Formation of the neuromuscular junction: molecules and mechanisms

The vertebrate skeletal neuromuscular junction is the site at which motor neurons communicate with their target muscle fibers. At this synapse, as at synapses throughout the nervous system, efficient and appropriate communication requires the formation and precise alignment of specializations for transmitter release in the axon terminal with those for transmitter detection in the postsynaptic cell. Classical developmental studies demonstrate that synapse formation at the neuromuscular junction is a mutually inductive event; neurons induce postsynaptic differentiation in muscle cells and myofibers induce presynaptic differentiation in motor axon terminals. More recent experiments indicate that Schwann cells, which cap axon terminals, also play an active role in the formation and maintenance of the neuromuscular junction. Here, we review recent advances in the identification of molecules mediating such inductive interactions and the mechanisms by which they produce their effects. Although our discussion concerns events at developing neuromuscular junctions, it seems likely that similar molecules and mechanisms may act at neuron–neuron synapses in the peripheral as well as the central nervous system. BioEssays 20 :819–829, 1998. © 1998 John Wiley & Sons, Inc.     

Structure, bioactivity, and cellular localization of myomodulin B: a novel Aplysia peptide.

Important insights into mechanisms by which neuromuscular activity can be modulated have been gained by the study of experimentally advantageous preparations such as the ARC neuromuscular system of Aplysia. Previous studies have indicated that one source of modulatory input to the ARC muscle is its own two motor neurons, B15 and B16. Both of these neurons synthesize multiple peptide cotransmitters in addition to their primary neurotransmitter acetylcholine (ACh). Peptides present in the ARC motor neurons include SCPA, SCPB, buccalin A and B, and myomodulin A. We have now purified a novel neuropeptide, myomodulin B, which is structurally similar to myomodulin A. Myomodulin B is present in two identified Aplysia neurons that contain myomodulin A; the ARC motor neuron B16 and the abdominal neuron L10. Ratios of myomodulin A to myomodulin B are approximately 6:1 in both cells. Like myomodulin A, myomodulin B potentiates ARC neuromuscular activity; it acts postsynaptically, and increases the size and relaxation rate of muscle contractions elicited either by motor neuron stimulation or by direct application of ACh to the ARC. When myomodulin A is applied to the ARC in high doses (e.g., at about 10(-7) M), it decreases the size of motor neuron-elicited muscle contractions. This inhibitory effect is never seen with myomodulin B. Thus, despite the structural similarity between the two myomodulins, there exists what may be an important difference in their bioactivity.     

Peptides in the Hydrozoa: are they transmitters?

A family of peptides with the carboxy-terminus Arg-Phe-amide has been localized to specific subpopulations of neurons in every cnidarian species examined. These neurons are typically sensory in character or are associated with smooth muscle. Although a transmitter role for these peptides has been suggested for anthozoans at neuromuscular synapses, no such evidence is available for hydrozoans. Instead there is evidence that RF-amides can be modulators of neuronal activity which takes the form of a biphasic (inhibitory then excitatory) response in vivo, while in vitro only the inhibitory response is seen. Voltage clamp studies of identified motor neurons showed large, transitory outward currents when Pol-RF-amide peptide was applied.     

Sexual differentiation and hormonal regulation of the laryngeal synapse in Xenopus laevis

