The effect of activity during early postnatal development on motor unit size.

At early stages of neuromuscular development, motor unit territory is expanded, with each muscle fibre being supplied by several axons. During postnatal development, some synapses are eliminated, motor unit size decreases, and the adult distribution of motor unit sizes emerges. This process depends on activity, since it proceeds more rapidly when the nerve is activated and is slower when activity is reduced. Here we studied whether, in addition to influencing the rate of retraction of motor unit territory, activity during the critical period of development affects the final outcome of the distribution of motor unit sizes. The sciatic nerve of 8- to 12-day-old rats was stimulated daily. One week later the tension of the extensor digitorum longus muscle and that of its individual motor units was recorded. The sizes of individual motor units were calculated and compared with those from animals that received no stimulation. The distribution of motor unit sizes from stimulated muscles was not significantly different from those from control muscles. Therefore, we conclude that although activity increases the rate at which motor units attain their adult size, it does not influence the final outcome of motor unit size distribution.

Rescue of the Friedreich's ataxia knockout mouse by human YAC transgenesis.

We have generated and characterised transgenic mice that contain the entire Friedreich's ataxia gene (FRDA) within a human YAC clone of 370 kb. In an effort to overcome the embryonic lethality of homozygous Frda knockout mice and to study the behaviour of human frataxin in a mouse cellular environment, we bred the FRDA YAC transgene onto the null mouse background. Phenotypically normal offspring that express only YAC-derived human frataxin were identified. The human frataxin was expressed in the appropriate tissues at levels comparable to the endogenous mouse frataxin, and it was correctly processed and localised to mitochondria. Biochemical analysis of heart tissue demonstrated preservation of mitochondrial respiratory chain function, together with some increase in citrate synthase and aconitase activities. Thus, we have demonstrated that human frataxin can effectively substitute for endogenous murine frataxin in the null mutant. Our studies are of immediate consequence for the generation of Friedreich's ataxia transgenic mouse models, and further contribute to the accumulating knowledge of human-mouse functional gene replacement systems.

Repeated injury to the sciatic nerve in immature rats causes motoneuron death and impairs muscle recovery.

Injury to the sciatic nerve of newborn rats causes motoneuron death, while the same insult inflicted 5 days later does not. In this study the effects of prolonging the period of target deprivation and axonal regeneration were investigated by inflicting a second nerve crush 6 days after the first, just before reinnervation of the muscle occurred. Two to 4 months later the number of motoneurons supplying soleus, tibialis anterior, and extensor digitorum longus muscles was established by retrograde labeling with horseradish peroxidase injected into the muscle. After nerve injury at 5 days there was no significant loss of motoneurons to any muscle. However, when the injury was repeated, the number of labeled motoneurons was reduced, suggesting that a significant proportion had died. Motoneurons to soleus were affected more than those to the fast muscles, reflecting their lesser maturity. Moreover, motoneurons to soleus that survived both injuries to their axon failed to grow to their full size. The relative impairment of recovery of the muscles, indicated by weight and maximal tetanic tension, mirrored the loss of motoneurons in each case. Previous studies have suggested that repeated nerve injuries in adult animals can enhance reinnervation. However, the present results along with those of other recent studies suggest that immature motoneurons that are repeatedly induced to support growth of their axons are at greater risk of death and can result in poorer reinnervation of the muscles.

Cell death of spinal interneurones.

The occurrence of neuronal death during development is well documented for some neuronal populations, such as motoneurones and dorsal root ganglion cells, whose connecting pathways are clearly defined. Cell survival is thought to be regulated largely by target and input connections, a process that serves to match the size of synaptically linked neuronal populations. Far less is known about interneurones. It is assumed that most interneurone populations are excluded from this process because their connections are more diffuse. Recent studies on the rat spinal cord have indicated that interneurone death does occur, both naturally during development and induced following peripheral nerve injury. Here the evidence for spinal interneurone death is reviewed and the factors influencing it are discussed. There are many functional types of interneurones in the spinal cord that may differ in vulnerability to cell death, but it is concluded that for most spinal interneurones the traditional view of target regulation is unlikely. Instead it is proposed that developmental interneurone death in the spinal cord forms part of a plastic response to altered sensory activation rather than a size-matching exercise. There is also emerging evidence that interneurone death may play a more direct role in some neurodegenerative diseases than hitherto considered.

