Properties of motor units of the frog sartorius muscle

1. The mechanical properties of single motor units in the sartorius muscle of the frog Litoria aurea were examined during single shock and repetitive stimulation of motor axons. 2. The tetanic tension developed by motor units lay in the range 1-40% of whole muscle tension with two peaks in the distribution, in the range 5-10% and 25-30%. The large units had briefer times-to-peak for the twitch than the small units and were more readily fatigued during prolonged repetitive stimulation. 3. Histological examination of the muscle gave a count of 620 muscle fibres with a diameter range of 28-128 mum. Cholinesterase stained preparations showed that the majority of muscle fibres had several nerve terminals (mean 3, range 1-5). 4. Muscle fibres received their multiple innervation from different axons (polyneuronal) or branches of the same axon (multiterminal). The presence of polyneuronal innervation of muscle fibres was confirmed by a comparison of the tensions when each of a pair of motor units was stimulated alone and when they were stimulated together. The tension excess, or overlap, was up to 60% when expressed in terms of the tension developed by either unit alone. Motor units developing similar amounts of tension tended to show more overlap in their innervation than units with very different tensions. 5. An estimate of the amount of multiterminal innervation gave variable results but could account for up to 60% of a motor unit's tension. No correlation could be detected between the values for multiterminal innervation and any other measured parameter. However, it is argued that because of the limitations of the measurements the existence of a relationship between the extent of multiterminal or polyneuronal innervation and the mechanical properties of the motor unit cannot be excluded.     

Self-reinnervated cat medial gastrocnemius muscles. II. analysis of the mechanisms and significance of fiber type grouping in reinnervated muscles

1. The technique of glycogen depletion was used to determine whether regenerating motor axons reestablish the normal regionalization of motor units (MUs) in the cat medial gastrocnemius (MG) muscle, 2) whether the extent of clumping between MU fibers and/or type grouping of muscle fibers progressively increases with a decrease in reinnervated MU numbers, and 3) whether the pattern of innervation can explain why MUs fail to increase significantly in size when the cut nerve is sutured directly to the muscle, even when few axons make functional connections. 2. Distributions of MU fibers were analyzed in 5 normal and 14 reinnervated cat MG muscles 4.5-16 mo after sectioning of its nerve and suturing of the proximal end to the distal nerve sheaths (N-N suture) or directly to the muscle fascia (N-M suture). Muscle unit distributions were quantified according to location, territory size, density, and extent of clumping between fibers from the same MU. 3. Normal MU fibers were regionalized within five regions along the muscle's longitudinal and transverse axes. Reinnervated MUs were located within similar regions, indicating that regenerating axons follow the major proximal nerve branches to restore normal compartmentalization. 4. Muscle unit fibers were diffusely scattered within discrete MU territories in normal muscles. Territory size tended to increase with MU size, whereas density of muscle unit fibers within the territory decreased. 5. Territories increased with MU size after N-N suture but were smaller and showed little size variation after N-M suture. The extent of muscle unit fiber clumping was inversely related to the number of reinnervated MUs. On average, the extent of clumping was substantially higher in muscles reinnervated after N-M suture. These results indicate that distal nerve sheaths facilitate proximal axon branching, which establishes MU territory size. Once the territory is established, motor axons branch distally to increase MU size, which in turn compensates for reduced MU numbers. 6. Muscles reinnervated by < 80% of the MUs exhibited fiber type grouping of type I fibers, and on average the extent of clumping was substantially higher in muscles reinnervated after N-M suture. With less innervation, type grouping increased inversely with the number of reinnervated MUs. However, for a similar number of MUs, type I fiber type grouping was substantially higher in muscle reinnervated after N-M suture. Type grouping therefore reflects muscle unit fiber clumping under conditions where MU size increased (N-N suture) or MU territory size decreased (N-M suture).     

