The neuron-specific phosphoprotein B-50/GAP43 has been implicated in axonal outgrowth, since high levels of B-50/GAP43 are found in growth cones and during development of the nervous system. In adult brain, the B-50 levels are decreased. B-50 is primarily found in axons and presynaptic terminals. It is phosphorylated by protein kinase C, and this process has been implicated in the modulation of membrane signal transduction. During the outgrowth of the pyramidal tract, high levels of B-50 have been reported, whereas a low amount of B-50 persists into the adult stage. By immunoelectron microscopy, using immunogold labeling on cryosections and pre-embedding peroxidase labeling, we examined the distribution of B-50 in the pyramidal tract at the third cervical segment in developing 2-d-old and adult 90-d-old rats. B-50 immunoreactivity was found in axons and growth cones of the outgrowing tract. In the adult pyramidal tract, both unmyelinated and myelinated axons contained B-50 immunoreactivity. The immunogold label was predominantly located at the plasma membrane. Since the peroxidase reaction product was observed exclusively intracellularly, we conclude that the B-50 immunoreactivity is predominantly located at the cytoplasmic side of the plasma membrane of axons and growth cones. The high immunoreactivity in growth cones and axons of the outgrowing pyramidal tract further supports the hypothesis that B-50 plays a role in neurite outgrowth. The presence of B-50 in the adult pyramidal tract cannot merely be attributed to transport to the synapse. Therefore, it is suggested that B-50 plays, in addition, a local, growth-associated role in the adult tract.

The neuron-specific phosphoprotein B-50 is a major substrate of kinase C in fetal nerve growth cones, neonatal neural and synaptosomal plasma membranes. B-50 is identical to a growth-associated protein GAP43. Similarly, increases in B-50 occur during rat brain development, neuronal differentiation and axon regeneration. To document the relation between the expression of B-50 and the outgrowth of central axons, we studied B-50 in the developing pyramidal tract in rats at postnatal days 2, 7 and 90 (P2, P7 and P90), at the third cervical spinal segment C3, using affinity-purified antibodies to B-50. At P2 and P7, when outgrowth of pyramidal tract fibers is occurring, B-50 immunoreactivity (BIR) is intense in these fibers. BIR is reduced from P2 to P7 in the ascending fiber tracts of the cuneatus and the gracilis, which develop earlier. At P90 when most of the dorsal funiculus fibers have reached their targets and many are myelinated, BIR is dramatically reduced. In agreement, a 10-fold decrease in B-50 content was measured at P90, as compared to P7. Therefore, our results indicate that B-50 is only expressed relatively abundant in axons of the funiculus posterior during outgrowth. By inference, B-50 may be a differentiating marker to detect elongating fibers.The ontogenesis of the spinal pyramidal tract in the rat occurs during the postnatal development. Shortly after birth, the leading corticospinal fibers are entering the upper cervical segments of the spinal cord [21], where they occupy the most ventral part of the dorsal funiculus. The spatial and temporal outgrowth of these corticospinal fibers in the spinal white and gray matter in the neonatal rat have been described by Gribnau et al. [8]. They demonstrated that the corticospinal fibers have reached the upper thoracic segments of the spinal cord at postnatal day 2 (P2) and the sacral segments at P7. A delay of two days was observed between the arrival of the corticospinal axons at a given spinal cord level and their outgrowth into the adjacent spinal

In order to examine the mode of outgrowth of the pyramidal tract in the rat, the ultrastructure of its pathway in the dorsal funiculus of the spinal cord was analysed. The analysis was performed by means of serial sections of the third cervical segment before and during the arrival of pyramidal tract axons, and focussed on the morphology and microenvironment of the growth cones. Growth cones appear as elongated terminal enlargements without side branches. Two zones could be discerned: the distal, usually lamellipodial fine granular zone, containing no organelles, except for an occasional clear vesicle; and the proximal organelle-rich zone, which contains various organelles, such as agranular reticulum and vesicular structures. In addition, the proximal organelle-rich zone contains round or elliptic structures, limited by two concentric membranes, that enclose reticular and vesicular elements. The electron density of these structures varied from as low as the surrounding growth cone matrix to as dark as lysosomal structures, suggesting their involvement in turnover processes. At embryonic day 20, the most ventral part of the dorsal funiculus, where the first pyramidal tract axons are due to arrive within two days, is populated by axons that are relatively small compared to those in the rest of the dorsal funiculus. At birth, the arrival of the first pyramidal tract axons is marked by the presence of numerous large growth cone profiles in between small axons in the most ventral part of the dorsal funiculus; no circumscript bundle separated from the ascending sensory fiber tracts is present yet. The growth cones descend, club-shaped and 1 to 2 microns in diameter, without lamellipodia or filopodia. Within the same area a second growth cone type is present, which contains dense-core vesicles and has spread-out lamellipodia. Most of these growth cones are ascending and they probably belong to primary afferent or propriospinal fibers. At postnatal day 2, the pyramidal tract can be readily delineated from the adjacent fasciculus cuneatus where myelination has already started, but no glial boundary is present. The abundant growth cones are 1-2 microns wide and extend single unbranched lamellipodia, up to 15 microns long, which often enfold parallel axons or other growth cones. At postnatal day 4, growth cones are scarce in the tract. They measure 1 micron or less in diameter and each extends a single, straight lamellipodium or filopodium over 1 to 7 microns in the caudal direction.(ABSTRACT TRUNCATED AT 400 WORDS)

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