Cellular mechanisms of cortical myelin plasticity and regeneration in vivo

描述(申请人提供)：髓鞘是成熟神经网络的基本组成部分，在中枢神经系统(CNS)的大量病理条件下受到影响。更好地在体内了解少突胶质细胞及其各自的髓鞘如何发育、在整个生命周期中保持并对损伤做出反应，是推进对这些情况的了解的关键。越来越多的证据表明，神经元活动和适当的星形胶质细胞功能可能在这些过程中发挥关键作用。我们已经开发了一种新的技术，可以在体内进行高分辨率的无标记有髓轴突成像。这项技术利用了富含脂质的多层髓鞘的高折射率，并基于光谱共聚焦反射(SCORE)显微镜。利用SCORE成像技术，我第一次获得了活体动物皮质髓鞘在细胞尺度上的长期动态图像。初步数据显示，这项技术以及对少突胶质细胞、轴突和星形胶质细胞的荧光成像是一套强大的工具，它们结合在一起提供了关于这些结构在体内的精细结构动力学的丰富信息。除了记录单个少突胶质细胞和星形胶质细胞消融后脱髓鞘的时间动力学外，我还证明了追踪节间长度长期变化的可行性。我建议使用这些强大的技术来解决关于髓鞘和少突胶质细胞的体内可塑性和再生的三个基本问题。首先，我将确定小鼠皮质中髓鞘和少突胶质细胞的长期可塑性。接下来，我将确定神经元活动对髓鞘形成、可塑性和少突胶质细胞的影响。 再生。最后，我将使用单细胞消融技术来确定是否需要星形胶质细胞或改变体内少突胶质细胞重新髓鞘形成的时间动力学。这些实验将首次在细胞尺度上描述少突胶质细胞和髓鞘的纵向动态和再生能力。此外，这些实验将揭示在活着的大脑中，轴突和星形胶质细胞的变化如何在几周到几个月的时间里影响这些动力学。

DESCRIPTION (provided by applicant): Myelin is a fundamental component of mature neural networks that is affected in a large number of pathological conditions of the central nervous system (CNS). Critical for advancing knowledge about these conditions would be a better in vivo understanding of how oligodendrocytes and their respective myelin sheaths develop, are maintained throughout life and respond to injury. Growing evidence suggests neuronal activity and proper astrocyte function may play crucial roles in these processes. We have developed a new technique that allows high resolution label-free in vivo imaging of myelinated axons. This technique takes advantage of the high refractive index of lipid rich multilayered myelin and is based on spectral confocal reflectance (SCoRe) microscopy. Using SCoRe imaging I have obtained for the first time long-term images of the dynamics of cortical myelin on the cellular scale in a living animal. Preliminary data shows this technique as well as fluorescence imaging of oligodendrocytes, axons and astrocytes are a powerful set of tools that in combination provide a wealth of information about fine structural dynamics of these structures in vivo. I demonstrate the feasibility to track long- term changes in internode length in addition to documenting the temporal dynamics of demyelination after single oligodendrocyte and astrocyte ablation. I propose to use these powerful techniques to address three fundamental questions concerning the in vivo plasticity and regeneration of myelin and oligodendrocytes. First, I will determine the long term plasticity of myelin and oligodendrocytes in the mouse cortex. Next I will determine the effects of neuronal activity on myelin formation, plasticity and oligodendrocyte regeneration. Finally I will use single cell ablation techniques to determine if astrocytes are required for or alter the temporal dynamics of oligodendrocyte remyelination in vivo. Together these experiments will describe for the first time the longitudinal dynamics and regeneration capabilities of oligodendrocytes and myelin on the cellular scale. Furthermore these experiments will reveal how changes in axons and astrocytes influence these dynamics over weeks to months in the living brain.