Developmental changes in GAP-43 expression in primary cultures of rat cerebellar granule cells

Abstract

GAP-43 is a growth-associated protein that has been implicated in the developmental outgrowth of axons. We have examined the profile of GAP-43 levels in rat cerebellar granule cells during their development in vitro. During the first 1–2 days after plating, the majority of cells expressed neurites and after 8 days a complex neuronal network had developed. In situ hybridization studies showed that GAP-43 mRNA levels rapidly increased to peak at 1–2 days and gradually returned to initial values after 7–8 days. Analysis of GAP-43 protein levels followed a similar transient profile. Initially, granule cell perikarya and structures associated with neuritogenesis all displayed GAP-43 immunoreactivity. In older cultures, perikaryal labelling was lost after 10 days whilst process staining decreased more gradually. During the first 48 hours detailed analysis of GAP-43 mRNA revealed two populations of granule cells. It was suggested that cells with significant label originated from the external germinal layer which displays much GAP-43 mRNA in cerebellar sections. Cells with little or no GAP-43, however, probably originated from the internal granular layer since this region displayed no specific labelling. Granule cells within clumps expressed more GAP-43 mRNA compared to isolated cells perhaps indicating cell-cell regulation of expression. These results describe the transient rise in GAP-43 protein and mRNA levels expressed by developing cerebellar granule cell neurons in vitro and provide further evidence for the role GAP-43 plays during neuritogenesis.