Developmental changes in B-50 (GAP-43) in primary cultures of cerebral cortex: B-50 immunolocalization, axonal elongation rate and growth cone morphology

Changes in neurite outgrowth parameters and in the immunolocalization of the neuronal growth-associated protein B-50 (GAP-43) were studied in cultured neocortex as a function of development. In addition, we studied the effects of chronic blockade of bioelectric activity (BEA) with tetrodotoxin (TTX) on these parameters. Axonal outgrowth rate in control cultures reached a maximum at 8 days in vitro (DIV) and declined to a low level at 21 DIV. B-50 staining shifted from the perikaryon to the axons and growth cones during the first 3 DIV. In axons the intensity of B-50 staining increased towards the growth cone. Within growth cones, the central/basal region and filopodia were intensely stained, whereas lamellipodia showed only marginal staining. Growth cone size gradually decreased after 3 DIV, due to the successive loss of lamellipodia and filopodia, and became club-shaped during the second week, until by 21 DIV growth cones were completely lost, and axons started retracting and degenerated. In the central area of the cultures, growth cones also decreased in size with time, but became stabilized as presynaptic elements onto other neurons. Acute addition of TTX did not affect the outgrowth rate at 6 DIV. Chronic TTX treatment led to an earlier retraction and degeneration of axons than in control cultures and to a loss of B-50-stained cells and varicosities during the third week, but did not affect growth cone morphology or B-50 staining. The regressive phenomena are probably due to an increased neuronal cell death shown to occur after chronic TTX treatment. The developmental changes in axonal elongation rate and growth cone morphology may be related to developmental changes in the content and/or phosphorylation of B-50 (GAP-43, which are studied in the same cultures in the following paper (Ramakers et al. (1991) Int. J. Devl Neurosci. 9, 231-241].