THE CREEPER STAGE IN CEREBELLAR CLIMBING FIBER SYNAPTOGENESIS PRECEDES THE PERICELLULAR NEST - ULTRASTRUCTURAL EVIDENCE WITH PARVALBUMIN IMMUNOCYTOCHEMISTRY

In perinatal rats, neurons in the dorsal cap of the inferior olivary complex transiently express parvalbumin-immunoreactivity (PA-IR). Their terminal axenic fields, particularly in the flocculonodular lobe, appear as precise bands of fine and convoluted immunostained fibers extending over the Purkinje plate and the nascent molecular layer. Thicker PA-IR fibers, corresponding to vestibular fibers, are observed only under the Purkinje plate. Electron microscopic analysis of the PA-IR climbing fibers within the bands allowed us to study their synaptogenesis with Purkinje cells. At birth (PO), thin PA-IR climbing fiber axons creep over these immature neurons following the contours of their perikarya and dendrites. They establish a few synaptic contacts, some of them of mature appearance, upon the smooth surface of Purkinje cell apical dendrites. PA-IR axonal growth cones are observed in the upper portion of the molecular layer. This precocious stage of climbing fiber/Purkinje cell synaptogenesis has been named here the 'creeper' stage. After the regression of the Purkinje cell apical dendrites (by P5), PA-IR climbing fibers are perisomatically located and synapse on Purkinje cell somatic protrusions, forming the classical pericellular 'nests'. The presence of mature synapses at PO indicates the precocity of climbing fiber/Purkinje cell synaptogenesis and suggests its fetal onset. Therefore, this process of synaptogenesis occurs in two steps: (i) an early transient one, simultaneous with the initiation of the formation of the olivocerebellar map, that could be involved in the maintenance of the nascent topography of the projection and (ii) a latter step which concerns the refinement of the projection within a given PA-IR band through the regression of multiple innervation of Purkinje cells by climbing fibers, step which brings the required synaptic specificity to the adult olivocerebellar system.