We have studied the form and fine structure of developing afferent axons in postnatal mouse cerebellum, before and during the formation of synaptic connections. In slices of fresh brain, bundles of axons were injected with horseradish peroxidase (HRP), and individual axons were examined in the light and electron microscopes. At birth, before formation of cortical layers, axons with growing tips are rare in the peduncular tracts but instead ramify throughout the cerebellar anlage. All axons have similar structures; they branch infrequently and terminate in bud-like tips and/or small growth cones. Growth cones contain small and large vesicles in the flank and small vesicles in filopodia. Typical mossy and climbing fiber branching patterns and bouton shapes are recognizable after postnatal day (P) 5, even though fibers are still intermingled in a plexus beneath the newly formed Purkinje cell layer. Climbing fiber-like axon arbors are highly branched and covered with small foliate growing tips that contact Purkinje cells. Mossy fiber-like branches have large irregular expansions that give rise to long filopodia and resemble growth cones seen in vitro. The flanks of these growth cones contact granule cell dendrites and form glomeruli typical of mossy fibers, whereas the filopodia make primitive contacts or are associated with coated vesicles in adjacent profiles. A novel finding is the occurrence during the second postnatal week of many single axons that simultaneously have the morphology and synaptic connections of both climbing and mossy fibers. These "combination" axons have some branches that extend into the granule cell layer and others that enter the Purkinje cell layer, with the shape and synaptic connections of terminals on each branch type corresponding to the respective layer. Climbing fiber-like branches, including those on combination fibers, extend over several adjacent Purkinje cells. Combination fibers are rare in late postnatal or adult stages. These results suggest that long after arrival in the cerebellum, afferent axons have similar elementary forms and overlap in their projections. Mature axonal forms are not exhibited until cellular layers develop. During a limited period of postnatal maturation, some axons have dual morphologies and synaptic relations with appropriate and inappropriate partners. These aspects of cerebellar axonal development, particularly the transient exuberant branching onto two types of target cells, offer a valuable opportunity to examine, in developing cerebellum, the sorting out of afferents and the formation of specific synaptic connections.
