Manipulating transmitter release at the neuromuscular junction of neonatal rats alters the expression of ChAT and GAP-43 in motoneurons.

During early postnatal development, motoneurons innervating rat hindlimb muscles die following injury to the sciatic nerve. However, prematurely enhancing transmitter release from nerve terminals of neonatal rats renders motoneurons less vulnerable to nerve injury, whereas reducing transmitter release increases their susceptibility to injury. Thus, alterations in transmitter release may have an influence on motoneuron phenotype. Here we investigated the relationship between the vulnerability of motoneurons to injury, and the expression of proteins associated with axonal growth and neuromuscular transmission. We examined the effect of agents that affect transmitter release from nerve terminals and that have been shown to influence the expression of transmitter and growth related proteins in developing motoneurons in response to nerve injury. In newborn rats, implants containing either 4-aminopyridine (4-AP), to increase transmitter release, or magnesium sulphate (MgSO4), to decrease release, were applied to the soleus muscle in one hindlimb. The effect of these treatments on the activity of choline acetyltransferase (ChAT) in nerve terminals in the soleus muscle was measured using a radiochemical assay. Levels of GAP-43 in sciatic nerve were also assessed, by Western blot analysis. The results showed that during normal development, there was a gradual increase in ChAT activity during the second week of postnatal development, whereas GAP-43 levels declined sharply between postnatal days 12-14. However, following 4-AP treatment, there was a dramatic increase in ChAT activity in nerve terminals contacting the treated soleus muscles and the levels of GAP-43 in the sciatic nerve declined at an earlier age than normal. Conversely, following treatment with MgSO4 the normal increase in ChAT activity that occurs during the second postnatal week was delayed, and GAP-43 levels in the sciatic nerve were maintained for significantly longer than normal. Thus, manipulating transmitter release from nerve terminals in neonatal rats alters the normal pattern of expression of transmitter and growth related proteins in developing motoneurons. This alteration in protein expression may influence both the maturation of motoneurons and their ability to withstand nerve injury.

Changes in the expression of parvalbumin immunoreactivity in the lumbar spinal cord of the rat following neonatal nerve injury.

Nerve injury in newborn animals results in the loss of motoneurons and dorsal root ganglion neurons and long-term changes in reflex activation of surviving motoneurons. Parvalbumin has been previously shown to be found in large-diameter primary afferent axons and interneurons in the spinal cord, and was used here to study the changes in parvalbumin-immunoreactive appositions onto identified tibialis anterior/extensor digitorum longus (TA/EDL) motoneurons, during both normal development and following neonatal nerve injury in the rat spinal cord. During normal development, there was a decrease in the number of parvalbumin-immunoreactive appositions onto TA/EDL motoneurons. Thus, at postnatal day 7 (P7), there were 72.8 +/- 17.5 (mean +/- SD) appositions per motoneuron and by P14, it had decreased to 38.8 +/- 13.2 (mean +/- SD; p > 0.05). Following neonatal nerve injury at P2, there were fewer parvalbumin-positive afferent appositions close to the TA/EDL motoneurons than normal, so that at P7, there were 53.5 +/- 17.1 (mean +/- SD), and at P14, it further decreased to 25.8 +/- 8.6 (mean +/- SD; p > 0.05). This injury-induced reduction in the number of parvalbumin-immunoreactive boutons apposing TA/EDL motoneurons may result, at least in part, from the death of dorsal root ganglion cells with the consequent loss of their central projections. The alterations in the number of parvalbumin-positive appositions close to motoneurons observed in this study may contribute to the changes in the pattern of reflex activity observed in the developing spinal cord both during normal development and following neonatal injury.