In Xenopus laevis frogs, sex differences in adult laryngeal synapses contribute to sex differences in vocal behavior. This study explores the development of sex differences in types of neuromuscular synapses and the development and hormone regulation of sex differences in transmitter release. Synapses in the juvenile larynx have characteristics not found in adults: juvenile muscle fibers can produce subthreshold or suprathreshold potentials in response to the same strength of nerve stimulation and can also produce multiple spikes to a single nerve stimulus. Juvenile laryngeal muscle also contains the same synapse types (I, II, and III) as are found in adult laryngeal muscle. The distribution of laryngeal synapse types in juveniles is less sexually dimorphic than the distribution in adults. Analysis of quantal content indicates that laryngeal synapses characteristically release low amounts of transmitter prior to sexual differentiation. Quantal content values from male and female juveniles are similar to values for adult males and are lower than values for adult females. When juveniles are gonadectomized and treated with exogenous estrogen, quantal content values increase significantly, suggesting that this hormone may increase transmitter release at laryngeal synapses during development. Gonadectomy alone does not affect quantal content of laryngeal synapses in either sex. Androgen treatment decreases quantal content in juvenile females but not males; the effect is opposite to and smaller than that of estrogen. Thus, muscle fiber responses to nerve stimulation and transmitter release are not sexually dimorphic in juvenile larynges. Transmitter release is strengthened, or feminized, by the administration of estradiol, an ovarian steroid hormone. © 1995 John Wiley & Sons, Inc.     

Strength of synaptic transmission at neuromuscular junction of crustaceans and insects in relation to calcium entry

Crustacean and insect neuromuscular junctions typically include numerous small synapses, each of which usually contains one or more active zones, which possess voltage-sensitive calcium channels and are specialized for release of synaptic vesicles. Strength of transmission (the number of quantal units released per synapse by a nerve impulse) varies greatly among different endings of individual neurons, and from one neuron to another. Ultrastructural features of synapses account for some of the physiological differences at endings of individual neurons. The nerve terminals that release more neurotransmitter per impulse have a higher incidence of synapses with more than one active zone, and this is correlated with more calcium build-up during stimulation. However, comparison of synaptic structure in neurons with different physiological phenotypes indicates no major differences in structure that could account for their different levels of neurotransmitter release per impulse, and release per synapse differs among neurons despite similar calcium build-up in their terminals during stimulation. The evidence indicates differences in calcium sensitivity of the release process among neurons as an aspect of physiological specialization.     

Synaptic development: insights from Drosophila

In Drosophila, the larval neuromuscular junction is particularly tractable for studying how synapses develop and function. In contrast to vertebrate central synapses, each presynaptic motor neuron and postsynaptic muscle cell is unique and identifiable, and the wiring circuit is invariant. Thus, the full power of Drosophila genetics can be brought to bear on a single, reproducibly identifiable, synaptic terminal. Each individual neuromuscular junction encompasses hundreds of synaptic neurotransmitter release sites housed in a chain of synaptic boutons. Recent advances have increased our understanding of the mechanisms that shape the development of both individual synapses--that is, the transmitter release sites including active zones and their apposed glutamate receptor clusters--and the whole synaptic terminal that connects a pre- and post-synaptic cell.     

Structural plasticity at crustacean neuromuscular synapses

Crustacean motor axons innervate muscle fibers via a multiplicity of synaptic terminals which release small but variable amounts of transmitter. Differences in release performance appear to be correlated with the size of synaptic contacts and presynaptic dense bars (active zones). These structural parameters proliferate via sprouting from existing synaptic terminals and relocate to ever more distal sites during development and growth of an identified axon. Moreover, alterations in number of synaptic contacts and active zones occur in adults following stimulation or decentralization, demonstrating structural plasticity of crustacean neuromuscular synapses.     

Postsynaptic regulation of the development and long-term plasticity of Aplysia sensorimotor synapses in cell culture

