ASTROCYTES AND MICROGLIAL CELLS INCORPORATE DEGENERATING FIBERS FOLLOWING ENTORHINAL LESION - A LIGHT, CONFOCAL, AND ELECTRON-MICROSCOPIC STUDY USING A PHAGOCYTOSIS-DEPENDENT LABELING TECHNIQUE

Entorhinal lesion leads to anterograde degeneration of perforant path fibers in their main termination zone in the outer molecular layers of the dentate gyrus. Concomitantly, astrocytes become hypertrophic, and microglial cells alter their phenotype, suggesting participation in a nterograde degeneration. This study analyzes the involvement of these lesion-induced activated glial cells in the process of phagocytosis of degenerated axonal debris. We established a phagocytosis-dependent la beling technique that allows for direct and simultaneous visualization of both labeled incorporated axonal debris and incorporating glial ce lls. Stereotaxic application of small crystals of the biotin-and rhoda mine-conjugated dextran amine Mini Ruby (MR) into the entorhinal corte x led to strong and stable axonal staining of perforant path axons. Fo llowing entorhinal lesion, labeled terminals and fibers condensed and formed small granules. Incorporation of these rhodamine-fluorescent gr anules resulted in a phagocytosis-dependent cell labeling. During the first 3 days, we were able to identify these cells as microglia by usi ng double-fluorescence and confocal microscopy. The first unequivocall y double-labeled astrocytes were found 6 days post lesion (dpl). Where as in all stages a subpopulation of microglial cells remained devoid o f MR-labeled granules, all astrocytes in the middle molecular layer we re double-labeled after long survival times (20 dpl). On the ultrastru ctural level, labeled granules appeared to be perforant path axons con taining the tracer. Both terminals and myelinated fibers could be seen inside the cytoplasm of microglial cells and astrocytes. Thus, antero grade degeneration is a sufficient stimulus to induce axon incorporati on by both astrocytes and a subpopulation of microglial cells. (C) 199 7 Wiley-Liss, Inc.