M.L. Reynolds

The growth-associated protein GAP-43 appears in dorsal root ganglion cells and in the dorsal horn of the rat spinal cord following peripheral nerve injury.

When adult dorsal root ganglion cells are dissociated and maintained in vitro, both the small dark and the large light neurons show increases in the growth-associated protein GAP-43, a membrane phosphoprotein associated with neuronal development and plasticity. Immunoreactivity for GAP-43 appears in the cytoplasm of the cell bodies as early as 3.5 h post axotomy and is present in neurites and growth cones as soon as they develop. At early stages of culture (4 h to eight days) satellite/Schwann cells are also immunoreactive for GAP-43. Neurons in isolated whole dorsal root ganglion maintained in vitro become GAP-43-immunoreactive between 2 and 3 h after axotomy. It takes three days however, after cutting or crushing the sciatic nerve in adult rats in vivo, for GAP-43 immunoreactivity to appear in the axotomized dorsal root ganglion cells. GAP-43 immunoreactivity can be detected in the central terminals of primary afferent neurons in the superficial laminae of the dorsal horn of the lumbar enlargement four days after sciatic cut or crush. The intensity of the GAP-43 staining reaches a peak at 21 days and becomes undetectable nine weeks following crush injury and 36 weeks following sciatic nerve cut. The pattern of GAP-43 staining is identical to the distribution of sciatic small-calibre afferent terminals. Little or no staining is present in the deep dorsal horn, but GAP-43 does appear in the ipsilateral gracile nucleus 22 days after sciatic injury. In investigating the mechanism of GAP-43 regulation, blockade of axon transport in the sciatic nerve with vinblastine (10(-5) M-10(-4) M) or capsaicin (1.5%) was found to produce a pattern of GAP-43 immunoreactivity in the dorsal horn identical to that found with crush, while electrical stimulation of the sciatic nerve had no effect. Axotomy of primary sensory neurons or the interruption of axon transport in the periphery therefore acts to trigger GAP-43 production in the cell body. The GAP-43 is transported to both the peripheral and the central terminals of the afferents. In the CNS the elevated GAP-43 levels may contribute to an inappropriate synaptic reorganization of afferent terminals that could play a role in the sensory disorders that follow nerve injury.

Terminal schwann cells elaborate extensive processes following denervation of the motor endplate

SummaryTerminal Schwann cells, when stained for S100 (a calcium binding protein), can be seen to cap motor axons at the neuromuscular junction. Within days of denervation the Schwann cells begin to stain for the low affinity nerve growth factor receptor, but remain Thy-1 negative, and elaborate fine processes. These processes become longer and more disorganized over weeks, and cells positive for S100 and nerve growth factor receptor migrate into the perisynaptic area. Reinnervation results in a withdrawal of the processes. The morphology and location of terminal Schwann cells seems to depend on axonal contact. The spread of Schwann cells and their processes away from the synaptic zone following denervation, implies that these cells do not target axons directly to the endplate.

GAP-43 expression in primary sensory neurons following central axotomy

Primary sensory neurons are capable of successful regenerative growth in response to peripheral nerve but not dorsal root injury. The present study is concerned with the differential expression of the mRNA for GAP- 43, a growth-associated protein, in these sensory neurons, in response to injury of their central or peripheral axonal branches. Peripheral axotomy resulted in an elevation in message detectable within 24 hr, using Northern blot and in situ hybridization, which was maintained for 30 d, whereas dorsal root section produced no change except a transient and small increase if the axotomy was immediately adjacent to the dorsal root ganglia (DRG). Dorsal root section had no effect on GAP-43 mRNA levels in the dorsal horn or in neighboring intact DRG. It also failed to alter the laminar boundaries of the GAP-43 central terminal labeling produced by peripheral nerve section, even though vacant synaptic sites were produced in unstained laminae by this procedure. This indicates that the location of GAP-43 immunolabeling in the central terminals of primed sensory cells may not depend only on the location of vacant synaptic sites. We conclude that distinct control mechanisms regulate the response of DRG neurons to peripheral nerve and dorsal root injury, and these may be related both to the glial environment and the particular target influences exerted on the central and peripheral branches of the primary sensory neuron. Central denervation alone is insufficient to upregulate GAP-43 levels, and this may explain the relative absence of collateral sprouting after the production of central vacant synaptic sites. The failure of dorsal root section to increase GAP-43 expression may contribute to the poor regenerative response initiated by such lesions.

