Synaptic localization of growth-associated protein 43 in cultured hippocampal neurons during synaptogenesis

Abstract

Growth-associated protein 43 (GAP-43), a novel axonal phosphoprotein, is originally identified as a growth-cone-specific protein of developing neurons in vitro. The expression of GAP-43 is also shown to be up-regulated concomitant with increased synaptic plasticity in the brains in vivo, but how GAP-43 is concerned with synaptic plasticity is not well understood. In the present study, therefore, we aimed to elucidate subcellular localization of GAP-43 as culture development of rat hippocampal neurons. Western blotting showed that the expression of GAP-43 in the cerebral and hippocampal tissues was prominently high at postnatal days 14 and 21 or the active period of synaptogenesis. Double-labelling immunohistochemistry with an axonal marker Tau revealed that the immunoreactivity of GAP-43 was seen throughout axons of cultured hippocampal neurons but stronger at axonal puncta of developing neurons than axonal processes. Double-labelling immunohistochemistry with presynaptic terminal markers of synapsin and synaptotagmin revealed that the immunoreactivity of GAP-43 was observed mostly at weak synapsin- and synaptotagmin-positive puncta rather than strong ones. The quantitative analysis of immunofluorescent intensity showed a clear inverse correlation between GAP-43 and either synapsin or synaptotagmin expression. These data indicate that GAP-43 is highly expressed at immature growing axonal terminals and its expression is decreased along with the maturation of synaptogenesis. © 2012 John Wiley & Sons, Ltd.