The role of synapse elimination in the establishment of neuromuscular compartments

To investigate the specificity of development of initial neuromuscular connections, we examined the compartmental distribution of synapses in neonatal rat lateral gastrocnemius (LG) muscle. Initial neuromuscular connections might be restricted to the compartmental territories present in adults; alternatively, synapse elimination could establish the compartments from a less precise pattern of innervation. We examined 46 pups of ages 0 to 14 postnatal days using a variety of techniques. The principle method was evoked electromyographic (EMG) activity in response to nerve stimulation. The nerve branch to one neuromuscular compartment was cut and the remainder of the nerve was stimulated. The presence of EMG activity was used to identify the areas of muscle contracting in response to nerve stimulation. After cutting a particular branch, EMG activity generally could not be recorded from the denervated compartment. These results indicate that the pattern of innervation at birth is essentially compartment-specific, and that neuromuscular compartments are not shaped from some less precise pattern by postnatal synapse elimination. The factors which operate prenatally to determine this high degree of specificity in neuromuscular connectivity seen at the time of birth, however, remain unknown.     

Activity and synapse elimination at the neuromuscular junction

The neuromuscular junction undergoes a loss of synaptic connections during early development. This loss converts the innervation of each muscle fiber from polyneuronal to single. During this change the number of motor neurons remains constant but the number of muscle fibers innervated by each motor neuron is reduced. Evidence indicates that a local competition among the inputs on each muscle fiber determines which inputs are eliminated. The role of synapse elimination in the development of neuromuscular circuits, other than ensuring a single innervation of each fiber, is unclear. Most evidence suggests that the elimination plays little or no role in correcting for errant connections. Rather, it seems that connections are initially highly specific, in terms of both which motor neurons connect to which muscles and which neurons connect to which particular fibers within these muscles. A number of attempts have been made to determine the importance of neuromuscular activity during early development for this rearrangement of synaptic connections. Experiments reducing neuromuscular activity by muscle tenotomy, deafferentation and spinal cord section, block of nerve impulse conduction with tetrodotoxin, and the use of postsynaptic and presynaptic blocking agents have all shown that normal activity is required for normal synapse elimination. Most experiments in which complete muscle paralysis has been achieved show that activity may be essential for the occurrence of synapse elimination. Furthermore, experiments in which neuromuscular activity has been augmented by external stimulation show that synapse elimination is accelerated. A plausible hypothesis to explain the activity dependence of neuromuscular synapse elimination is that a neuromuscular trophic agent is produced by the muscle fibers and that this production is controlled by muscle-fiber activity. The terminals on each fiber compete for the substance produced by that fiber. Inactive fibers produce large quantities of this substance; on the other hand, muscle activity suppresses the level of synthesis of this agent to the point where only a single synaptic terminal can be maintained. Inactive muscle fibers would be expected to be able to maintain more nerve terminals. The attractiveness of this scheme is that it provides a simple feedback mechanism to ensure that each fiber retains a single effective input.     

Development and ultrastructure of the neuromuscular junction in bronchial smooth muscle tissue

At various stages of pre- and postnatal ontogenesis ultrastructure of contacts of smooth myocytes and nervous terminals in the white mice bronchial wall has been investigated. Nervous fibers grow into the forming tissue of the lung beginning from the 11th day of embryogenesis. By the end of the prenatal development the nervous fibers fasciculi with varicosites and having vesicles are localized at the distance of 100-300 nm from the developing myocytes. Formation of dense neuromuscular junctions with the distance of 35-60 nm between the axonal membranes and the myocyte is observed on the 10-15 day after birth. In mature animals combination of various types of neuromuscular connections is revealed; they ensure local and distant neurotrophic regulation. In the bronchial smooth musculature afferent connections are revealed, as well as connections of myocytic processes with the effectors. Terminals of the cholinergic type predominate, adrenergic effectors occur very seldom. There are terminals, in which combination of vesicles having various structure and diameter are observed.     

Nerve Stump Length and Membrane Changes in Denervated Skeletal Muscle

THE effect of nerve stump length on the rate of failure of neuromuscular transmission and on the degeneration of the neuromuscular apparatus has been studied in some detail^1–4 and there is general agreement that neuromuscular transmission fails and the nerve terminals degenerate more rapidly if the nerve is cut close to the muscle than if it is cut far away.     