Motoneurones that innervate the rat soleus muscle mature later than those to the tibialis anterior and extensor digitorum longus muscles.

The response of motoneurones that innervate either the soleus or tibialis anterior (TA) and extensor digitorum longus (EDL) muscles to increased locomotor activity or to nerve injury at different stages after birth was examined. Increased locomotor activity of rat pups was induced by daily treatment with L-dopa during the first 12 days after birth, and the number of surviving motoneurones to the soleus or TA/EDL muscles was established by retrograde labelling. Treatment with L-dopa resulted in the loss of a significant number of motoneurones within the soleus motor pool but had no effect on the survival of those motoneurones innervating the TA/EDL. Furthermore, following nerve injury during the first few days postnatally, more motoneurones within the soleus motor pool die than in the TA/EDL pool. These results indicate that motoneurones to the soleus muscle mature later than those to the TA/EDL muscles.

The role of apoptosis and excitotoxicity in the death of spinal motoneurons and interneurons after neonatal nerve injury.

There is evidence that motoneurons which die following neonatal nerve injury in rats do so through an excitotoxic mechanism. In this study, we have investigated whether this excitotoxicity induces motoneuron death by apoptosis. Sciatic motoneurons were prelabelled at birth with the retrograde tracing agent, Fast Blue, and the sciatic nerve was crushed in one leg two days later. At intervals up to 12 days, sections of the lumbar enlargement were analysed for apoptosis using propidium iodide and terminal deoxynucleotidyl transferase biotin-14-UTP nick end labelling techniques. A significant concentration of Fast Blue-labelled apoptotic motoneurons was seen in the area of the sciatic motor pool ipsilateral to the nerve injury, with the majority occurring in the first three days. Comparison of estimates of the time-course of apoptosis with that of motoneuron survival suggest that all motoneuron death induced during the first 12 days occurs by apoptosis and that the process is only recognizable for 2 h. Treatment with the N-methyl-D-aspartate receptor antagonist, dizocilpine maleate, reduced the level of apoptosis by 60%. Taken together, these data show that motoneurons which have been affected by an excitotoxic mechanism die by apoptosis. The apoptotic study also provides evidence, for the first time, that unilateral nerve injury induces motoneuron death in the contralateral sciatic motor pool. Apoptotic interneurons were also seen on both sides of the spinal cord as a result of nerve injury.

Motoneuron survival after neonatal peroneal nerve injury in the rat-evidence for the sparing effect of reciprocal inhibition.

Sciatic nerve crush at birth results in the death of most of the motoneurons in the sciatic motor pool. It has been proposed that these cells die through excessive activation which can be explained partly by an increased susceptibility to NMDA. However, it is also possible that decreased inhibitory mechanisms resulting from nerve injury may contribute to overactivation of the motoneurons. In this study we compared the survival of motoneurons innervating two muscles in the peroneal motor pool, tibialis anterior and extensor digitorum longus, after either sciatic or common peroneal nerve crush. These two procedures both axotomize the motoneurons but differ in their effects on afferent input. Sciatic nerve crush severely reduces the afferent input from the antagonist muscles innervated via the tibial nerve, whereas common peroneal nerve crush preserves them. Using retrograde labeling with horseradish peroxidase, we found that almost twice as many motoneurons survived common peroneal nerve crush than sciatic nerve crush and that muscle weight showed a corresponding significant improvement. A control experiment excluded the possible involvement of increased stretch of the muscles as a result of common peroneal nerve crush alone as an explanation for the improvement. We therefore suggest that the increased survival of motoneurons after peroneal nerve crush was due to the preservation of their reciprocal inhibitory input. However, since even with this improvement the majority of motoneurons still died, loss of reciprocal inhibition probably does not play a major role in the death of motoneurons induced by overactivation.