The monosynaptic component of the neuronal circuit that mediates the withdrawal reflex of Aplysia californica can be reconstituted in dissociated cell culture. Study of these in vitro monosynaptic connections has yielded insights into the basic cellular mechanisms of synaptogenesis and long-term synaptic plasticity. One such insight has been that the development of the presynaptic sensory neurons is strongly regulated by the postsynaptic motor neuron. Sensory neurons which have been cocultured with a target motor neuron have more elaborate structures—characterized by neurites with more branches and varicosities—than do sensory neurons grown alone in culture or sensory neurons that have been cocultured with an inappropriate target cell. Another way in which the motor neuron regulates the development of sensory neurons is apparent when sensorimotor cocultures with two presynaptic cells are examined. In such cocultures the outgrowth from the different presynaptic cells is obviously segregated on the processes of the postsynaptic cell. By contrast, when two sensory neurons are placed into cell culture without a motor neuron, thier processes readily grow together. In addition to regulating the in vitro development of sensory neurons, the motor neuron also regulates learning-related changes in the structure of sensory neurons. Application of the endogenous facilitatory trasmitter serotonin (5-HT) causes long-term facilitation of in vitro sensorimotor synapses due in part to growth of new presynatpic varicosities. But 5-HT applied to sensory neurons alone in cultuer does not produce structural changes in these cells. More recently it has been found that sensorimotor synapses in cell culture can exhibit long-term potentiation (LTP). Like LTP of some hippocampal synapses, LTP of in vitro Aplysia syanpses is regulated by the voltage of the postsynaptic cell. Pairing high-frequency stimulation of sensory neurons with strong hyperpolarization of the motor neuron blocks the induction of LTP. Moreover, LTP of sensorimotor synapses can be induced in Hebbian fashion by pairing weak presynaptic stimulation with strong postsynaptic depolarization. These findings implicate a Habbian mechanism in classical conditioning in Aplysia. They also indicate that Hebbian LTP is a phylogenetically ancient form of synaptic plasticity. 1994 John Wiley & Sons, Inc.     

Regulation of tetanus toxin-inhibited transmitter secretion in the neuromuscular junction in a calcium-free medium

The effect of Ca2+ removal from the external medium on regulation of the release of the synaptic transmitter in the tetanus toxin (TT)-inhibited neuromuscular junctions was studied on a rat phrenicodiaphragmal preparation with the aid of the conventional microelectrode technique of recording synaptic activity. As the external concentration of calcium was decreased from 2 to 0 mM, the frequency of miniature end plate potentials remained unchanged in the preparations isolated 3 to 3.5 h after intramuscular injection of TT (10(5) MLD for mouse). TT considerably reduced activation of the transmitter release, caused in intact synapses by ouabain (0.1 mM) and repetitive stimulation of the diaphragmatic nerve (50 imp/s). The data obtained indicate that in the TT-inhibited motor nerve terminals, the level of the transmitter release does not depend on the external concentration of calcium and that TT damages some of the intracellular sources of calcium.     

Effects of Noradrenaline on End-Plate Currents and the Kinetics of Transmitter Release under Long-Lasting Repetitive Stimulation

Noradrenaline causes a significant increase in the amplitude of multiquantum end-plate currents (EPC) and also diminishes the EPC rising phase vs the rising phase of the miniature EPC ratio in the frog neuromuscular junction under conditions of low-frequency long-lasting stimulation of the motor nerve. Noradrenaline changes the kinetics of transmitter release due to synchronization of the quantum transmitter secretion. The synchronizing action of noradrenaline can underlie its de-fatiguing effect in the neuromuscular junction.     

Changes in the Parameters of Quantal Acetylcholine Release after Activation of PAR1-Type Thrombin Receptors at the Mouse Neuromuscular Junctions

