Characterization of an axon inhibitor from immature target neurons

Axon inhibitors are essential developmental cues for axon guidance to target regions and interaction with appropriate target cells. Peripheral nervous system axons respond to growth inhibitors when avoiding inappropriate regions or contacting the proper target cell. Central nervous system axons also respond to inhibitors when avoiding inappropriate trajectories. However, the role of inhibitors in central afferent interactions with final targets is not clear.; Purified target cerebellar granule neurons inhibit extension of afferent pontine mossy fibers in coculture (Baird et al., 1992b). Inferior olivary and retinal explants grow long axons on purified granule neurons (Baird et al., 1992a). Work in this thesis shows that pontine neurons extend long axons on non-target neuron monolayers but short axons on living or fixed target granule neuron monolayers. Inhibition of pontine axon extension does not require target cell contact, as shown by gel coculture assays, and target cell plasma membranes do not inhibit afferent axons. Pons axons are inhibited in a choice assay by heat-sensitive granule cell factors extracted with high salt.; Purified early postnatal cerebellar granule cells incorporate bromodeoxyuridine and express a marker phenotype similar to external granular layer cells. More mature target granule cells, derived by immunopanning purified granule neurons or by aging granule neurons in culture, allow longer pontine axon extension in coculture than external granular layer cells. Pons axons branch extensively on granule cells aged in culture or on granule cell plasma membranes. Pontine mossy fiber inhibition in vitro reflects two different in vivo processes: afferent exclusion from immature targets of the external granular layer and extensive afferent branching among mature targets of the internal granular layer.; Salt extraction of granule cell cultures or cerebellar tissue yields an inhibitor of axon extension. Extracts of immature granule cells show higher specific activity than those from mature granule cells. Inhibitory activity is greatly enriched by sequential chromatography, including laminin affinity chromatography. Identification of the factor responsible for axon inhibition should elucidate mechanisms of afferent inhibition in coculture and afferent exclusion from immature target zones in vivo.