Motoneuron and muscle fibre counts in normal and bilaterally innervated Xenopus hindlimbs.

Motoneurons of the Lumbar Lateral Motor Column (LMC) and muscle fibres of gastrocnemius and tibialis anterior were counted in juvenile Xenopus frogs, including normal animals and those reared with a single bilaterally innervated hindlimb (monopodal frogs). In many monopodal frogs, the single hind limb becomes hyperinnervated by a large number of motoneurons on the contralateral side in addition to the normal ipsilateral number, even after the completion of cell death. In frogs with hyper-innervated limbs, muscle fibre number was evaluated beyond that expected for a normal population, but this increase was not commensurate with the quantity of extra innervation. When taken together with previous findings which showed that the supporting capacity of individual fibres was not elevated, the conclusion is that the ability of the single limb to support extra motoneurons cannot be completely explained by a commensurate increased proliferation of muscle fibres resulting from the operation or the bilateral innervation. The results give further evidence against the hypothesis that motoneuron numbers are controlled solely by peripheral competition. The study also provides evidence that muscle fibre numbers are regulated in part by the quantity of motor innervation received by the limb.

The clinical specialist-staff nurse research team: a model for clinical research.

Increasing sophistication in nursing research has created a demand for preliminary work before the completion of a design for a major research project. This report presents a method which meets this need and provides research experience to nurses working on the clinical units. The role of the doctorally prepared clinical nurse specialist (CNS) in promoting collaborative research is presented. For the staff nurse the purpose of this collaboration was to facilitate an introduction of nursing research during the completion of her BSN. A secondary purpose was to provide the doctorally prepared CNS with preliminary data on the effects of nursing care on preterm infants with bronchopulmonary dysplasia. Through collaborative effort, a research protocol was developed and implemented with four preterm infants. The successful completion of this project provided the CNS with preliminary data and the staff nurse with a better understanding of the complexities as well as the rewards of clinical nursing research.

Peripheral competition in the control of sensory neuron numbers in Xenopus frogs reared with a single bilaterally innervated hindlimb.

Sensory neurons were counted in the hind-limb innervating spinal ganglia on both sides of juvenile Xenopus frogs which, as tadpoles, had had one hind limb bud amputated prior to innervation, and a channel made to allow innervation of the remaining limb bud from both sides. The total number of sensory neurons surviving on the two sides approximated the number on one side of normal frogs, the ipsilateral and contralateral numbers being negatively correlated. These effects differ markedly from the effects on motoneuron numbers, suggesting different control mechanisms of cell death in the two neuronal classes.

Aspects of peripheral motor system development.

The adult motor system is precisely connected, topographically and functionally. This order is reached during embryonic development through a sequence of mechanisms which increasingly resolve the adult patterns of connectivity. First is axon guidance, which gives rise to a pattern of axon outgrowth leading to an approximation of the adult projections. Second there is a period of motoneuron death, which eliminates motoneurons which have projected to inappropriate regions of the limb, and, it is conjectured, motoneurons which are functionally less well connected in terms of the behaviour of the whole system. Third there is a period of retraction of axon terminals which refines the pattern of connectivity from polyneuronal to mononeuronal innervation of the muscle fibres. The patterns of retraction may be determined by the same functional criteria as are used in the control of motoneuron death.

Muscle fibre types and innervation of the chick embryo limb following cervical spinal cord removal.

Several segments of spinal cord were removed from the cervical regions of stage-13 or -14 (day-2) chick embryos. After further incubation to day 17 or 18, the patterns of end-plate distribution and ATPase typing of muscle fibres in the anterior and posterior latissimus dorsi and the ulnimetacarpalis dorsalis, and the ATPase typing of the forearm muscles were examined. No differences from control embryos were found. The embryos had normal numbers of lateral motor column motoneurons in both the brachial and lumbar enlargements and the positions of motoneurons supplying the biceps as identified with retrograde horseradish peroxidase labelling were consistent with the normal patterns of motor projection into the limb. These results show that the fibre typing of limb muscles and their patterns of innervation are independent of descending inputs until just before hatching in the chick.