Fusimotor-free spindles in reinnervated muscles of neonatal rats treated with nerve growth factor

Crushing the nerve to the medial gastrocnemius muscle in newborn rats and administering nerve growth factor afterwards results in a reinnervated muscle containing supernumerary muscle spindles. The structure and innervation of 88 spindles in the reinnervated muscles were reconstructed from serial thick and thin transverse sections at 30-35 days after the nerve crush, and compared to those of five control spindles. The spindles consisted of one to four small-diameter encapsulated fibers with features of nuclear chain intrafusal fibers, or infrequently a nuclear bag intrafusal fiber. Some of the spindles were located within a capsule that also contained an extrafusal fiber. Each spindle was innervated by an afferent with features of the primary afferent. The density of secondary afferents was lower in reinnervated muscles than in controls. Endplates were observed on extrafusal fibers in the experimental muscles, attesting to restoration of skeletomotor (alpha) innervation after the nerve crush. However, 78% of the experimental spindles were entirely devoid of efferent innervation. The remainder received either one or two fusimotor (gamma) axons or a skeletofusimotor (beta) axon, compared to the six to eight motor axons that innervated control spindles. The presence of supernumerary spindles composed of fibers that resemble normal intrafusal fibers in the absence of motor innervation suggests that afferents alone can induce the formation and subsequent differentiation of intrafusal fibers in nerve-crushed muscles of neonatal rats. In addition, the paucity of gamma innervation in nerve-crushed muscles suggests that immature gamma neurons are more susceptible than spindle afferents or alpha efferents to cell death after axotomy at birth.     

Laser ablation of Drosophila embryonic motoneurons causes ectopic innervation of target muscle fibers

We have tested the effects of neuromuscular denervation in Drosophila by laser-ablating the RP motoneurons in intact embryos before synaptogenesis. We examined the consequences of this ablation on local synaptic connectivity in both 1st and 3rd instar larvae. We find that the partial or complete loss of native innervation correlates with the appearance of alternate inputs from neighboring motor endings and axons. These collateral inputs are found at ectopic sites on the denervated target muscle fibers. The foreign motor endings are electrophysiologically functional and are observed on the denervated muscle fibers by the 1st instar larval stage. Our data are consistent with the existence of a local signal from the target environment, which is regulated by innervation and influences synaptic connectivity. Our results show that, despite the stereotypy of Drosophila neuromuscular connections, denervation can induce local changes in connectivity in wild-type Drosophila, suggesting that mechanisms of synaptic plasticity may also be involved in normal Drosophila neuromuscular development.     

A morphological study of cat dorsal spinocerebellar tract neurons after intracellular injection of horseradish peroxidase

This work represents an attempt to elucidate structural features of electrophysiologically characterized, individual cat dorsal spinocerebellar tract (DSCT) neurons by using intracellular application of horseradish peroxidase (HRP). Intracellular recordings and HRP injections were made in DSCT neurons of the Clarke's column in cat lumbar (L3) spinal cord. The units were identified by antidromic invasion following electrical stimulation of the ipsilateral dorsolateral funiculus at C1. In addition, sensory inputs to the DSCT neurons were determined by natural (adequate) stimuli applied to the hind limb with intact innervation. The morphological analysis is based on data obtained from 19 well-stained electrophysiologically identified neurons located in Clarke's column. Thirteen of these units received excitatory sensory inputs from muscle receptors, two were activated by cutaneous afferents only, and four had a convergent (muscle + cutaneous) input. The DSCT--muscle cells were equivalent to the large Clarke cells (class C of Leowy, '70). Their dendrites were oriented primarily in the rostro--caudal direction (up to 2500 micron) and appeared generally smooth except for some branchlets. In four of these cells, the axon was traced into the lateral funiculus. In light microscopic analysis there was no evidence that axon collaterals arose from these axons during the initial trajectory through the spinal grey matter. The four DSCT--convergent neurons were similar in shape to the DSCT--muscle units although they appeared to have somewhat smaller cell bodies. Of the two DSCT--cutaneous neurons one was found to be of the B type, with the dendritic tree having fewer branches and oriented mainly in the medio--lateral direction. The other cell, however, turned out to be similar in appearance to the C type Clarke neurons.     