In mature and newly formed neuromuscular synapses of mouse skeletal muscles, miniature endplate potentials (MEPPs) and multiquantal endplate potentials (EPPs) evoked by a single stimulation of the nerve were recorded using intracellular microelectrode technique. The mechanisms underlying the changes in spontaneous and evoked acetylcholine (ACh) release caused by the activation of PAR1-type muscle receptors induced by their peptide agonist TRAP6-NH₂ were studied. It has been shown for the first time that, in either mature or newly formed motor synapses, the activation of PAR1 that lack presynaptic localization causes a sustained increase in the MEPP amplitude due to the increase in the ACh quantal size at the presynaptic level. It was found that phospholipase C (PLC) participates in the signaling mechanism triggered by the PAR1 activation. Exogenously applied brain-derived neurotrophic factor (BDNF) mimics the effect of activation of PAR1 by TRAP6-NH₂. Moreover, an increase in the MEPP amplitude caused by the peptide PAR1 agonist was fully prevented by blocking the BDNF receptors–tropomyosin receptor kinases B (TrkB). Thus, it has been shown for the first time that the increase in ACh quantal size due to the activation of PAR1 in motor synapses is mediated by a complex signaling cascade that starts at the postsynaptic level of the motor synapse and ends at the presynaptic level. It is expected that the activation of PAR1 at the muscle fiber membrane followed by the PLC upregulation results in the release of neurotrophin BDNF as a retrograde signal. Its effect on the presynaptic TrkB receptors triggers the cascade leading to an increase in the quantal size of ACh.     

Lack of Correspondence between the Amplitudes of Spontaneous Potentials and Unit Potentials evoked by Nerve Impulses at Regenerating Neuromuscular Junctions

IT is known from earlier studies of regeneration of neuromuscular synapses in the frog¹ that the nerve fibres return to the region of the original end-plate and that there is a time after the ending has re-established synaptic contact during which a nerve impulse fails to evoke transmitter release, even though spontaneous release occurs. Even after neuromuscular transmission is restored, the response latency is longer than usual and the nerve is more liable to presynaptic failure of propagation¹. This study is part of an attempt to examine in more detail the characteristics of transmitter release during this period.     

Nonhomogeneous excitatory synapses of a crab stomach muscle

Neuromuscular synapses of pyloric muscle P1 in the blue crab Callinectes sapidus were examined using electrophysiological and electron microscopic methods. The muscle is innervated by a single excitatory axon of the stomatogastric ganglion. Excitatory postsynaptic potentials show striking facilitation at very low frequencies of stimulation, indicating very slow decay of the facilitation process after a single nerve impulse. Quantal content of transmitter release at a low frequency of stimulation averaged 1.5. Evidence was obtained that not all synapses on a muscle fiber are equivalent. This was particularly evident at the morphological level in serially sectioned nerve terminals. On each nerve terminal examined, a wide range of synapse sizes was found. Synaptic contact areas ranged from less than 0.5 micron2 to almost 10 micron2; the latter value is large compared with those obtained for other crustacean neuromuscular synapses. Most of the smaller synapses lacked the presynaptic dense bodies which are putative release sites for the transmitter substance. The larger synapses all had presynaptic dense bodies, and some showed evidence of splitting apart into smaller subunits. It is postulated that about half the morphologically identified synapses are relatively inactive.     

Transmitter release increases intracellular calcium in perisynaptic Schwann cells in situ.

Glial cells isolated from the nervous system are sensitive to neurotransmitters and may therefore be involved in synaptic transmission. The sensitivity of individual perisynaptic Schwann cells to activity of a single synapse was investigated, in situ, at the frog neuromuscular junction by monitoring changes in intracellular Ca2+ in the Schwann cells. Motor nerve stimulation induced an increase in intracellular Ca2+ in these Schwann cells; this increase was greatly reduced when transmitter release was blocked. Furthermore, local application of the cotransmitters acetylcholine and ATP evoked Ca2+ responses even in the absence of extracellular Ca2+. Successive trains of nerve stimuli or applications of transmitters resulted in progressively smaller Ca2+ responses. We conclude that transmitter released during synaptic activity can evoke release of intracellular Ca2+ in perisynaptic Schwann cells. This Ca2+ signal may play a role in the maintenance or modulation of a synapse. These data show that synaptic transmission involves three cellular components with both postsynaptic and glial components responding to transmitter secretion.     