Regeneration studies on a crayfish neuromuscular system. I. Connectivity changes after intersegmental nerve transplants

The superficial flexor muscles of the crayfish are a neuromuscular system of a few muscle cells innervated by six neurons in a precise position-dependent pattern. The neurons are capable of regenerating their normal connectivity patterns within a short span of time when conditions are favorable. The superficial flexor muscles of the second and third segments, despite their similarities in neuronal and muscle cell size and number, have distinctive connectivity patterns; some homologous neurons form similar patterns but other homologous neurons form patterns that are reversed between segments. We transplanted each segment's nerve into each other's muscle in order to observe regeneration of the nerves into a target area that differed in connectivity patterns from their original muscle. During the first weeks of regeneration all neurons formed a connectivity pattern with more connections medially and declining connections laterally, a pattern determined by the medial location of the nerve transplant. This pattern is maintained for most of the neurons, but for some there is an eventual reduction in medial connections as maximum synapse formation shifts to the lateral muscle fibers. Three of the eight neurons studied were able to regenerate connectivity patterns that corresponded to their segment of origin and not to their host muscle. This suggests that intersegmental muscle differences are not influencing the formation of these connectivity patterns, so the neurons will follow their inherent synaptogenesis program.     

Mutant human embryonic stem cells reveal neurite and synapse formation defects in type 1 myotonic dystrophy

Myotonic dystrophy type 1 (DM1) is a multisystem disorder affecting a variety of organs, including the central nervous system. By using neuronal progeny derived from human embryonic stem cells carrying the causal DM1 mutation, we have identified an early developmental defect in genes involved in neurite formation and the establishment of neuromuscular connections. Differential gene expression profiling and quantitative RT-PCR revealed decreased expression of two members of the SLITRK family in DM1 neural cells and in DM1 brain biopsies. In addition, DM1 motoneuron/muscle cell cocultures showed alterations that are consistent with the known role of SLITRK genes in neurite outgrowth, neuritogenesis, and synaptogenesis. Rescue and knockdown experiments suggested that the functional defects can be directly attributed to SLITRK misexpression. These neuropathological mechanisms may be clinically significant for the functional changes in neuromuscular connections associated with DM1.     

Neurobiology of Polyorchis. I. Function of effector systems

The electrical correlates of activity in the effector systems responsible for swimming, crumpling and postural changes have been recorded in the anthomedusan Polyorchis penicillatus. Motor spikes (pre-swim pulses), that initiate swimming contractions, appear without delay at distant sites on the inner nerve-ring in unstimulated preparations. Levels of Mg⁺⁺ anaesthesia which block the neuromuscular junctions between PSP giant neurons and swimming muscle do not affect PSP activity. Swimming muscle potentials can be recorded from subumbrella and velar muscle sheets using extra- and intracellular electrodes. These action potentials have a distinct plateau and are propagated in a myoid fashion. Resting potentials average ?70 mV with spikes overshooting zero by some 62 mV. The effects of repetitive stimulation are described. Extracellular recordings indicate that neuronal pathways may play a major role in mediating crumpling, unlike many other species where epithelial pathways are more important. Endodermal spikes recorded intracellularly from the radial and ring canals have amplitudes of some 92 mV arising from resting potentials that average ?55 mV. Repetitive stimulation causes a decrease in amplitude and increase in duration of epithelial action potentials. Tentacle length is controlled by a pacemaker system located in both nerve rings. The frequency of spikes (PTPs) generated by this system determines the length and tonus of tentacles. The neuromuscular junctions between the motor neurons and tentacle muscle are Mg⁺⁺ sensitive and show facilitating properties.     

Ultrastructural Evidence for Neuromuscular Systems in Coelenterates

Cellular interrelationships and synaptic connections in tentaclesof several species of coelenterates were examined by means ofelectron microscopy to determine if neuromuscular pathways werepresent. The presence of sensory cells, ganglion cells, epitheliomuscularcells, interneuronal synapses, and neuromuscular junctions suggeststhat neuromuscular pathways are present in coelenterates. Nakedaxons without sheath cells form several synapses en passantwith the same and with different epitheliomuscular cells aswell as with nematocytes and other neurons. Interneuronal synapsesand neuromuscular and neuronematocyte junctions have clear ordense-cored vesicles (700–1500 Å in diameter) associatedwith a dense cytoplasmic coat on the presynaptic membrane, acleft (100–300 Å in width) with intracleft filaments,and a subsynaptic membrane with a dense cytoplasmic coat. Atscyphozoan neuromuscular junctions there is a subsurface cisternaof endoplasmic reticulum, which is separated from the epitheliomuscularcell membrane by a narrow cytoplasmic gap (100–300 Åin width) . Neuromuscular junctions in coelenterates resembleen passant axonal junctions with smooth muscle in higher animals. Morphological evidence is presented for a simple reflex involvinga two-cell (sensory or ganglion-epitheliomuscular cell) or three-cell(sensory-ganglion-epitheliomuscular cell) pathway that may resultin the coordinated contraction of the longitudinal muscle intentacles of coelenterates.     