NMDA receptor-mediated control of presynaptic calcium and neurotransmitter release

Before action potential-evoked Ca2+ transients, basal presynaptic Ca2+ concentration may profoundly affect the amplitude of subsequent neurotransmitter release. Reticulospinal axons of the lamprey spinal cord receive glutamatergic synaptic input. We have investigated the effect of this input on presynaptic Ca2+ concentrations and evoked release of neurotransmitter. Paired recordings were made between reticulospinal axons and the neurons that make axo-axonic synapses onto those axons. Both excitatory and inhibitory paired-cell responses were recorded in the axons. Excitatory synaptic inputs were blocked by the AMPA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 microM) and by the NMDA receptor antagonist 2-amino-5-phosphonopentanoate (AP-5; 50 microM). Application of NMDA evoked an increase in presynaptic Ca2+ in reticulospinal axons. Extracellular stimulation evoked Ca2+ transients in axons when applied either directly over the axon or lateral to the axons. Transients evoked by the two types of stimulation differed in magnitude and sensitivity to AP-5. Simultaneous microelectrode recordings from the axons during Ca2+ imaging revealed that stimulation of synaptic inputs directed to the axons evoked Ca2+ entry. By the use of paired-cell recordings between reticulospinal axons and their postsynaptic targets, NMDA receptor activation was shown to enhance evoked release of transmitter from the axons that received axoaxonic inputs. When the synaptic input to the axon was stimulated before eliciting an action potential in the axon, transmitter release from the axon was enhanced. We conclude that NMDA receptor-mediated input to reticulospinal axons increases basal Ca2+ within the axons and that this Ca2+ is sufficient to enhance release from the axons.     

Polyneural innervation in the psoas muscle of the developing rat

Polyneural innervation was studied in the psoas muscle in developing rats from P4 till P25 and at adult age, with the combined silver-acetylcholinesterase technique. Nerve endings were counted, and end-plates were measured. These data were compared with such data in the human. The end of polyneural innervation in the rat (around P20) and in the human (around 12 weeks postterm age) in both cases coincides with a transformation in motor behavior and postural control. The rat's psoas muscle at early stages is less heavily innervated than this muscle in the human. Up to three axons per motor end-plate were counted at P4, but in the human up to five axons at 25 weeks of post menstrual age. This difference might be related to the lower percentage of type I muscle fibers in the rat.     

Synaptic competition during the reformation of a neuromuscular map

We have been studying the mechanisms whereby pools of motor neurons establish a rostrocaudal bias in the position of their synapses in some skeletal muscles. The serratus anterior (SA) muscle of the rat displays a rostrocaudal topographic map before birth, and the topography is re-established after denervation. In this report, we explore the potential role of synaptic competition between innervating axons as a means of generating topographic specificity. We followed the progress of the reformation of this map in neonatal animals under conditions that enhanced the likelihood of observing synaptic competition. This was accomplished by forcing caudal axons to regenerate ahead of rostral axons onto a surgically reduced SA muscle. In this way, caudal (C7) motor neurons had unopposed access to vacated synaptic sites on the remaining rostral half of the SA before the return of the rostral (C6) axons. Intracellular recording revealed that 2 d after the second denervation, most of the reinnervated end plates contained only axons from the C7 branch; the remaining reinnervated end plates received input from C6 only or were multiply innervated by C6 and C7 axons. After 6 d, the pattern was reversed, with most end plates innervated exclusively by C6. After 17 d, axons from C6 were the sole input to reinnervated end plates. During the transition from C7- to C6-dominated input, at end plates coinnervated by C6 and C7 axons, the average quantal content from C6 was the same as that from C7; after 7 d, the quantal content of C6 was greater than that of C7. We have thus developed an experimental situation in which the outcome of synaptic competition is predictable and can be influenced by the positional labels associated with axons from different levels in the spinal cord.     

