B50/GAP43 is a neuron-specific phosphoprotein whose expression is associated with neural development and synaptic plasticity. Its postnatal ontogeny was investigated in the primary olfactory pathway of the rat using immunohistochemical methods. The unique ability of the olfactory neuroepithelium to generate new neurons from a population of precursor cells present in the basal cell layer of this tissue makes it a valuable model in the study of neural development. In newborn rats B50/GAP43 is present throughout the entire population of olfactory receptor neurons. These cells are stained throughout, from the ciliated dendritic knob to their axon terminals in the bulb. This appears to be the first example of unambiguous B50/GAP43 expression in dendritic processes. With increasing age the distribution of this protein becomes progressively restricted to a subpopulation of olfactory neurons. Comparison of the expression of B50/GAP43 and the olfactory marker protein (OMP), a polypeptide only present in mature olfactory neurons, revealed that during postnatal development of the olfactory system these 2 proteins are expressed in a nearly reciprocal fashion. In adult animals (3.5 months-6 months of age), B50/GAP43-positive cells are exclusively present adjacent to the basal cell layer of the neuroepithelium. Basal cells appear to be unstained. The region of the epithelium containing the B50/GAP43-positive cells is virtually devoid of OMP-positive neurons. A significant fraction of these B50/GAP43-containing cells bear dendritic and neuritic processes. However, these cells do not express olfactory cilia. It is probable that the olfactory neurons expressing the growth-associated B50/GAP43 protein may correspond to a particular subset of olfactory neurons at an intermediate state of maturation.

The regeneration of the olfactory neuroepithelium following olfactory bulbectomy or peripheral deafferentation was studied with mRNA probes and antibodies for B-50/GAP43 and for olfactory marker protein (OMP). Two stages in the regeneration of the olfactory epithelium could be discerned with these reagents. The first stage occurs following either peripheral deafferentation of the olfactory epithelium with Triton X-100 (TX-100) or after bulbectomy and is characterized by the formation of a large population of immature olfactory receptor neurons. These newly formed neurons express B-50/GAP43, a phosphoprotein related to neuronal growth and plasticity. During the second stage of the regeneration process the newly formed olfactory neurons mature, as evidenced by a decrease in their expression of B-50/GAP43 and an increase in the expression of OMP. This stage is only manifested if the developing neurons have access to the target olfactory bulb. Formation of a full complement of OMP-expressing neurons occurs only after peripheral lesion with TX-100. In contrast, following bulbectomy the reconstituted olfactory epithelium lacks its normal target and is compromised in its ability to recover from nerve damage, as evidenced by the presence of a large number of B-50/GAP43-expressing neurons up to 3 months after the lesion and its failure to establish a full complement of OMP-expressing neurons. These results demonstrate that the olfactory epithelium is capable of replacing its sensory neurons independently of the presence of its target, the olfactory bulb. However, the differential patterns of expression of B-50/GAP43 and OMP at long times after peripheral lesion with TX-100 or bulbectomy illustrate the profound effect the olfactory bulb has on neuronal maturation in reconstituted olfactory neuroepithelium.

Protein kinase C (PKC) is believed to have a crucial role in synaptic transmitter release and long-term potentiation. An important substrate of PKC in the brain is the neuron-specific presynaptically localized protein B-50 (also termed GAP-43, F1, pp46 or P-57). B-50 has been implicated in the regulation of polyphosphoinositide metabolism and calmodulin binding, and in the mechanisms of neurite outgrowth, long-term potentiation and transmitter release. It is still unknown, however, whether B-50 (and/or its phosphorylation) is essential to any of these processes. Here we report the results of studies in which antibodies to B-50, which interfere with B-50 phosphorylation, were introduced into rat cortical synaptosomes that were permeabilized with streptolysin-O (SL-O). We found that the release of [3H]noradrenaline, induced by increasing the Ca2+ concentration in the buffer, is inhibited completely by the antibodies. These results provide the first demonstration of a causal relationship between the PKC substrate B-50 and the release of neurotransmitter.

Recently it has been shown that B-50 is identical to the neuron- specific, growth-associated protein GAP43. The present study reports on the fate of B-50/GAP43 mRNA and B-50/GAP43 protein, determined by radioimmunoassay, in a rat model of peripheral nerve regeneration (sciatic nerve crush) over a period of 37 and 312 d, respectively. Moreover, the effects of repeated subcutaneous injection of the neurotrophic peptide Org.2766 (an ACTH4–9 analog) and of a conditioning lesion on B-50/GAP43 protein levels in the regenerating nerve and dorsal root ganglia (DRG) were investigated. Both treatments enhanced the functional recovery as evidenced by a foot-flick withdrawal test. Immunocytochemical analysis using antineurofilament antibodies revealed a peptide-induced increase in the number of outgrowing sprouts in the sciatic nerve. Both the peptide and the conditioning lesion amplified the crush lesion-induced increase in B-50 protein content in the nerve as determined by radioimmunoassay. B-50 protein levels seem to correlate proportionally with the number of sprouts. In the DRG of the crushed sciatic nerve, the time course of B-50 expression was studied. B-50 mRNA was quantified from Northern blots. A linear increase up to 10 times the basal level of B-50 mRNA was observed 2 d postsurgery, followed by a gradual decline to normal levels at day 37. The first significant rise in B-50 mRNA level became apparent between 8 and 16 hr after placement of the crush lesion. The first significant rise in B-50 protein level occurred 40 hr after the crush lesion, reaching a plateau of 3 times the basal level between day 6 and 20. B-50 protein levels in DRG cell bodies remained elevated up to 60 d after crush, a period much longer than that observed for B-50 mRNA. Thus, during a later phase of peripheral axonal regeneration, the presence of B-50 appears to be prolonged, probably by an increase in half-life and not so much by enhanced transcription. Treatment with Org.2766 did not affect the B- 50/GAP43 levels in DRG cell bodies during the first 6 d following crush. Conditioning lesion resulted in a DRG B-50/GAP43 protein amount at the same level as in rats 14 d after the test lesion. B-50/GAP43 levels in DRG are probably influenced by the rapid axonal transport of the protein, as has been reported by others.

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