Evidence for two populations of N-methyl-D-aspartate receptors in neonatal rat spinal cord. The effect of peripheral nerve axotomy.

Quantitative autoradiography was used to characterise the binding of the N-methyl-D-aspartate (NMDA) receptor antagonist [3H]dizocilpine maleate (MK801) in the white matter and dorsal, intermediate and ventral subregions of the grey matter in the lumbar spinal cord of neonatal rats. The effect on the binding of unilateral sciatic nerve section on the day of birth was examined. In unoperated animals the Bmax and Kd of the binding had decreased in all subregions by two weeks, when the values were similar to those in the adult. After axotomy the Bmax values declined during the first 14 days in all subregions although the density appeared higher in the grey matter in the ventral horn compared to sham operated control. In the axotomised animals, the Kd values for the white matter and ventral horn grey matter had declined by two weeks but in the dorsal and intermediate subregions of the grey matter the values remained elevated. The results are consistent with the presence of two populations of NMDA receptor at birth. In the normal animals the lower affinity receptor disappears in all subregions, but after axotomy it is retained in the dorsal and intermediate subregions for at least 2 weeks.

N-methyl-D-aspartate receptors in motoneurones after unilateral axotomy in the neonatal rat.

Quantitative autoradiography was used to characterise the binding of the N-methyl-D-aspartate receptor antagonist [3H]dizocilpine maleate in the ventral horns of the lumbar spinal cord of normal, sham-operated, and axotomized neonatal rats. Specific binding sites were revealed on the cell membranes of the motoneurones. In the normal neonate both the Bmax and the Kd values for the binding declined over the first 14 days of life. At 14 days the Kd value was similar to that in adult rats. Unilateral sciatic nerve section was performed in neonates on the day of birth. Axotomy caused the death of approximately 53% of motoneurones. The Bmax and Kd values for [3H]dizocilpine maleate binding declined in the first 2 weeks on operated and contralateral sides in both axotomized and sham-operated animals. However, the specific binding per motoneurone was significantly higher on the sectioned side in axotomized animals at all times examined after the day of birth. The results are consistent with two populations of NMDA receptors with different binding affinities being present in motoneurones at birth, and the lower affinity receptor gradually disappearing over the first few days. The lower affinity receptor may be responsible for the plasticity of motoneurones during embryonic and neonatal life, and for determining which motoneurones die after axotomy.

Evidence that spinal interneurons undergo programmed cell death postnatally in the rat.

Programmed cell death has been demonstrated in several specific neuronal populations as a mechanism for modulating the population size following differentiation, but its applicability to all neuronal types is unclear. Evidence for programmed cell death in some populations such as the numerous spinal interneurons has been lacking. We have studied the incidence of apoptosis in the rat spinal cord with three different methods and found a previously undocumented wave of apoptosis occurring in spinal grey matter shortly after birth. The apoptotic morphology was confirmed ultrastructurally. Dying cells were identified as neurons by immunocytochemical labelling for neuronal markers and had an anatomical distribution which indicated that most of the apoptotic cells were interneurons not motoneurons. This wave of apoptosis has the characteristics of a discrete developmental process and occurs later than that of either ventral horn motoneurons or dorsal root ganglion cells, to which most spinal interneurons are connected. These findings indicate that interneurons do undergo programmed cell death, and we suggest that this occurs in response to the earlier reduction in size of their main synaptic targets.

The effects of neonatal dorsal root section on the survival and dendritic development of lumbar motoneurons in the rat.

Peripheral nerve crush during the early neonatal period results in the death of a large proportion of affected motoneurons and abnormal dendritic development in those which survive. The present study reports the effects of neonatal dorsal root section on motoneurons supplying the extensor digitorum longus muscle of the rat. This lesion did not result in motoneuron death, but did disrupt subsequent dendritic development. In cells retrogradely labelled with cholera toxin subunit B conjugated to horseradish peroxidase, there was little change in adult dendritic morphology in the transverse plane, where abnormalities associated with loss of efferent contact and cell death have been found. However, there was a caudal expansion of the dendritic field, an effect seen following nerve crush but not after blockade of neuromuscular transmission alone. The results show that disruption of dorsal root sensory inputs alone can affect the dendritic development of motoneurons but does not cause their death. In conjunction with our earlier findings, it is clear that both afferent and efferent connections are required for normal dendritic development, and disruption of either has a characteristic effect on survival and dendritic morphology.

