Long Term 3D Imaging of Mouse Brain In Vivo to Study Glial Cells and Gliogenesis

描述（申请人提供）：神经胶质细胞在大脑中数量远远超过神经元，在发育、神经传递调节、健康和疾病中发挥积极作用。然而，与神经元相比，人们对神经胶质细胞的了解相对较少。对神经胶质细胞的了解主要来源于对游离细胞或活脑切片的研究。然而，这些方法还没有阐明一些现象，比如活体大脑中星形胶质细胞和神经元之间细微的动态变化。多光子荧光显微镜可以促进对活的、完整的小鼠大脑（体内）的研究，但只有当细胞在脑表面以下小于~0.5 mm时。因此，大多数胶质细胞不能在体内观察到。细光纤已被植入小鼠大脑，可以深入到很远的地方，在不破坏太多脑组织的情况下观察荧光标记的细胞体。然而，当它们与神经元相互作用时，它们不能分解胶质细胞的精细膜过程。这对于研究正常发育和衰老、药物使用、神经变性或损伤的影响将是有用的信息。最近，内窥镜已经小型化，用于小鼠大脑的体内研究。高分辨率的版本被植入玻璃鞘，当与多光子显微镜一起使用时，可以分辨精细的细胞过程。然而，它的直径为1800微米，明显比光纤宽，光纤的总直径约为300微米。因此，它取代的脑组织比纤维多25倍以上，并且需要在植入前切除脑组织。因此，这些应用有限，不应该植入大脑的非常深的地方。该项目将开发一种可植入的直径为350微米的透镜，用于多光子荧光显微镜，它像光纤一样薄，具有高分辨率，可以观察体内精细的细胞过程。它将具有光纤和小型化内窥镜的最佳特性，而没有它们的缺点。为了证明它的实用性，一个长版本的晶状体将被植入足够深的地方，在三个月的时间里观察成人出生的神经胶质细胞。这将对目前使用大脑切片进行短期研究的方法提供重大改进。切片研究的观察结果高度依赖于技术，并产生了相互矛盾的迁移率估计。在对其能力的另一项测试中，一个用于注射钙敏感染料的小端口将与植入物结合在一起。染料将在稍后的时间点注射以观察

DESCRIPTION (provided by applicant): Glial cells greatly outnumber neurons in the brain and have active roles in development, modulation of neurotransmission, health and disease. Yet, relatively little is known about glial cells compared to neurons. What is known about glial cells i derived largely from studies of dissociated cells or live brain slices. Yet these methods have not elucidated phenomena such as the nuanced dynamics between astrocytes and neurons in the living brain. Multiphoton fluorescence microscopy can facilitate studies in the living, intact mouse brain (in vivo), but only when the cells are less than ~0.5-mm below the brain surface. Thus, most glial cells cannot be observed in vivo. Thin optical fibers have been implanted in the mouse brain that can reach great depths to visualize fluorescently labeled cell bodies without disrupting much brain tissue. However, they cannot resolve fine membrane processes of glial cells as they interact with neurons. This would be useful information to study normal development and aging, or the effects of drug use, neurodegeneration, or injury. More recently, endoscopes have been miniaturized for in vivo studies in mouse brain. The high-resolution version is implanted in a glass sheath and can resolve fine cellular processes when used with a multiphoton microscope. Yet, it's 1800-um diameter is markedly wider than fiber optics, which are on the order of 300-um in overall diameter. Therefore, it displaces over 25 times more brain tissue than fibers and requires brain tissue removal prior to implantation. Thus, these have limited applications and should not be implanted very deep into the brain. This project will develop an implantable, 350-um diameter lens to use with multiphoton fluorescence microscopy that is thin, like an optical fiber, and has high resolution to observe fine cellular processes in vivo. It will have the best attributes of optical fibers and miniaturized endoscopes without their drawbacks. To demonstrate its utility, a long version of the lens will be implanted deeply enough to observe adult-born glial cells in vivo over a period of three months. This will offer a major improvement over the current method of using brain slices for short-term studies. The slice study observations are highly dependent on technique and have produced conflicting estimates of migration rates. In another test of its ability, a small port for injection of a calcium-sensitie dye will be incorporated with the implant. The dye will be injected at later time points to observe the release of calcium inside glial cells. Calcium release is one measure of glial function and may provide important clues to their modulation of neuron function. This tool is expected to have numerous other uses because of the expansion of fluorescent labeling tools, including promoter-directed expression of fluorescent proteins in mice that could label subpopulations of glial cells, and the ability to image the same brain region over hours, days or months. PUBLIC HEALTH RELEVANCE: This project will create a tool for researchers to discover how glial cells in the brain function and how they are involved in aging and disorders, such as Alzheimer's and Parkinson's diseases. A tiny glass lens with needle-like diameter will be implanted in the brain of laboratory mice that have a fluorescent dye (or protein) in their glial cells. Using a microscope to look into the lens, researchers will be able to record the numbers and shapes of the cells by illuminating the fluorescent dye and determine if there are major changes in aging or certain diseases, and if potential treatments return them to a normal state.