Neuromuscular development in the avian paralytic mutant crooked neck dwarf (cn/cn): further evidence for the role of neuromuscular activity in motoneuron survival.

Neuromuscular transmission and muscle activity during early stages of embryonic development are known to influence the differentiation and survival of motoneurons and to affect interactions with their muscle targets. We have examined neuromuscular development in an avian genetic mutant, crooked neck dwarf (cn/cn), in which a major phenotype is the chronic absence of the spontaneous, neurally mediated movements (motility) that are characteristic of avian and other vertebrate embryos and fetuses. The primary genetic defect in cn/cn embryos responsible for the absence of motility appears to be the lack of excitation-contraction coupling. Although motility in mutant embryos is absent from the onset of activity on embryonic days (E) 3-4, muscle differentiation appears histologically normal up to about E8. After E8, however, previously separate muscles fuse or coalesce secondarily, and myotubes exhibit a progressive series of histological and ultrastructural degenerative changes, including disarrayed myofibrils, dilated sarcoplasmic vesicles, nuclear membrane blebbing, mitochondrial swelling, nuclear inclusions, and absence of junctional end feet. Mutant muscle cells do not develop beyond the myotube stage, and by E18-E20 most muscles have almost completely degenerated. Prior to their breakdown and degeneration, mutant muscles are innervated and synaptic contacts are established. In fact, quantitative analysis indicates that, prior to the onset of muscle degeneration, mutant muscles are hyperinnervated. There is increased branching of motoneuron axons and an increased number of synaptic contacts in the mutant muscle on E8. Naturally occurring cell death of limb-innervating motoneurons is also significantly reduced in cn/cn embryos. Mutant embryos have 30-40% more motoneurons in the brachial and lumbar spinal cord by the end of the normal period of cell death. Electrophysiological recordings (electromyographic and direct records form muscle nerves) failed to detect any differences in the activity of control vs. mutant embryos despite the absence of muscular contractile activity in the mutant embryos. The alpha-ryanodine receptor that is genetically abnormal in homozygote cn/cn embryos is not normally expressed in the spinal cord. Taken together, these data argue against the possibility that the mutant phenotype described here is caused by the perturbation of a central nervous system (CNS)-expressed alpha-ryanodine receptor. The hyperinnervation of skeletal muscle and the reduction of motoneuron death that are observed in cn/cn embryos also occur in genetically paralyzed mouse embryos and in pharmacologically paralyzed avian and rat embryos. Because a primary common feature in all three of these models is the absence of muscle activity, it seems likely that the peripheral excitation of muscle by motoneurons during normal development is a major factor in regulating retrograde muscle-derived (or muscle-associated) signals that control motoneuron differentiation and survival.

Morphology of single vestibulospinal collaterals in the upper cervical spinal cord of the cat: III collaterals originating from axons in the ventral funiculus ipsilateral to their cells of origin.

Some vestibulospinal pathways are composed of a homogeneous collection of axons with similar intraspinal collaterals. Other pathways contain axons whose collaterals vary in terms of shape, distribution, and complexity. The purpose of the present study was to extend the study of homogeneity versus heterogeneity of vestibulospinal axons to vestibulospinal axons that travel in the ventral funiculus ipsilateral to their cells of origin. Collaterals of these axons were stained following extracellular injections of Phaseolus vulgaris-leucoagglutinin in rostral parts of the medial and descending vestibular nuclei. All collaterals found in C2 and C3 were reconstructed. Collaterals arising from small diameter (0.5 to 2.9 microns) axons usually consisted of a single main branch with short side branches. The termination zones of most of these collaterals formed a narrow path in lamina VIII, but the location of this pathway was highly variable. Collaterals arising from large-diameter (3.0-6.1 microns) axons were usually more complex and consisted of many branches with en passant and terminal boutons that were located in motoneuron nuclei as well as laminae VIII and VII. Despite a relationship between termination zone and the position of the parent axon in the ventral funiculus, the variability in collaterals from large-diameter axons precluded a simple classification scheme. These results demonstrate that diversity, instead of homogeneity, is a characteristic feature of vestibulospinal axons that originate from the medial and descending vestibular nuclei and travel in the ipsilateral ventral funiculus. This pathway is therefore composed of multiple anatomical subunits that, as individuals, may selectively coordinate the activity of specific combinations of interneurons and motoneurons.

Morphology of single vestibulospinal collaterals in the upper cervical spinal cord of the cat: I. Collaterals originating from axons in the ventromedial funiculus contralateral to their cells of origin.