Intricate effects of primary motor neuronopathy on contractile proteins and metabolic muscle enzymes as revealed by label-free mass spectrometry

While the long-term physiological adaptation of the neuromuscular system to changed functional demands is usually reflected by unilateral skeletal muscle transitions, the progressive degeneration of distinct motor neuron populations is often associated with more complex changes in the abundance and/or isoform expression pattern of contractile proteins and metabolic enzymes. In order to evaluate these intricate effects of primary motor neuronopathy on the skeletal muscle proteome, label-free MS was employed to study global alterations in the WR (wobbler) mouse model of progressive neurodegeneration. In motor neuron disease, fibre-type specification and the metabolic weighting of bioenergetic pathways appear to be strongly influenced by both a differing degree of a subtype-specific vulnerability of neuromuscular synapses and compensatory mechanisms of fibre-type shifting. Proteomic profiling confirmed this pathobiochemical complexity of disease-induced changes and showed distinct alterations in 72 protein species, including a variety of fibre-type-specific isoforms of contractile proteins, metabolic enzymes, metabolite transporters and ion-regulatory proteins, as well as changes in molecular chaperones and various structural proteins. Increases in slow myosin light chains and the troponin complex and a decrease in fast MBP (myosin-binding protein) probably reflect the initial preferential loss of the fast type of neuromuscular synapses in motor neuron disease.     

Inhibitory innervation of a lobster muscle

Summary Inhibitory neuromuscular synapses formed by the common inhibitor (CI) neuron on the distal accessory flexor muscle (DAFM) in the lobster, Homarus americanus, were studied with electrophysiological and electron-microscopic (thin-section and freeze-fracture) techniques. Postsynaptic inhibition as indicated by inhibitory junctional potentials was several-fold stronger on distal compared to proximal muscle fibers. This difference correlated with the results of serial thin-section studies, which showed more inhibitory synapses on distal fibers than on their proximal counterparts. Effects of postsynaptic inhibition on excitatory junctional potentials via current shunting had a morphological correlate in the spatial relationship between inhibitory and excitatory synapses on the distal fibers. Inhibitory synapses were larger than their excitatory counterparts and had fewer glial processes. In freeze-fracture views, inhibitory synapses did not appear as raised plateaus in the P-face as do excitatory synapses, and their active zones were more widely scattered. The intramembrane particles in the inhibitory postsynaptic membrane-representing neurotransmitter receptors-are arranged in parallel rows in the sarcolemmal P-face and have complementary furrows in the sarcolemmal E-face. Altogether, our findings help to describe a population of inhibitory neuromuscular synapses formed by the CI neuron in lobster muscle.     

Long term facilitation of tension in crustacean muscle and its modulation by temperature,activity and circulating amines

Summary Prolonged stimulation of the motor axon of the opener and stretcher muscles of the crayfish claw leads to long-term facilitation (LTF) of transmitter release at the neuromuscular junction. This facilitation is correlated with enhancement of tension development. Factors shown to enhance LTF of transmitter release, such as increased frequency of excitation, lower temperature, and exposure to ouabain also enhance tension development (Figs. 1, 2 and 4). Prolonged stimulation delivered in a bursting pattern enhances the development of tension more than an equivalent amount of stimulation delivered in a regular pattern (Fig. 3).Two circulating neurohormones, serotonin and octopamine, were examined for their effect on the development of tension during short and long periods of muscle activation. Serotonin and LTF of transmitter release appear to have an additive effect on the development of tension. The threshold for a detectable serotonin effect is 10^–10 M. The effect of octopamine on the development of tension appears to be enhanced by longer periods of maintained muscle activation. LTF of transmitter release resulting from 5 min of continuous activation at 15 Hz is accompanied by a drop in the threshold of an observable octopamine effect on tension from 10^–9Mto 10^–10 M. It is proposed that octopamine's trophic effects on metabolism in muscle act to sustain muscular performance during maintained activity.Abbreviations LTF long term facilitation - ec Membrane potential threshold for contraction - STF short term facilitation - e.j.p. excitatory junction potential This work was supported by a N.S.E.R.C. grant to H.L.A.     