Myelinated sensory and alpha motor axon regeneration in peripheral nerve neuromas

Histochemical staining for carbonic anhydrase and cholinesterase (CE) activities was used to analyze sensory and motor axon regeneration, respectively, during neuroma formation in transected and tube-encapsulated peripheral nerves. Median-ulnar and sciatic nerves in the rodent model permitted testing whether a 4 cm greater distance of the motor neuron soma from axotomy site or intrinsic differences between motor and sensory neurons influenced regeneration and neuroma formation 10, 30, and 90 days later. Ventral root radiculotomy confirmed that CE-stained axons were 97% alpha motor axons. Distance significantly delayed axon regeneration. When distance was negligible, sensory axons grew out sooner than motor axons, but motor axons regenerated to a greater quantity. These results indicate regeneration differences between axon subtypes and suggest more extensive branching of motor axons within the neuroma. Thus, both distance from injury site to soma and inherent motor and sensory differences should be considered in peripheral nerve repair strategies.     

Early events in myofibrillogenesis and innervation of skeletal, sound-generating muscle in a teleost fish

The plainfin midshipman, Porichthys notatus, generates acoustic communication signals through the rapid contraction of a pair of vocal (sonic) muscles attached to the walls of the swimbladder. Light and electron microscopic methods were used to study two aspects of sonic muscle ontogeny: 1) the development and transformation of myotubes into muscle fibers and 2) innervation, including the formation of sonic neuromuscular junctions and the myelination of sonic motor axons. Sonic motor axons are associated with sonic mesenchyme during its initial migration away from occipital somites. However, myofibrillogenesis, the formation of neuromuscular junctions, and axon myelination do not occur until sonic mesenchyme reaches its final destination (i.e., the swimbladder). A continuum of developing myotubes is present rather than two temporally distinct populations of primary and secondary myotubes as observed for skeletal muscles in mammalian and avian species. Potential reasons for the lack of primary and secondary myotubes are considered, including the functional homogeneity of the sonic motor system and the sonic muscle's unique architecture, namely its direct attachment to the wall of the swimbladder.     

Differential survival of isolated portions of crayfish axons

Electron microscopic studies show that transplanted segments of sensory axons of varying lengths degenerate within 7-14 days whereas transplanted segments of crustacean motor axons survive morphologically intact for 20-30 days. The middle portion of an isolated motor axon segment degenerates less rapidly than portions of the same axon located nearer the periphery or nearer the ventral nerve cord. One week after transplantation, glial cells appear to phagocytize sensory axons whereas glial cells around motor axons appear to hypertrophy and to have more rough endoplasmic reticulum. After three weeks, motor axons also appear to be phagocytized by glial cells. These data suggest that the glia surrounding isolated motor axons can change from a supportive to a destructive function, whereas glial cells surrounding severed sensory axons primarily have a destructive function. These and other data also indicate that crustacean motor axons receive significant trophic inputs from their own perikaryon, from post-synaptic contacts, and from adjacent glial cells. The possibility that adjacent healthy cells may supply metabolically deficient cells with needed substances could be a significant adaptive advantage for the evolution of multicellular organisms.     

Hypomyelination alters K+ channel expression in mouse mutants shiverer and Trembler

Voltage-gated K+ channels are localized to juxtaparanodal regions of myelinated axons. To begin to understand the role of normal compact myelin in this localization, we examined mKv1.1 and mKv1.2 expression in the dysmyelinating mouse mutants shiverer and Trembler. In neonatal wild-type and shiverer mice, the focal localization of both proteins in axon fiber tracts is similar, suggesting that cues other than mature myelin can direct initial K+ channel localization in shiverer mutants. In contrast, K+ channel localization is altered in hypomyelinated axonal fiber tracts of adult mutants, suggesting that abnormal myelination leads to channel redistribution. In shiverer adult, K+ channel expression is up-regulated in both axons and glia, as revealed by immunocytochemistry, RNase protection, and in situ hybridization studies. This up-regulation of K+ channels in hypomyelinated axon tracts may reflect a compensatory reorganization of ionic currents, allowing impulse conduction to occur in these dysmyelinating mouse mutants.     