Reciprocal Schwann cell-axon interactions

This article describes the reciprocal interactions between neurones and Schwann cells with particular reference to the role of growth factors and neurokines as signalling molecules between these cells and of the extracellular matrix as a conduit for such signalling. Major recent advances have identified molecules produced by neurones that are responsible for Schwann cell proliferation, as well as some of the Schwann cell factors regulating the expression of molecules shown to play an important role in neuronal survival and differentiation.

GAP-43 expression in developing cutaneous and muscle nerves in the rat hindlimb.

The expression of the growth associated protein, GAP-43, in developing rat hindlimb peripheral nerves has been studied using immunocytochemistry. GAP-43, is first detected in lumbar spinal nerves at embryonic day (E)12 as the axons grow to the base of the hindlimb. It is expressed along the whole length of the nerves as well as in the growth cones. GAP-43 staining becomes very intense over the next 36 h while the axons remain in the plexus region at the base of the limb bud before forming peripheral nerves at E14. It remains intense along the length of the growing peripheral nerves, the first of which are cutaneous, branching away from the plexus and growing specifically to the skin, their axon tips penetrating the epidermis of the proximal skin at E15 and the toes at E19. GAP-43-containing terminals form a dense plexus throughout the epidermis which subsequently withdraws subepidermally in the postnatal period. GAP-43 staining is also evident along the growing muscle nerves during muscle innervation, which follows behind that of skin. Axons branch over the surface of proximal muscles at E15 but do not form terminals until E17. As target innervation proceeds, GAP-43 staining declines in the proximal part of the nerve but remains intense in the distal portions. Overall GAP-43 expression in the hindlimb decreases in the second postnatal week as axon growth and peripheral terminal formation decline.

GAP-43 expression in the developing rat lumbar spinal cord

The expression of the growth-associated protein GAP-43, detected by immunocytochemistry, has been studied in the developing rat lumbar spinal cord over the period E11 (embryonic day 11), when GAP-43 first appears in the spinal cord, to P29 (postnatal day 29) by which time very little remains. Early GAP-43 expression in the fetal cord (E11-14) is restricted to dorsal root ganglia, motoneurons, dorsal and ventral roots and laterally positioned and contralateral projection neurons and axons. Most of the gray matter is free of stain. The intensity of GAP-43 staining increases markedly as axonal growth increases, allowing clear visualization of the developmental pathways taken by different groups of axons. Later in fetal life (E14-19), as these axons find their targets and new pathways begin to grow, the pattern of GAP-43 expression changes. During the period, GAP-43 staining in dorsal root ganglia, motoneurons, and dorsal and ventral roots decreases, whereas axons within the gray matter begin to express the protein and staining in white matter tracts increases. At E17-P2 there is intense GAP-43 labelling of dorsal horn neurons with axons projecting into the dorsolateral funiculus and GAP-43 is also expressed in axon collaterals growing into the gray matter from lateral and ventral white matter tracts. At E19-P2, GAP-43 is concentrated in axons of substantia gelatinosa. Overall levels decline in the postnatal period, except for late GAP-43 expression in the corticospinal tract, and by P29 only this tract remains stained.

Nerve growth factor levels in developing rat skin: upregulation following skin wounding.

Levels of nerve growth factor (NGF) in rat hindpaw skin, measured with a sensitive two-site enzyme-linked immunosorbent assay, show two peaks during normal development. The first (57 +/- 5 pg mg-1) occurs at embryonic days (E) 18-20 and coincides with the arrival of axon terminals into the hindpaw skin. The second, larger peak (132 +/- 10 pg mg-1), occurs later, around postnatal day (P) 21 and may be involved in maintenance of neuronal phenotype. Levels outside the two peaks stay relatively constant throughout development (30 pg mg-1). Skin wounding at birth produces a marked increase in NGF levels (149 +/- 25 pg mg-1) which declines after 4 days. This large increase is not observed if wounding is performed at older ages and may underlie the sensory hyperinnervation that accompanies neonatal wounds.

