Developmental changes in B‐50 (GAP‐43) in primary cultures of cerebral cortex: Content and phosphorylation of B‐50

The content and phosphorylation of the neuronal growth‐associated protein B‐50 (GAP‐43) were studied in cultured neocortex as a function of normal development and development in the presence of tetrodotoxin (TTX), a blocker of bioelectric activity (BEA). The observations were correlated with previous morphological findings on neurite outgrowth and B‐50 immunolocalization in the same cultures. In control cultures, the concentration of B‐50 reached a maximum at 7 days in vitro (DIV) and decreased thereafter, whereas the concentration of neuron specific enolase (NSE), which was used as a neuronal reference marker, rose till 28 DIV and leveled off towards 42 DIV. The degree of basal phosphorylation of B‐50 (relative to that of total protein) decreased after the first week in vitro. Stimulation of B‐50 phosphorylation by phorbol ester also decreased with age in vitro, indicating that changes in B‐50 phosphorylation were mainly due to changes in protein kinase C (PKC) activity. The chronic presence of TTX led to a reduced content of B‐50 and NSE after 14 DIV. The basal phosphorylation of B‐50 was neither affected by acute nor chronic TTX treatment. However, upon stimulation of PKC with phorbol esters, some alterations of B‐50 phosphorylation were revealed in cultures grown in TTX. These biochemical observations are in line with the absence of effects of TTX on neurite outgrowth during the first 2 weeks in culture, and later effects of TTX on neuronal survival. The developmental changes in B‐50 concentration and phosphorylation largely correlate with previous morphological observations on axonal outgrowth and growth cone shape in the same cultures. We suggest that B‐50 phosphorylation plays an important role in transducing extracellular signals into directed neurite outgrowth.