Both basic fibroblast growth factor and ciliary neurotrophic factor promote the retention of polyneuronal innervation of developing skeletal muscle fibers

At birth, nearly all rat muscle fibers receive synaptic inputs from more than one motoneuron at a single end-plate site. By the end of the third postnatal week all but one of these inputs has been eliminated. During this loss of polyneuronal innervation, developing neuromuscular synapses compete with one another. Although the nature of this competition is not known, it is commonly assumed that it is mediated through differential activity of the competing inputs. One means by which such differential activity might be translated into a biological signal would be if the synapses compete in an activity-dependent manner for a scarce supply of neurotrophic molecules. A prediction of this hypothesis is that excess quantities of such trophic molecules will reduce competition and thereby slow down or abolish the normal loss of polyneuronal innervation. In newborn rats, the effects of injection of either basic fibroblast growth factor (bFGF) or ciliary neurotrophic factor (CNTF) on the outcome of neuromuscular synapse elimination were investigated. Daily injections of either bFGF or CNTF were made for 1 week into the lateral gastrocnemius muscle beginning at the postnatal age of 2 days. The amount of polyneuronal innervation of fibers in trophic molecule-injected muscles and saline-injected contralateral muscles was assayed using intracellular recording methods. For both bFGF- and CNTF-injected muscles, an increase in the percentage of polyneuronally innervated fibers relative to saline-injected muscles was noted. For bFGF-injected muscles, the amount of polyneuronal innervation remained at nearly 60% as late as the postnatal age of 14 days (P14). This is the amount of polyneuronal innervation found at age 6 days in normal animals. Nearly 40% of the fibers of CNTF-injected muscles remained polyneuronally innervated at age P14, the amount expected at age 9 days. These results indicate that both bFGF and CNTF exert powerful and long-lasting effects on developing neuromuscular synapses.     

Differential production of IL-10 by T cells and monocytes of HIV-infected individuals: association of IL-10 production with CD28-mediated immune responsiveness

The present study determines the proportions of unmyelinated cutaneous axons at the dermal-epidermal junction in glabrous skin and of myelinated and unmyelinated axons in the sural and medial plantar nerves that immunostain for subunits of the ionotropic glutamate receptors. Approximately 20% of the unmyelinated cutaneous axon profiles at the dermal-epidermal junction immunostain for either N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), or kainate receptor subunits. These findings are consistent with previous observations that NMDA and non-NMDA antagonists ameliorate nociceptive behaviors that result from noxious peripheral stimulation. In the sural nerve, where the large majority of myelinated fibers are sensory, approximately half of the myelinated axon profiles immunostain for the NMDA receptor 1 (R1) subunit, 28% immunostain for the glutamate receptor 1 (GluR1) AMPA subunit, and 11% for the GluR5,6,7 kainate subunits. Even higher proportions immunostain for these receptors in the medial plantar nerve, a mixed sensory and motor nerve. In the sural nerve, 20% of the unmyelinated axon profiles immunostain for NMDAR1 and only 7% label for GluR1 or GluR5,6,7. Because the sural nerve innervates hairy skin, these data suggest that glutamate will activate a higher proportion of unmyelinated axons in glabrous skin than in hairy skin. Measurements of fiber diameters indicate that all sizes of myelinated axon profiles, including Adelta and Abeta, are positively labeled for the ionotropic receptors. The presence of glutamate receptors on large-diameter myelinated axons suggests that these mechanosensitive receptors, presumably transducing touch and pressure, may also respond to local glutamate and thus be chemosensitive.     

Sequelae of massive fluid resuscitation in trauma patients

Morphological and physiological characteristics of the two major motor axons supplying the commonly studied ventral longitudinal muscle fibers (6 and 7) of third-instar Drosophila melanogaster larvae were investigated. The innervating terminals of the two motor axons differ in the size of their synapse-bearing varicosities. The terminal with the larger varicosities also fluoresces more brightly when stained with the vital fluorescent dye 4-(4-diethylaminostyryl)-N-methylpyridinium iodide (4-Di-2-Asp) and occupies a larger total contact area on the muscle fiber. Through selective simultaneous recording of synaptic currents from identified boutons in living preparations during elicitation of synaptic potentials, it was shown that the axon with the smaller varicosities generates a large excitatory junction potential (EJP) in muscle 6 and that the axon with the larger varicosities generates a smaller EJP. Short-term facilitation is more pronounced for the smaller EJP. In preparations treated with 4-Di-2-Asp, the fluorescence of smaller varicosities increases with stimulation that elicits the large EJPs, indicating an activity-dependent entry of calcium that enhances mitochondrial fluorescence. The differences in morphology and physiology of the two axons are similar to, though less pronounced than, those observed in "phasic" and "tonic" motor axons of crustaceans.     

