Confocal laser scanning microscopy of hamster cerebellum using FM4-64 as intracellular staining

The FM4-64, a member of the family of fluorescent dyes, has been applied to the cerebellar cortex to evaluate its properties as an intracellular stain and intracortical tracer. Slabs of hamster cerebellum 1-2 mm thick, were i ncubated in 10, 30, and 100 mu m solutions of FM4-64, in sodium phosphate b uffer and observed in a slow scan confocal laser scanning micro scope. Moss y and climbing fibers were traced in the cerebellar white and gray substanc es. They exhibited a high fluorescence signal at the level of the myelin sh eath. Mossy fibers were identified in the granular layer by their typical r osette formation and dichotomous bifurcation pattern. Climbing fil,er bundl es were observed crossing the granular layer and giving collateral branches around Golgi cell bodies. They ascend to the Purkinje cell layer on their way to the molecular layer. Cerebellar macroneurons (Golgi and Purkinje cel l!;) and microneurons (granule, basket, and stellate cells) showed optimal intracellular staining of cell soma, axonal, and dendritic processes. The z -series of stacks of optodigital sections allowed us to explore in depth th e cyroarchitectonic arrangement, nerve and glial cell morphology, and the t opographic relationship with the afferent fibers.