Peripheral Actions of the SCPs in Aplysia and Other Gastropod Molluscs

The SCPs are a family of neuropeptides found in many gastropodspecies. Two SCPs with similar sequences have been characterizedin Aplysia. These peptides are potent modulators of centraland peripheral synapses. They also enhance ongoing contractileactivity in spontaneously active tissues such as heart and gut.Their distribution in central ganglia suggests that their predominantrole is in the regulation of feeding behavior. There is goodevidence that the identified SCP-containing neurons, B1 andB2, provide the major central regulation of gut motility duringfeeding through the release of the SCPs from their terminalsin gut. The SCPs have also been localized to motor neurons thatinnervate buccal muscles which generate biting and swallowingmovements. In many of these neurons, the SCPs have been shownto coexist with conventional transmitters such as ACh, or otherpeptides such as FMRFamide. The SCPs appear to be released alongwith conventional transmitters from these neurons to modulatethe effectiveness of the conventional transmitter. In all cases,the SCPs cause an enhancement of the amplitude of contractionsproduced by motor neuron stimulation. The precise mechanismsunderlying this effect vary from muscle to muscle. All of theeffects of the SCPs are mediated by increased cAMP levels intarget tissue. At many sites of action, serotonin produces actionsthat are qualitatively similar to those of the SCPs. This islikely to involve a convergence at the level of the adenylylcyclase. In addition to these peripheral effects, the SCPs alsohave multiple central effects on feeding and other behaviorsin gastropods.     

Differences in synaptic output between excitatory and inhibitory motoneurons in a crayfish muscle

A pair of antagonistic motoneurons, one excitatory and one inhibitory, innervates the distal accessory flexor muscle in the walking limb of the crayfish Procambarus clarkii. The number and size of synapses formed by these two axons on the muscle fibers (neuromuscular synapses) and on each other (axo-axonal synapses) were estimated using thin-section electron microscopy. Although profiles of nerve terminals of the two axons occur in roughly equal proportions, the frequency of occurrence of neuromuscular synapses differed markedly: 73% were excitatory and 27% were inhibitory. However, inhibitory synapses were 4–5 times larger than excitatory ones, and consequently, the total contact areas devoted to neuromuscular synapses were similar for both axons. Axo-axonal synapses were predominantly from the inhibitory axon to the excitatory axon (86%), and a few were from the excitatory axon to the inhibitory axon (14%). The role of the inhibitory axo-axonal synapse is presynaptic inhibition, but that of the excitatory axo-axonal synapse is not known. The differences in size of neuromuscular synapses between the two axons may reflect intrinsic determinants of the neuron, while the similarity in total synaptic area may reflect retrograde influences from the muscle for regulating synapse number.     

The nerve-muscle synapse of the garter snake

Snake nerve-muscle preparations are well-suited for study of both motor innervation patterns at the systems level and NMJ function at the cellular level. Their small size (～100 myofibers) and thinness (one fiber) allows access to all NMJs in one muscle. Snake NMJs are of three types, two twitch subtypes and a single tonic type. Properties of the NMJs supplied by a particular motor neuron, and of the motor unit fibers they innervate, are precisely regulated by the motor neuron in a manner consistent with the Henneman Size Principle. Unlike its amphibian or mammalian cousins, the snake NMJ comprises ～50 (twitch) or ～20 (tonic) individual one-bouton synapses, similar to synapses found in the central nervous system. Each bouton releases a few quanta per stimulus. Larger fibers, which require more synaptic current to initiate contraction, receive nerve terminals that contain more boutons and express receptor patches with higher sensitivity to transmitter. Quantal analysis suggests that transmitter release sites in one bouton do not behave independently; rather, they may cooperate to reduce fluctuations and enhance reliability. After release, two mechanisms coexist for retrieval and reprocessing of spent vesicles–one involving clathrin-mediated endocytosis, the other macropinocytosis. Unanswered questions include how each mechanism is regulated in a use-dependent manner.