IDENTIFICATION OF PHAGOCYTIC GLIAL-CELLS AFTER LESION-INDUCED ANTEROGRADE DEGENERATION USING DOUBLE-FLUORESCENCE LABELING - COMBINATION OF AXONAL TRACING AND LECTIN OR IMMUNOSTAINING

Retrograde and anterograde degeneration have been reported to be suffi cient stimuli to activate glial cells, which, in turn, are involved in phagocytosis of degenerating material. Here we describe a double-fluo rescence technique which allows for direct and simultaneous visualizat ion of both labeled incorporated axonal debris and incorporating glial cells in the course of anterograde degeneration. Stereotaxic applicat ion of small crystals of biotinylated and tetramethylrhodamine (TRITC) -conjugated dextran amine Mini Ruby into the medial entorhinal cortex resulted in a stable rhodamine fluorescence confined to fibers and ter minals in the middle molecular layer of the dentate gyrus, the stratum lacunosum-moleculare, and the crossed temporo-hippocampal pathway. Su bsequent stereotaxic lesion of the entorhinal cortex induced transform ation of rhodamine-fluorescent fibers and terminals into small granule s. Incorporation of these granules by microglial cells [labeled by flu orescein isothiocyanate (FITC)-coupled Bandeiraea simplicifolia isolec tin B-4] or astrocytes (labeled by FITC-coupled glial fibrillary acidi c protein antibodies) resulted in phagocytosis-dependent labeling of t hese non-neuronal cells, which could be identified by double-fluoresce nce microscopy. Electron microscopical analysis revealed that, followi ng lesion, the tracer remained confined to entorhinal axons which were found to be incorporated by glial cells. Our data show that TRITC- an d biotin-conjugated dextran amines are versatile tracers leading to Ph aseolus vulgaris leucoagglutinin-like axonal staining. Lesion-induced phagocytosis of anterogradely degenerating axons by immunocytochemical ly identified glial cells can be directly observed by this technique o n the light and electron microscopical levels.