Contralateral projections of the optic tectum in the zebra finch (Taenopygia guttata castanotis)

Summary Efferent projections of the optic tectum of zebra finches were investigated by injection of the radioactive anterograde tracer ³H-proline. In addition to a variety of ipsilateral projections, some contralateral connections were found. Quantitative evaluation of the recrossing tecto-rotundal and nucleus subpraetectalis/nucleus interstitio-praetecto-subpraetectalis projection revealed that these connections are much stronger than previously believed. In contrast, the tecto-tectal projection is very weak, as has been shown previously. Further support for this comes from results obtained using injections of retrograde tracers. The role of the different projections in conveying information from the ipsilateral eye to the ectostriatum, the telencephalic end-station of the tectofugal pathway, is discussed.     

Innervation and acetylcholine sensitivity of skeletal muscle cells differentiated in vitro from chick embryo

Trypsin-dissociated myoblasts from leg muscle of 12-day chick embryos have been cultured in monolayers. After four days the muscle cultures have been confronted with fragments of the spinal cord of six-day chick embryos. Electrophysiological and morphological analysis demonstrate that characteristic neuromuscular transmission can develop in these cultures. Electrical stimulation of the cord fragment evokes contractions of innervated muscle fibers, from which end plate potentials and miniature end plate potentials with average frequency around one per second or more can be recorded. D-tubocurarine (1 μg/ml) suppresses reversibly these synaptic potentials. Non-innervated muscle fibers are sensitive to acetylcholine over all their surface, while innervated muscle fibers are sensitive at the regions where structures suggestive of motor end plate (“bulb-type”) are found. We can conclude that neuromuscular connections developed in vitro in our experiments are functional in respect of transmission of impulses but also in respect of neurotrophic influences for restriction of chemosensitivity.     

Extreme Levels of Noise Constitute a Key Neuromuscular Deficit in the Elderly

Fluctuations during isometric force production tasks occur due to the inability of musculature to generate purely constant submaximal forces and are considered to be an estimation of neuromuscular noise. The human sensori-motor system regulates complex interactions between multiple afferent and efferent systems, which results in variability during functional task performance. Since muscles are the only active component of the motor system, it therefore seems reasonable that neuromuscular noise plays a key role in governing variability during both standing and walking. Seventy elderly women (including 34 fallers) performed multiple repetitions of isometric force production, quiet standing and walking tasks. No relationship between neuromuscular noise and functional task performance was observed in either the faller or the non-faller cohorts. When classified into groups with either nominal (group NOM, 25^th –75^th percentile) or extreme (either too high or too low, group EXT) levels of neuromuscular noise, group NOM demonstrated a clear association (r²>0.23, p<0.05) between neuromuscular noise and variability during task performance. On the other hand, group EXT demonstrated no such relationship, but also tended to walk slower, and had lower stride lengths, as well as lower isometric strength. These results suggest that neuromuscular noise is related to the quality of both static and dynamic functional task performance, but also that extreme levels of neuromuscular noise constitute a key neuromuscular deficit in the elderly.     

Morphine impairs Acetylcholine Release but facilitates Acetylcholine Action at a Skeletal Neuromuscular Junction

THERE is considerable evidence that morphine impairs the release of acetylcholine (ACh) at cholinergic synapses in the brain^1–5, although there are considerable problems in determining the exact site and mechanism of this action. A simple synaptic model would be useful for pursuing this problem and the question arises whether this action of morphine is universal for cholinergic synapses or is restricted to particular sites. Morphine impairs the release of ACh at peripheral muscarinic sites^6–8 but there are no reports about the effects of morphine on ACh release at nicotinic neuromuscular sites. We have reported that both morphine and nalorphine block neuromuscular transmission in amphibian and mammalian skeletal neuromuscular preparations^9,10, apparently as a result of impairment of ACh release. We have now determined by direct measurement that morphine impairs ACh release at a skeletal neuromuscular junction.     