Time-dependent differences in the increase in GAP-43 expression in dorsal root ganglion cells after peripheral axotomy

Peripheral axotomy of primary afferent neurons results in the up-regulation of the growth-associated phosphoprotein GAP-43, by dorsal root ganglion cells. We have studied the temporal sequence of GAP-43 expression in those dorsal root ganglion neurons with unmyelinated axons (the small dark cells) and in those with myelinated axons (the large light cells) after sciatic nerve section in the adult rat. Immunoreactivity for the RT 97 neurofilament epitope, which is detectable only in large light dorsal root ganglion cells, was used to differentiate the two types of dorsal root ganglion cell. Within two days of a sciatic nerve section the number of GAP-43-immunoreactive profiles in the ipsilateral ganglion had increased five-fold and this increase persisted for 80 days post-section. While 50% of the small numbers of GAP-43-positive cells in control ganglia were RT 97 positive, only 8% of the large number of GAP-43-immunoreactive cells four days post-section, were RT 97 positive. By 14 days the number of RT 97-positive/GAP-43-positive cells had increased to 29%. This was paralleled by an increase in GAP-43 immunoreactivity in large diameter profiles at 14 days. The signals that alter GAP-43 expression in unmyelinated (small, RT 97 -ve) and myelinated (large, RT 97 +ve) afferents after peripheral nerve injury appear to operate with different time-courses.

Intracranial haemorrhage in the preterm sheep fetus

The germinal layer in the brain of the sheep fetus at 58--85 days of gestation was found to resemble that of the human infant at 28--30 wk of gestation. Experiments were done on 65 exteriorized fetuses to explore the effect of various combinations of asphyxia and raised intravascular pressures in causing bleeding into the germinal layer, ventricles and other parts of the brain. Asphyxia by itself did not produce an increase in the incidence of intracranial hemorrhages when compared with control fetuses. The combination of asphyxia with intermittent increases in arterial or venous pressure, or both, did cause haemorrhages. Large increases in arterial pressure without asphyxia also caused intracranial haemorrhages, whereas increases in venous pressure without asphyxia did not. The types of haemorrhage observed closely resembled those seen in the preterm human infant, although massive intraventricular haemorrhages (IVHs) were rare. We conclude that: (1) the sheep fetus can be used for investigating factors associated with intracranial haemorrhage in the preterm brain; (2) the most effective method of producing haemorrhages into the germinal layer was by a combination of asphyxia with intermittent increases in cerebral intravascular pressure. Similar mechanisms may be at work in the newborn human infant, and could lead to IVH.

Postnatal development of the telencephalon of the tammar wallaby (Macropus eugenii). An accessible model of neocortical differentiation.

SummaryThe sequence of development of cell layers in the neocortex of the tammar has been followed from 24 days gestation to 213 days postnatal. The tammar is born at 27 days gestation and the major period of its development occurs during the subsequent 250 days, most of this time being spent within the pouch. Although the pattern of differentiation of the cell layers appears to resemble that described for many Eutherian mammals, the neocortex is at an embryonic 2 layered stage at birth and a cortical plate is not present throughout the telencephalon until 10–15 days postnatal. A transient subplate zone, presenting a characteristic appearance with widely spaced rows of cells aligned parallel to the cortical surface, develops between 20 and 70 days postnatal, but no secondary proliferative region is seen in the subventricular zone of the dorso-lateral wall.Preliminary experiments with (3H)-thymidine injections indicate that the cortical plate follows the “inside-out” pattern of development described in many Eutherian mammals and that the oldest neurons are found in the parallel cell rows of the subplate zone. The importance of the late differentiation of the neocortex in relation to the time of birth and the resulting usefulness of the tammar as an experimental model of cortical development is discussed.