Innervation pattern of muscles of one-legged Xenopus laevis supplied by motoneurons from both sides of the spinal cord.

In Xenopus tadpoles one limb bud was removed before innervation and motoneurons from both sides of the spinal cord were induced to innervate the remaining limb. When examined after metamorphosis the motor innervation of the limb had the following characteristics. In agreement with previous findings a large proportion of contralateral motoneurons survived (51-82% of the ipsilateral numbers) and sent axons to the limb. By acetylcholinesterase staining and intracellular recording from muscle fibers of the response to electrical stimulation of the two limb innervations, the neuromuscular junctions from contralateral motoneurons were indistinguishable from those from the ipsilateral side in their morphology, spacing along the fiber, and physiological properties. Many single muscle fibers shared innervation from both sides of the cord by symmetrically placed spinal nerves. By the same techniques junctions in one-legged frogs were morphologically indistinguishable from those in normal frogs, but the quantal content of transmitter release was increased by up to 63%. Recording twitch and tetanic tensions from individual motor units from the gastrocnemius muscle showed that the one-legged animals had many more and smaller motor units than do normal frogs. We confirm that the hind-limb musculature has the ability, normally unexpressed, to sustain, through the period of normal developmental cell death, up to twice the usual number of motoneurons. In maturity, motoneurons accommodate themselves to the limb muscles by making fewer than the normal number of synapses. The above suggests that developmental motoneuron death is not primarily a mechanism for adjusting the number of motoneurons to the size of the peripheral musculature and is likely to be related to mechanisms for securing specific neuromuscular connections.

Motoneuron death in the embryo.

In the developing embryo, 50% or more of the young motoneurons supplying the limb die. Amputation studies suggest that motoneurons depend on acquiring trophic factors from the limb. However, access to the trophic factors is under complex control since all motoneurons, whether destined to live or die, send axons into the limb. Axon invasion is not random but under the control of an axon guidance mechanism. Motoneurons probably compete for access to trophic factors at the neuromuscular junction. In order to compete, the motor axons must first be guided to specific limb regions. Failure causes death. Recent evidence suggests that motoneurons are specified for small localities within individual muscles and that the failure rate may be high enough to account for a large proportion of motoneuron deaths. The mechanisms of the implied recognition between motoneurons and muscles are unknown but may depend on positionally determined cell surface markers, histochemical compatibility, and functional congruence.

The distribution of muscle fibre types in chick embryo wings transplanted to the pelvic region is normal.

Chick embryo wing buds were transplanted to the pelvic region in place of, or in addition to, the hindlimb bud prior to innervation. The wrist muscle ulnimetacarpalis dorsalis (umd) was innervated by middle-dorsal or middle-ventral motoneurons in the lumbar lateral motor column (LMC) in a rostrocaudal position which varied with the rostrocaudal position of the wing. Despite the heterotopic innervation the subsequent development of the distributions of fast and slow muscle fibres, as judged by ATPase staining, was normal in all muscles examined. The pattern of innervation in the umd, as judged by acetylcholinesterase staining also developed normally. It is probable that muscle fibre type is intrinsically, not neurogenically, determined.

Development and motor innervation of a distal pair of fast and slow wing muscles in the chick embryo.

The chick wrist muscle ulnimetacarpalis dorsalis (umd) has two heads. Using myosin ATPase and acetylcholinesterase (ACh.E) staining it was shown that one of the heads is composed almost entirely of acid-stable muscle fibres with multiple end plates (slow muscle fibres) and the other of acid-labile fibres with single end plates (fast muscle fibres). The development of the muscle was traced from E7 (Stage 32-33) when it is a relatively homogeneous mass, to E18. The two heads of the muscle are first distinguishable, by ATPase staining, at E8 (Stage 33-34) prior to their cleaving. Both heads of the muscle are innervated by motoneurons positioned laterally in the lateral motor column in spinal segments 15 and 16. There is no observable difference in the positions of the motoneuron pools to the two heads. At E18 the motoneurons innervating the fast head tend to be slightly larger than those innervating the slow head.