Reformation of specific neuromuscular connections during axolotl limb regeneration: evidence that the first contacts are correct

Retrograde neuronal tracing with horseradish peroxidase was used to determine the position in the spinal cord of the motor neurone pools of a proximal (biceps) and a distal (extensor digitorum) limb muscle at various times during axolotl limb regeneration. It was found that from the earliest stages of muscle redifferentiation (as judged by light and electron microscopic analysis) the vast majority of axons innervating the regenerating muscles came from cells within the bounds of the normal motor neurone pool for each muscle. A few incorrect projections were noted in that the regenerating proximal muscle was sometimes innervated by some cells caudal to its normal motor neurone pool. The results are discussed in terms of mechanisms that may be operating in the regenerating limb to ensure that specific neuromuscular connections are made.     

The structure of the nervous system of the nematode Caenorhabditis elegans

The structure and connectivity of the nervous system of the nematode Caenorhabditis elegans has been deduced from reconstructions of electron micrographs of serial sections. The hermaphrodite nervous system has a total complement of 302 neurons, which are arranged in an essentially invariant structure. Neurons with similar morphologies and connectivities have been grouped together into classes; there are 118 such classes. Neurons have simple morphologies with few, if any, branches. Processes from neurons run in defined positions within bundles of parallel processes, synaptic connections being made en passant. Process bundles are arranged longitudinally and circumferentially and are often adjacent to ridges of hypodermis. Neurons are generally highly locally connected, making synaptic connections with many of their neighbours. Muscle cells have arms that run out to process bundles containing motoneuron axons. Here they receive their synaptic input in defined regions along the surface of the bundles, where motoneuron axons reside. Most of the morphologically identifiable synaptic connections in a typical animal are described. These consist of about 5000 chemical synapses, 2000 neuromuscular junctions and 600 gap junctions.     

New Step in Transmitter Release at the Myoneural Junction

QUANTAL release of acetylcholine from vesicles in the presynaptic terminals of neuromuscular synapses is well established^1–3, even if some doubts persist⁴. The mechanism by which acetylcholine (or any other transmitter at other synapses) is transferred from the vesicles into the synaptic gap, however, is unknown. A calcium influx into the terminal is associated with release of transmitter⁵, as is an electrical field change⁶.     

Modulation of neurotransmission by reciprocal synapse-glial interactions at the neuromuscular junction

Perisynaptic Schwann cells are glial cells that are closely associated with pre- and postsynaptic elements of the neuromuscular junction. Recent evidence shows that these cells detect and modulate neurotransmission in an activity-dependent fashion. Through G-protein signalling and Ca²⁺ released from internal stores they can decrease or increase neurotransmitter release, respectively. Thus, they help to establish the level of neurotransmission associated with activity dependent short-term synaptic plasticity. We discuss evidence implicating perisynaptic Schwann cells as being active partners in neurotransmission at the neuromuscular junction, with emphasis on the modulation of short-term plasticity and potential implications for long-term changes.     

Regulation of synaptic vesicle docking by different classes of macromolecules in active zone material

The docking of synaptic vesicles at active zones on the presynaptic plasma membrane of axon terminals is essential for their fusion with the membrane and exocytosis of their neurotransmitter to mediate synaptic impulse transmission. Dense networks of macromolecules, called active zone material, (AZM) are attached to the presynaptic membrane next to docked vesicles. Electron tomography has shown that some AZM macromolecules are connected to docked vesicles, leading to the suggestion that AZM is somehow involved in the docking process. We used electron tomography on the simply arranged active zones at frog neuromuscular junctions to characterize the connections of AZM to docked synaptic vesicles and to search for the establishment of such connections during vesicle docking. We show that each docked vesicle is connected to 10-15 AZM macromolecules, which fall into four classes based on several criteria including their position relative to the presynaptic membrane. In activated axon terminals fixed during replacement of docked vesicles by previously undocked vesicles, undocked vesicles near vacated docking sites on the presynaptic membrane have connections to the same classes of AZM macromolecules that are connected to docked vesicles in resting terminals. The number of classes and the total number of macromolecules to which the undocked vesicles are connected are inversely proportional to the vesicles' distance from the presynaptic membrane. We conclude that vesicle movement toward and maintenance at docking sites on the presynaptic membrane are directed by an orderly succession of stable interactions between the vesicles and distinct classes of AZM macromolecules positioned at different distances from the membrane. Establishing the number, arrangement and sequence of association of AZM macromolecules involved in vesicle docking provides an anatomical basis for testing and extending concepts of docking mechanisms provided by biochemistry.     