Expression of NGF in sympathetic neurons leads to excessive axon outgrowth from ganglia but decreased terminal innervation within tissues

The effects of nerve growth factor (NGF) on sympathetic axon growth were investigated by generating transgenic mice in which the beta subunit of NGF was expressed in sympathetic neurons using the human dopamine beta-hydroxylase (DBH) promoter. In DBH-NGF mice, the sympathetic trunk and nerves growing to peripheral tissues were enlarged and contained an increased number of sympathetic fibers. Although sympathetic axons reached peripheral tissues, terminal sympathetic innervation within tissues was decreased in DBH-NGF mice. This effect could be reversed in the pancreas by overexpression of NGF in pancreatic islets. The observations are consistent with a model in which NGF gradients are not required to guide sympathetic axons to their targets, but are required for the establishment of the normal density and pattern of sympathetic innervation within target tissues.     

Tyrosine hydroxylase- and nitric oxide synthase-immunoreactive nerve fibers in mitral valve of young adult and aged Fischer 344 rats

Using confocal fluorescence microscopy we studied, in whole mounts of heart mitral valves of young adult and aged Fischer 344 rats, the distribution of nerves containing the catecholamine marker tyrosine hydroxylase (TH) or the synthetic enzyme marker for nitric oxide, nitric oxide synthase (NOS). TH-IR was localized in two separate nerve plexuses which do not intermingle. The 'major' plexus arose from the annulus region, traversed the basal zone of the valve, and ramified in the intermediate zone to form a dense network of fine fibers. The 'minor' plexus was restricted to the distal zone and originated from bundles that ascended the chordae tendineae to enter the valve cusp. A concentric zone located between the major and minor plexuses was devoid of TH-IR nerve fibers. Both plexuses demonstrated (i) nerves that contained numerous varicosities along the length of each fiber, (ii) many terminal axons and (iii) different shaped terminal axon endings. With age, the density of TH-IR innervation in the mitral valve was markedly reduced; and nerve fibers of the minor plexus were limited to the chordae tendinae, without extending into the valve cusp itself. NOS-IR fibers in the mitral valve formed a loose network that extended from the annulus to more than halfway down the cusp. The varicose beads of the terminal NOS-IR axons appeared to become progressively smaller and less intensely fluorescent until they disappeared at the terminal endings, which showed no specializations. No NOS-IR fibers were observed in the distal zone of the valve leaflet or in the chordae. In the aged mitral valve, the density of NOS-IR nerves was decreased, as compared with NOS-IR innervation in the young adult valve. The existence of TH and NOS as well as other signal molecule markers in heart valve nerves and the disparate patterns of their distribution and localization provide evidence supporting the theory that heart valve nerves form a complex reflexogenic control system in the mitral heart valve. In summary, two distinct neural architectures are described for TH-IR and NOS-IR valve nerves, respectively. The former are believed to be axons dedicated to sympathetic motor functions. The NOS-IR valve nerves may have sensory and/or postganglionic parasympathetic motor functions. An implication of these findings is that different, but perhaps related, valve functions may be mediated by separate, dedicated circuits.     