Magnesium ions reduce motoneuron death following nerve injury or exposure to N-methyl-D-aspartate in the developing rat.

Developing motoneurons can be induced to die by target deprivation and there is evidence that this cell death involves the excitotoxic effects of N-methyl-D-aspartate. Treatment with dizocilpine maleate, an antagonist of this receptor, has been shown to rescue a proportion of those motoneurons destined to die following nerve injury at birth. However, this is a relatively toxic compound. In this study we examined whether systemic treatment with magnesium sulphate, a non-competitive antagonist of the N-methyl-D-aspartate receptor which is better tolerated than dizocilpine maleate, could prevent motoneuron death. Motoneurons were induced to die either by sciatic nerve injury at birth or by nerve injury at five days followed by exposure to N-methyl-D-aspartate. The number of surviving motoneurons reinnervating the tibialis anterior and extensor digitorum longus muscles were counted using retrograde labelling. Following nerve injury at birth and treatment with magnesium sulphate, there was a small increase in the survival of injured motoneurons, although this improvement was not significant. Nerve injury at five days does not result in motoneuron death, but when followed by treatment with N-methyl-D-aspartate, only 42 +/- 2.9% of motoneurons to these flexor muscles survived. Treatment with magnesium sulphate prior to injection of N-methyl-D-aspartate significantly increased motoneuron survival, so that 67 +/- 5.8% of motoneurons survived. Thus, systemic treatment with magnesium can prevent the death of motoneurons rendered susceptible to the excitotoxic effects of N-methyl-D-aspartate by nerve injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Nerve injury in adult rats causes abnormalities in the motoneuron dendritic field that differ from those seen following neonatal nerve injury.

Disruption of neuromuscular contact by nerve-crush during the early postnatal period causes increased activity and abnormal reflex responses in affected motoneurons, but such changes are not found after nerve-crush in adult animals. We found previously that neonatally lesioned cells develop an abnormal dendritic field, which may explain the functional changes. Here we have studied the dendritic morphology of the same motoneuron pool after nerve-crush at maturity in order to correlate the observed alterations in morphology with physiological findings. One to two months after sciatic nerve-crush in adult animals, motoneurons supplying the extensor hallucis longus muscles of the rat were retrogradely labelled with cholera toxin subunit-B conjugated to horseradish peroxidase. The dendritic tree of labelled cells was then analysed. Following adult nerve-crush, the dendritic tree of the motoneurons was smaller but did not display the localised increase in dendritic density seen after neonatal nerve-crush. These findings support the view that such specific morphological changes contribute to the physiological abnormalities seen only after neonatal nerve injury.

Dendritic development in normal lumbar motoneurons and following neonatal nerve crush in the rat.

Motoneurons from the rat were retrogradely labelled with cholera toxin-horseradish peroxidase at intervals during normal postnatal development and following nerve crush at birth. Normal cells displayed a relatively steady increase in total visible dendritic density which was largely confined to the dorsomedial direction. After nerve crush at birth, dorsomedially orientated dendrites failed to achieve normal density, resulting in a significantly smaller dendritic tree by adulthood. There was also a transient, abnormal extension of dendrites in the medioventral direction which had regressed to normal levels by maturity. The predominance of changes in the dorsally directed region of the dendritic tree suggests that dendritic development of motoneurons is influenced by synaptic inputs in the dorsal horn.

Time course of motoneurone death after neonatal sciatic nerve crush in the rat.

