Studies On Structure And Function Of Glial Cells By Optical Imaging

The central nervous system is consisted of neuron and glial cell. By recording electronic signals, the neural activities could be described precisely. As a non-excitatory cell in neural system, however, glial cell is hardly touched their functions by electronic signal; that is why glial cells have not received neuroscientists'attentions. Moreover, several to ten times to neuron in number in central nervous system, glial cell have implied to exert more functions in neural system. At a last decade, by advances in optical imaging and labeling techniques, the functions of glial cell will have been understood.The invention of microscope brings the life science research to cellular level. High resolution, contrast and penetration are the hot topics in development of optical imaging techniques. Here, by using two-photon laser excitation scanning microscopy and total- internal reflection microscopy, the morphology and function of glial cell were investigated.In CA1 region of hippocampal slices, four types of glial cell were identified by electrophysiological characteristics and immunocytochemical staining: astrocytes, oligodendrocyte, NG2 (a chondroitin sulfate proteoglycan) positive cells and microglia. By pipette-injected dyes and two-photon laser scanning microscopy (2PLSM), their shapes were observed. Further, their functions were explored : For astrocyte, by two-photon induced uncaging calcium, the astrocytic calcium elevation induced the vasodilatation. For oligodendrocyte in CA1 region of hippocampal slices, by 3-dimensional imaging, their processes showed distinct distribution: about two third of all the processes (67.5±12.0%) are parallel to Sch fiber and the others were vertical to them. For NG2 glial cell, the distinct mitosis mode was observed: the cell body divides into two, the processes belong to the daughter cells according to their localization in cell body.Stimulated astrocyte by femtosecond pulse laser induced the intracellular calcium elevation and followed intercellular calcium wave. Under the different powers, astrocytes displayed two modes of calcium elevation: calcium spike (named S-type in following) and higher and sustained calcium elevation (named H-type in following). Some evidences, from PI penetration, membrane reversible shift under the high numerical aperture objective and extracellular calcium-dependent results, demonstrated that photoporation effects were causes to intracellular calcium elevation. Photoporation induced calcium elevation could spread among astrocytes; from pharmacological results, this calcium wave is mediated by ATP from astrocyte released and activated the P2Y receptor in adjacent astrocytes. Moreover, in mix culture of neuron and astrocyte, the H-type calcium elevation in astrocyte could induce neuronal synchronized calcium oscillation, whereas no any responses to S-type。In conclusion, the photoporation based on femtosecond pulsed laser could generate calcium elevation in astrocyte, and induce P2Y receptor-dependent calcium wave. The calcium waves in astrocytes lead the neuronal synchronized calcium oscillation.Investigate lysosome exocytosis revealed by TIRFM. By FM dye labeled lysosome and TIRF imaging, three types of release modes of lysosomes in astrocyte were showed: full exocytosis from old puncta mode, full exocytosis from new puncta mode and"kiss and run"mode. Physiological stimulation by ATP could induce partial exocytosis, whereas pathological stimulation by an ischemic insult (KCN) induced full exocytosis of lysosome.