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 sufficient stimuli to activate glial cells, which, in turn, are involved in phagocytosis of degenerating material. Here we describe a double-fluorescence technique which allows for direct and simultaneous visualization of both labeled incorporated axonal debris and incorporating glial cells in the course of anterograde degeneration. Stereotaxic application 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 terminals in the middle molecular layer of the dentate gyrus, the stratum lacunosum-moleculare, and the crossed temporo-hippocampal pathway. Subsequent stereotaxic lesion of the entorhinal cortex induced transformation of rhodamine-fluorescent fibers and terminals into small granules. Incorporation of these granules by microglial cells [labeled by fluorescein isothiocyanate (FITC)-coupled Bandeiraea simplicifolia isolectin B-4] or astrocytes (labeled by FITC-coupled glial fibrillary acidic protein antibodies) resulted in phagocytosis-dependent labeling of these non-neuronal cells, which could be identified by double-fluorescence microscopy. Electron microscopical analysis revealed that, following lesion, the tracer remained confined to entorhinal axons which were found to be incorporated by glial cells. Our data show that TRITC- and biotin-conjugated dextran amines are versatile tracers leading to Phaseolus vulgaris leucoagglutinin-like axonal staining. Lesion-induced phagocytosis of anterogradely degenerating axons by immunocytochemically identified glial cells can be directly observed by this technique on the light and electron microscopical levels.