Vestibulospinal neurons in the medial and descending vestibular nuclei have widespread bilateral terminations in the upper cervical spinal cord. These terminations arise from axons travelling in several funiculi, including the ventromedial, ventrolateral, lateral, and dorsolateral funiculi in addition to the dorsal columns. The purpose of the present study was to examine the morphology of single vestibulospinal collaterals which terminate in the upper cervical spinal cord and which originate from axons located in one of these funicular pathways, the ventromedial funiculus, contralateral (cVMF) to their cells of origin in the vestibular nuclei. The 32 collaterals described were selected from two separate sets of experiments which took advantage of different techniques. Nineteen of the collaterals were labelled following Phaseolus vulgaris leucoagglutinin (PHA-L) injections into the medial vestibular nucleus and medial regions of the descending vestibular nucleus. The remaining 13 collaterals originated from physiologically identified vestibulospinal axons that were stained after intra-axonal injections of horseradish peroxidase (HRP). The combined projection of all cVMF axon collaterals spread from laminae V to IX, and included the central cervical nucleus. There was a high degree of variability in the pattern of terminations of individual collaterals. This variability was more pronounced among PHA-L-labelled collaterals than HRP-labelled collaterals whose terminations were restricted to laminae VIII and IX. Some PHA-L-labelled collaterals had terminations which were focused within a single lamina, whereas others had termination zones spanning as many as four laminae. The differences between collaterals were compounded when the characteristics of branching patterns were considered. Some collaterals which occupied similar termination zones had different branching structures.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphology of single vestibulospinal collaterals in the upper cervical spinal cord of the cat. II. Collaterals originating from axons outside the ventral funiculi.

Recent studies have shown that vestibulospinal axons reach the upper cervical spinal cord of the cat via several different funicular routes. The purpose of this study was to describe the projections of those axons travelling outside the well-recognized pathways in the ventral funiculi. These axons are located in the dorsal columns, dorsolateral funiculi, and lateral funiculi. Collaterals of these axons were stained following extracellular injections of Phaseolus vulgaris leucoagglutinin in the medial and descending vestibular nuclei. The trajectories of individual collaterals were reconstructed from serial histological sections. Collaterals arising from axons in the same funiculus usually had the same characteristic appearance. Axons in the lateral funiculi, ipsilateral or contralateral to their cells of origin, gave rise to collaterals that had a simple structure and usually followed a horizontal trajectory across laminae VII and VIII. The boutons of these collaterals were distributed throughout the mediolateral extent of laminae VI and VII and the dorsal half of lamina VIII. In contrast, axons in the dorsolateral funiculi, ipsilateral or contralateral to their cells of origin, terminated primarily in laminae IV and V. Many collaterals of these axons projected either rostrally or caudally and had a narrow mediolateral distribution. The combined distribution of boutons from collaterals originating from axons in the dorsal columns included the dorsal horn and intermediate zone. Although these collaterals were less common and formed a heterogeneous group, they were easily distinguished from collaterals originating from axons travelling in other funiculi. These results indicate that vestibulospinal axons travelling outside the ventral funiculi comprise several distinct systems. Each system travels by a different funicular route and is distinguished by differences in collateral morphology and termination zones.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiplicity of vestibulospinal projections to the upper cervical spinal cord of the cat: a study with the anterograde tracer Phaseolus vulgaris leucoagglutinin.

The distribution and frequency of vestibulospinal axons and boutons in the upper cervical spinal cord of the cat were investigated. The anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L) was injected into discrete regions of the vestibular nuclei, including the medial and descending nuclei, as well as small regions of the lateral vestibular nucleus along its medial border with the medial vestibular nucleus. In contrast to previous reports, labelled vestibulospinal axons were not found to be restricted to the ventromedial and ventrolateral funiculi, but were also observed bilaterally in the lateral funiculi, the dorsolateral funiculi and the dorsal columns. The diameter of these axons ranged from 0.5 to 7.4 microns. Labelled boutons were found bilaterally from lamina IV to IX as well as in lamina X. Contralateral to the injection site, boutons were frequently found as far dorsal as lamina II. Ipsilaterally, boutons were found this far dorsal in only one experiment. There was a dense projection to the contralateral central cervical nucleus, while very few, if any, boutons were observed in the ipsilateral central cervical nucleus. In each experiment, the density of boutons was greater in the rostral cervical segments than in more caudal segments. The "new" vestibulospinal projections to the dorsal horn and central cervical nucleus were confirmed in separate experiments using retrograde transport of horseradish peroxidase. These results show that vestibulospinal axons project to the upper cervical spinal cord via multiple funicular paths. The rich terminations of these axons outside of the ventral horn, as well as in the neck motoneuron nuclei, indicate that vestibulospinal projections must play a wide variety of functions in addition to their well-documented role in the direct control of head movement.