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.
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.