B-50/GAP43 LOCALIZATION IN POLARIZED HIPPOCAMPAL-NEURONS INVITRO - AN ULTRASTRUCTURAL QUANTITATIVE STUDY

Hippocampal pyramidal neurons cultured in vitro gradually develop morphologically and biochemically distinct axons and dendrites, resulting in functional neuronal polarization [Dotti C. G. et al. (1988) J. Neurosci. 8, 1454-1468]. We have studied the distribution of the growth-associated protein B-50 in hippocampal neurons of the rat at stage 3 of development by means of light and electron microscopic immunocytochemistry. Hippocampal neurons grown for two to three days in vitro were aldehyde fixed and immunolabelled using polyclonal rabbit antibodies to B-50 and goat anti-rabbit immunoglobulins tagged with 1 nm gold particles. In order to permit visualization by both light and electron microscopy, the gold probes were silver intensified. Light microscopy demonstrated the absence of B-50 immunostaining in living neurons and the presence after permeabilization by fixation and subsequent treatment of the neurons with sodium borohydride, indicating that B-50 is located intracellularly. Both immunofluorescence and immunogold-silver labelling revealed that B-50 immunoreactivity outlined all neurites of the morphologically polarized neurons. For quantitative electron microscopy, six morphologically polarized neurons (developmental stage 3) were carefully selected from immunolabelled Epon-embedded neurons and processed completely to ultrathin sections. In this way the ultrastructural localization of B-50 has been studied in the cell body, the neurites and their growth cones. For each sectioned neuron, the relative distribution of the gold-silver deposits (representing B-50) over the plasma membrane of various cellular compartments was quantitated. B-50 is located at the plasma membrane of the neuronal cell body and all neurites including their growth cones. The density of B-50 on the plasma membrane of growth cones is not different from that of the neuritic shaft. In addition, B-50 is present on the cytosolic side of the membrane of small electron-lucent vesicles (average diameter 102.7 +/- 2.5 nm) resembling transport vesicles. These vesicles are present in the cell body and the neurites. A two-fold concentration is found in the central region of the growth cones, suggesting a role of these vesicles in axonal transport, membrane insertion and (or) recycling. Since, at the onset of neuronal polarization, B-50 is present at the plasma membrane in all compartments of the hippocampal neuron, we suggest that at this stage of development B-50 does not participate directly in the processes leading to morphological polarization. The findings of a lack of B-50 enrichment on the plasma membrane of growth cones and the presence of B-50 on putative transport vesicles may indicate that B-50 plays a general role, not selective for the growth cone, at the neuronal plasma membrane during neuritogenesis.