有助于完善中枢神经系统髓鞘化的成像研究，进一步揭示髓鞘化的动态过程及相关机制。

通过药理学或者光遗传手段干预， 探究神经元活动性对斑马鱼髓鞘形成的影响。利用单细胞电转技术标记神经元及少突胶质细胞亚细胞器线粒体并对其动态行为进行观察，探究线粒体在髓鞘化过程及神经元活动性影响髓鞘化过程中发挥的作用，丰富斑马鱼中枢神经系统的髓鞘化动态发育过程的理论知识。.实现了药理学和光遗传学手段干预，神经元活动性增强能够增加单个少突胶质细胞髓鞘包裹的形成，而这一过程中，我们发现环化腺嘌呤核苷酸信号调控神经钙粘蛋白堆积的过程，从而影响到了毛特纳神经元轴突发生髓鞘包裹的过程；接下来我们成功利用斑马鱼幼鱼毛特纳神经元构建了一种新的非侵入式活体成像模型来研究线粒体的动态运动，并据此发现神经元活动性增强也会导致线粒体运动性的提高，同时神经元轴突再生能力也会随之提高，确定了线粒体在神经元活动性影响髓鞘化过程中发挥的作用；最后我们又建立了低氧诱导斑马鱼幼鱼PVL的疾病模型，低氧作用下线粒体的运动性较低，而我们发现低氧可以显著抑制bmp2b信号通路，从而影响少突胶质细胞前体细胞的迁移，最终影响了髓鞘形成的过程。综上所述，项目中的发现，有效地丰富了斑马鱼中枢神经系统的髓鞘化动态发育过程的理论知识。因此，本项目在实际执行过程中，很好地完成了项目制定的研究目标。