Target dependency of developing motoneurons in Xenopus laevis.

The effect of complete and partial limb bud removal on motoneuron survival was studied in Xenopus laevis tadpoles. Amputation of both hind limb buds at stage 49 before limb innervation begins caused the subsequent death of all motoneurons. This result confirms that there are no exceptions to the rule that developing motoneurons must contact the limb to survive. Partial removals of the limb at early stages of innervation caused the subsequent death of motoneurons that normally project to the deleted segments while motoneurons for the remaining segments survived. Before dying, the motoneurons deprived of their normal targets invaded the remaining limb segments thereby forming erroneous projections. Since the motoneurons died despite contacting limb tissue, it is concluded that they must be dependent on contact with specific limb regions and that errors of projection lead to death. Since large-scale errors of the type induced by this experiment affect only a minority of developing motoneurons in normal embryos, the possibility of small-scale errors is discussed in an attempt to equate all naturally occurring motoneuron death with error correction.

Selective bilateral motor innervation in Xenopus tadpoles with one hind limb.

Bilateral innervation of a single hindlimb bud was induced by amputating the other limb bud and disrupting the barriers between the two sides. Though the routes of the crossed nerves were necessarily abnormal, the motor projections that developed subsequently were normal as determined by horseradish peroxidase tracing. The limb therefore appears to be innervated selectively, each region being invaded and/or synapsed with only by motoneurones at particular locations. The numbers of motoneurones surviving after metamorphosis were almost normal on both sides provided the operation was done before motor invasion of the limb bud begins. From this it is argued that the axons were probably guided actively to their correct destinations. Without such guidance, axons would probably not have been able to find their correct termination sites and motoneurons survival would therefore have been depressed. The normal motoneurone numbers also imply that the single limb was supporting twice its usual quota of motoneurones. The hypothesis that motoneurones compete in the limb for survival is therefore not supported.

Axon regeneration by developing limb motoneurones in Xenopus laevis.

Newly generated motoneurones invading the limb bud were labelled with horseradish peroxidase to identify them later in development. They were then axotomized by amputating the limb bud. The limb bud was replaced immediately in some animals. After the period of normal motoneurone death, which falls a few days after the operation, many horseradish peroxidase labelled motoneurones were still alive provided the limb bud had been replaced. Without a limb bud, labelled motoneurones were few or non-existent. The conclusion is that in spite of their immaturity, the labelled motoneurones must have grown new axons into the limb for their death to have been avoided. Axon regeneration therefore seems to be inherent to motoneurones of any age.

Ventral horn cell counts in a Xenopus with naturally occurring supernumerary hind limbs.

In a Xenopus toad, two partially functioning supernumerary hind limbs developed naturally on the right side and were innervated by the ipsilateral side of the spinal cord. The number of ventral horn cells on the right was 18% higher than on the left while the combined mass of limb muscle on the right was estimated to be a 100% greater. This result corroborates studies with transplanted supernumerary limbs.

Retrograde axonal transport of horseradish peroxidase for determining motor projection patterns to the developing limb in Xenopus.

Horseradish peroxidase (HRP) injected into developing limb buds of Xenopus laevis tadpoles is carried by retrograde axonal transport to the somata of motoneurones in the ventral horn. Small injection of 10% HRP were found to remain well localised to specified sites in the limb bud. Two types of labelled cells were found: diffusely labelled and granular labelled. Diffusely labelled cells result from axonal damage in the presence of HRP. Granular labelled cells result only from uptake of HRP from the region of the axon endings. No gradular uptake was found from axon shafts. It is concluded that the distribution of granular labelled cells accurately reflects the region of the ventral horn projecting to the site of injection in the limb.