Sciatic motoneurones were retrogradely labelled with long-lasting fluorescent dyes prior to unilateral nerve crush in either 3-day-old or adult rats. The number of surviving labelled motoneurones at intervals after nerve injury were compared to the number in the contralateral control ventral horn and in unoperated animals. Following adult nerve crush there was no significant reduction in the number of labelled motoneurones, but after neonatal nerve crush the count was reduced to about 35%. Most of the cell death occurred during the first 6 days after nerve injury, mainly from the lower half of the motor column, but about one third died between 6 and 12 days, mainly from the upper part. These results suggest that less mature motoneurones tend to die earliest, before the muscle is reinnervated. Those in the upper, more mature part of the motor pool survive longer but may still die during reinnervation. At least two types of glial cell were secondarily labelled by this method, distinguished by their response to nerve injury.

Both afferent and efferent connections influence postnatal growth of motoneuron dendrites in the rat.

Spinal motoneurons from mature rats, which had received one of 5 different surgical procedures neonatally, were retrogradely labelled with a cholera toxin-horseradish peroxidase conjugate and their dendritic morphology was analysed. The motoneurons studied were those innervating extensor digitorum longus and the procedures disrupted their motor and sensory connections to varying degrees. Disruption of motor contact with the target muscle retarded dendritic growth in the transverse plane, particularly in the dorso-medial direction. Disruption of sensory as well as motor contact resulted additionally in an increase in dendritic density in the longitudinal plane, largely along the rostral-caudal axis. The findings suggest that dendritic development of motoneurons is influenced by both afferent and efferent target contacts and that these effects can be differentiated.

Evidence for age-dependent changes in motoneuron dendritic morphology following neonatal nerve-crush in the rat.

Motoneurons supplying the extensor hallucis longus muscle of the rat were temporarily separated from the muscle by sciatic nerve-crush at five days postnatally. Such treatment permanently alters the reflex response of the affected motoneurons without the large-scale cell death associated with nerve-crush at birth. After reinnervation, the motoneurons were retrogradely labelled with cholera toxin subunit-B conjugated to horseradish peroxidase and the dendritic tree of each labelled cell was analysed. When compared to normal data, significantly higher levels of dendritic density were observed in the rostrodorsally orientated parts of the dendritic field. This was similar to that found previously for the same motor pool after nerve-crush at birth. However, in other parts of the field where a lower dendritic density was found after nerve-crush at birth, no change was seen after nerve-crush at five days. These data present evidence for the influence of sensory afferents on the development of motoneuron dendrites. Taken together with the previous findings after nerve-crush at birth, we suggest that the differential dendritic changes caused by neonatal nerve lesion contribute to an imbalance in the pattern of excitatory and inhibitory inputs to the motoneuron, which results either in cell death, or the abnormal activity seen in those motoneurons which survive.

Neonatal nerve injury causes long-term changes in growth and distribution of motoneuron dendrites in the rat.

Disruption of neuromuscular contact by nerve-crush during the early postnatal period results in the death of a large proportion of affected motoneurons. Increased activity and abnormal reflex responses are evident in those that survive. We have studied the aberrant dendritic morphology of surviving cells and have attempted to correlate the observed alterations in morphology with the above experimental findings. Motoneurons supplying the extensor hallucis longus muscles of the rat were retrogradely labelled with cholera toxin subunit-B conjugated to horseradish peroxidase. The dendritic tree of labelled cells was analysed in adult animals having undergone unilateral sciatic nerve-crush at birth. Unoperated control animals were also examined. Following nerve-crush at birth, total visible dendritic length was more than 30% smaller than control cells in the transverse plane. This decrease was confined largely to the medially directed segments of the dendritic field and appeared to be due to a reduction in dendritic branching combined with a failure to achieve the correct branch length. There was no overall change in total visible dendritic length in the longitudinal plane, but a reorientation of dendrites in favour of rostrodorsal regions was observed. There was no alteration in dendritic length in cells contralateral to the nerve injury. These results show that nerve injury during early postnatal development produces lasting changes in the distribution of motoneuron dendrites. The localized nature of these changes may explain the altered activity and induced death of motoneurons seen after neonatal nerve-crush.