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

描述（由申请人提供）：神经胶质细胞在脑中的数量大大超过神经元，并且在发育、神经传递的调节、健康和疾病中具有积极作用。然而，与神经元相比，对神经胶质细胞的了解相对较少。我们对神经胶质细胞的了解主要来自于对分离细胞或活脑切片的研究。然而，这些方法还没有阐明诸如活脑中星形胶质细胞和神经元之间的细微动力学等现象。多光子荧光显微镜可以促进活的完整小鼠大脑（体内）的研究，但只有当细胞低于大脑表面小于0.5 mm时。因此，大多数神经胶质细胞不能在体内观察到。细光纤已经被植入小鼠大脑，可以到达很深的地方，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，它们不能分辨神经胶质细胞的精细膜过程。这将是研究正常发育和衰老，或药物使用，神经变性或损伤的影响的有用信息。最近，内窥镜已经小型化，用于小鼠大脑的体内研究。高分辨率版本植入玻璃鞘中，当与多光子显微镜一起使用时，可以解析精细的细胞过程。然而，它的1800微米直径明显比光纤宽，光纤的总直径为300微米。因此，它取代的脑组织比纤维多25倍，并且在植入之前需要切除脑组织。因此，这些具有有限的应用，并且不应该植入大脑非常深。该项目将开发一种可植入的、直径为350 μ m的透镜，用于多光子荧光显微镜，该透镜像光纤一样薄，具有高分辨率，可观察体内精细的细胞过程。它将具有光纤和微型内窥镜的最佳属性，而没有它们的缺点。为了证明它的实用性，一个长型的透镜将被植入足够深的地方，以便在三个月的时间内观察体内的成人神经胶质细胞。这将为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估计迁移率。在另一项测试中，植入物中将加入一个用于注射钙敏感染料的小孔。将在稍后的时间点注射染料以观察 神经胶质细胞内钙的释放。钙释放是胶质细胞功能的一种量度，并可能为它们调节神经元功能提供重要线索。由于荧光标记工具的扩展，该工具预计将具有许多其他用途，包括在小鼠中启动子指导的荧光蛋白表达，可以标记神经胶质细胞亚群，以及在数小时，数天或数月内对同一大脑区域进行成像的能力。 公共卫生相关性：该项目将为研究人员创造一种工具，以发现大脑中的神经胶质细胞如何发挥作用，以及它们如何参与衰老和疾病，如阿尔茨海默氏症和帕金森氏症。一个直径像针一样的小玻璃透镜将被植入实验室老鼠的大脑中，老鼠的神经胶质细胞中有荧光染料（或蛋白质）。使用显微镜观察透镜，研究人员将能够通过照射荧光染料来记录细胞的数量和形状，并确定衰老或某些疾病是否有重大变化，以及潜在的治疗是否使它们恢复正常状态。

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.