A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

Human primary muscle cells cultured aneurally in monolayer rarely contract spontaneously because, in the absence of a nerve component, cell differentiation is limited and motor neuron stimulation is missing¹. These limitations hamper the in vitro study of many neuromuscular diseases in cultured muscle cells. Importantly, the experimental constraints of monolayered, cultured muscle cells can be overcome by functional innervation of myofibers with spinal cord explants in co-cultures.Here, we show the different steps required to achieve an efficient, proper innervation of human primary muscle cells, leading to complete differentiation and fiber contraction according to the method developed by Askanas². To do so, muscle cells are co-cultured with spinal cord explants of rat embryos at ED 13.5, with the dorsal root ganglia still attached to the spinal cord slices. After a few days, the muscle fibers start to contract and eventually become cross-striated through innervation by functional neurites projecting from the spinal cord explants that connecting to the muscle cells. This structure can be maintained for many months, simply by regular exchange of the culture medium.The applications of this invaluable tool are numerous, as it represents a functional model for multidisciplinary analyses of human muscle development and innervation. In fact, a complete de novo neuromuscular junction installation occurs in a culture dish, allowing an easy measurement of many parameters at each step, in a fundamental and physiological context.Just to cite a few examples, genomic and/or proteomic studies can be performed directly on the co-cultures. Furthermore, pre- and post-synaptic effects can be specifically and separately assessed at the neuromuscular junction, because both components come from different species, rat and human, respectively. The nerve-muscle co-culture can also be performed with human muscle cells isolated from patients suffering from muscle or neuromuscular diseases³, and thus can be used as a screening tool for candidate drugs. Finally, no special equipment but a regular BSL2 facility is needed to reproduce a functional motor unit in a culture dish. This method thus is valuable for both the muscle as well as the neuromuscular research communities for physiological and mechanistic studies of neuromuscular function, in a normal and disease context.     

Biochemical,Pharmacological and Structural Characterization of a New PLA<Subscript>2</Subscript> from <Emphasis Type="Italic">Crotalus durissus terrificus</Emphasis> (South American Rattlesnake) Venom

A new PLA₂ (F16) was purified from Crotalus durissus terrificus venom by molecular exclusion chromatography followed by analytical reverse phase HPLC. The PLA₂ (14.86 kDa by MALDI-TOF mass spectrometry) had an amino acid sequence of SLLQFNKMIKFETRKNAVPFYAFYGCYCGWGGRRRPKDATDRCCFVHDCCYEKVTKCNTKWDIYRYSLKSGYITCGKGTWCKEQICECDRVAAECLRRSLSTYKNGYMFYPDSRCRGPSETC, and showed highly conserved Ca²⁺-binding and catalytic sites. F16 showed allosteric behavior with 10 mM Ca²⁺ and had temperature and pH optima of 25°C and 7.9, respectively. F16 (10 μg/ml) produced neuromuscular blockade in chick biventer cervicis preparations in the absence and presence of crotapotin, indicating that crotapotin was not essential for neuromuscular action in this preparation. In contrast, in mouse phrenic nerve-diaphragm preparations, the neuromuscular blockade produced by the same concentration of toxin was dependent on crotapotin. Pre-incubation with heparin markedly reduced the neurotoxicity of F16. These results show that the biochemical and structural properties of F16 are similar to those of the PLA₂ isoforms F15 and F17, but that the neurotoxicity and the requirement for crotapotin to form the crotoxin complex varies according to the neuromuscular preparation.     

THE FUNCTION OF THE BRAIN IN LOCOMOTION OF THE POLYCLAD WORM,YUNGIA AURANTIACA

Coordinated swimming movements in Yungia are not dependent upon the presence of the brain. The neuromuscular mechanism necessary for spontaneous movement and swimming is complete in the body of the animal apart from the brain. Normally this mechanism is set in motion by sensory stimulation arriving by way of the brain. The latter is a region of low threshold and acts as an amplifier by sending the impulses into a great number of channels. When the head is cut off these connections with the sensorium are broken, consequently peripheral stimulation does not have its usual effect. If, however, the motor nerves are stimulated directly as by mechanical stimulation of the median anterior region, then swimming movements result. Also if the threshold of the entire nervous mechanism is lowered by phenol or by an increase in the ion ratios See PDF for Equation and See PDF for Equation then again peripheral stimulation throws the neuromuscular mechanism into activity and swimming movements result.