Regulation of synaptic position, size, and strength in anuran skeletal muscle

An analysis of the physiology, morphology, and position of endplates on identified fibers in the Xenopus laevis pectoralis muscle has revealed the following. 1. The percentage of fibers with one endplate is lower in large muscles, and within the same muscle, singly innervated fibers are smaller than dually innervated fibers. 2. Single junctions tend to be stronger than junctions on dually innervated fibers. 3. Single junctions typically are located near the middle of their fibers, while the endplates on dually innervated fibers are located toward either end and usually are separated by at least 20% of the total fiber length. A significant proportion of dually innervated fibers appears to be innervated by the same axon at both junctions. 4. Junctions on the same dually innervated fiber tend to be more similar in length than do junctions on different fibers of the same input resistance. This observation is the same whether both junctions on a given fiber are formed by the same or different axons. There is no corresponding tendency for greater similarity in physiological strength of paired junctions, which frequently show large differences in endplate potential amplitude. 5. The total terminal length on dually innervated fibers of equivalent input resistance is inversely correlated with the mean release per unit length and total release of both junctions. There is no apparent correlation between the distance separating endplates and their strength or length. The data support a model of synaptic regulation in which nerve terminals are attracted, grow, and are maintained in proportion to the amount of a substance supplied by muscle fibers. Our findings suggest that such a substance is produced or distributed uniformly throughout each fiber in amounts proportional to the fiber size and inversely proportional to the total transmitter output of all junctions innervating the fiber. A form of competitive interaction between the terminals which helps to determine synaptic spacing may involve local depletion or inactivation of this substance.     

BEN/SC1/DM-GRASP expression during neuromuscular development: a cell adhesion molecule regulated by innervation

BEN/SC1/DM-GRASP is a cell adhesion molecule belonging to the Ig superfamily that is transiently expressed during avian embryogenesis in a variety of cell types, including the motoneurons of the spinal cord. We have investigated the pattern of BEN expression during neuromuscular development of the chick. We show that both motoneurons and their target myoblasts express BEN during early embryonic development and that the protein becomes restricted at neuromuscular contacts as soon as postsynaptic acetylcholine receptor clusters are observed in muscle fibers. Muscle cells grown in vitro express and maintain BEN expression even when they fuse and give rise to mature myotubes. When embryos are deprived of innervation by neural tube ablation, BEN expression is observed in muscle fibers, whereas, in control, the protein is already restricted at neuromuscular synaptic sites. These results demonstrate that all myogenic cells intrinsically express BEN and maintain the protein in the absence of innervation. Conversely, when neurons are added to myogenic cultures, BEN is rapidly downregulated in muscle cells, demonstrating that innervation controls the restricted pattern of BEN expression seen in innervated muscles. After nerve section in postnatal muscles, BEN protein becomes again widely spread over muscle fibers. When denervated muscles are allowed to be reinnervated, the protein is reexpressed in regenerating motor axons, and reinnervation of synaptic sites leads to the concentration of BEN at neuromuscular junctions. Our results suggest that BEN cell adhesion molecule acts both in the formation of neuromuscular contacts during development and in the events leading to muscle reinnervation.     

Axon projections of reciprocal inhibitory interneurons in the spinal cord of young Xenopus tadpoles and implications for the pattern of inhibition during swimming and struggling

We have examined the morphology and longitudinal axon projections of a population of spinal commissural interneurons in young Xenopus tadpoles. We aimed to define how the distribution of axons of the whole population constrains the longitudinal distribution of the inhibition they mediate. Forty-three neurons at different positions were filled intracellularly with biocytin and processed with avidin-conjugated horseradish peroxidase. Soma size did not vary longitudinally and only one ipsilateral axon was found. Contralateral axons ascended, descended, or usually branched to do both. Total axon length and the extent of dendritic arborisation decreased caudally. The distributions of ascending and descending axon lengths were different; there were more long ascending (mean 737 +/- standard deviation 365 microm) than long descending (447 +/- 431 microm) axons. We used the axon length distribution data with existing data on the distribution of commissural interneuron somata to calculate the overall longitudinal density of these inhibitory axons. Axon numbers showed a clear rostrocaudal gradient. Axon length distributions were then incorporated into a simple spatiotemporal model of the forms of inhibition during swimming and struggling motor patterns. The model predicts that the peak of inhibition on each cycle will decrease from head to tail in both motor patterns, a feature already confirmed physiologically for swimming. It also supports a previous proposal that ascending inhibition during struggling shortens cycle period by shortening rostral motor bursts, whereas descending inhibition could delay subsequent burst onset.