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Neuromuscular Junction (NMJ): Mammalian Development
Neuromuscular Junction (NMJ): Mammalian Development 585
Neuromuscular Junction (NMJ): Mammalian Development C R Slater, University of Newcastle, Newcastle upon Tyne, UK ã 2009 Elsevier Ltd. All rights reserved.
Introduction The neuromuscular junction (NMJ) is an example of a highly specialized, asymmetric, cell-cell junction. The speed with which fast chemical transmission occurs requires that the specializations of the postsynaptic muscle cell surface that allow it to respond to the transmitter released from the nerve are less than a micrometer from the presynaptic release sites. An important question concerning the development of the NMJ is therefore, what are the mechanisms that account for the alignment and proximity of the distinctive specializations of its pre- and postsynaptic components? A second important question concerns the specificity of the mature nerve-muscle contacts. What part does the process of NMJ formation play in achieving functional matching of individual motor neurons to the muscle fibers they innervate? This article gives an overview of the way mammalian NMJs develop. Most of the information has come from rats and mice, in which the most detailed studies of NMJ formation in vertebrates have been made. The article starts with the origin of motor neurons and skeletal muscle fibers and then considers how the NMJ itself is formed.
Development of Motor Neurons Birth
Motor neurons are among the earliest nerve cells to be born, that is, to complete their final round of DNA synthesis. Soon after cell birth, the motor neurons begin to extend an axon that leaves the spinal cord in the nascent ventral roots. In mammals, the motor neurons that innervate an individual muscle are usually grouped into a longitudinally oriented column that extends over two to three spinal segments. Axon Outgrowth and Motor Neuron Identity
The immature motor axons leave the spinal cord even before their target muscles have formed. As they grow, the axons select paths that lead to the muscles they are destined to innervate. The ability of an immature motor neuron to make such decisions indicates that it has some knowledge of its identity, and that different motor neurons therefore have different identities. In adults, motor neurons that innervate
slowly contracting nonfatigable muscle fibers tend to be rich in oxidative enzymes that can be visualized by appropriate histochemical techniques. Well before the first NMJs are formed, embryonic motor neurons already differ in their oxidative enzyme profiles. This supports the idea that the motor neurons that innervate a single muscle differ in their properties and that those differences arise before any interaction with the muscle occurs. Once contact with the appropriate premuscle mass has been established, but not before, the axons branch extensively. In rats and mice, functional contacts with newly formed limb muscles are first present around embryonic day 14 (E14), a week before birth. A similar stage occurs in humans at about week 9 of gestation. Release of Acetylcholine from Growth Cones
The terminals of cultured motor neurons can release acetylcholine (ACh), the chemical transmitter at neuromuscular junctions, even before they make contact with muscle. This suggests that motor neurons in vivo also synthesize ACh and have the necessary specializations for its activity-dependent release at an early stage of their development.
Development of Skeletal Muscle Fibers Myotube Formation
Most vertebrate skeletal muscle fibers arise during development from the fusion of many mononucleated postmitotic myoblasts. These spindle-shaped cells line up and fuse to form the primitive myotubes. As the myotubes grow and mature into muscle fibers, they incorporate additional myoblasts. In adult muscle fibers, there is typically one nucleus for every 10 mm of length. Thus, a single fiber in a large human muscle, such as vastus lateralis, which has fibers up to 20 cm long, has up to 20 000 nuclei. Kinetics of Primary and Secondary Myotube Formation
In mammalian muscles, myotube formation occurs in two phases. The first involves the formation of an initial cohort of relatively few primary myotubes. As the muscle elongates, further myoblasts line up along the primary myotubes and eventually fuse to form secondary myotubes. These form initially roughly midway between the ends of the primary myotubes and then grow rapidly in length as they add new myoblasts. The two populations of myotubes, and the mature muscle fibers that form from them, have
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somewhat different functional properties. The great majority of fibers in the adult muscles are derived from secondary myotubes. Origin of Muscle Fiber Types
Most adult mammalian muscles contain fibers of different functional types. Some are specialized for relatively slow, sustained contraction whereas others are specialized for fast, high-powered contractions. At birth, the muscle fibers in rats and mice contract uniformly slowly. Differentiation into faster and slower contracting fibers begins soon after birth and is well established 2–3 weeks later. Most of the muscle fibers that develop from primary myotubes end up as slow in the adult, while secondary myotubes give rise to both fast and slow fibers. These distinctive properties arise from the pattern of genes expressed by the fibers. Most of the nuclei in each fiber express the same set of genes, raising the as yet unanswered question of how that homogeneity of gene expression comes about.
Polyaxonal Innervation
A distinctive feature of the early motor innervation of vertebrates is that several motor neurons initially innervate each muscle fiber. This innervation occurs at a single postsynaptic site that is thus contacted by the terminal axons of several motor neurons (Figure 1). Myotubes cultured in vitro can acquire polyneuronal innervation, but such multiple inputs are normally distributed over the myotube surface rather than focused on a single site. The forces that initially restrict the immature nerve terminals to this single site in vivo are not known. AChR Accumulation
A high density of AChRs is a hallmark of the postsynaptic membrane of the vertebrate NMJ. A distinct cluster of AChRs, detectable after labeling with fluorescent conjugates of the snake toxin a-bungarotoxin, is present from a very early stage of NMJ formation (Figure 2). Within the immature cluster, AChRs are often gathered into microclusters less than 1 mm across. The mean density of AChRs within the plaque
Early Appearance of Delocalized Postsynaptic Properties
Early Development of the NMJ Initial Nerve–Muscle Encounters
Muscle fibers become innervated very soon after they first form. In rats and mice, signs of functional innervation can be detected within a day or two of the earliest myotube formation at about E12–14, depending on the muscle. The early nerve-muscle contacts lack many of the structural features of mature NMJs but are characterized by a high density of AChRs in the muscle fiber membrane. Whether the AChR clusters form before or after nerve contact is a matter of continuing debate (see below). Indeed, there is evidence that NMJs in different muscles in the same species form by different sequences of events.
Axon Muscle fiber
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Number of terminals
Even before they are innervated, immature muscle fibers begin to express the receptor proteins that allow them to respond to ACh (acetylcholine receptors, or AChRs). AChRs of the fetal form, containing a2bdg subunits, are initially present over the whole fiber surface at a density of about 500 mm2. Other molecules that play roles in AChR localization and gene expression, such as rapsyn and muscle specific kinase (MuSK; see ‘AChR aggregation’ below), have a similar pattern of expression. As the muscle fibers become increasingly active, their activity suppresses the expression of all these proteins away from the NMJ.
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Figure 1 Development of muscle innervation. (a) Comparison of polyneuronal innervation of muscles in newborn rats or mice (left) with the adult state (right). (b) Changes in the number of axon terminals innervating neuromuscular junctions in rat diaphragm with age. (b) Reproduced from Bennett MR and Pettigrew AG (1974) The formation of synapses in striated muscle during development. Journal of Physiology 241: 515–545, with permission from Blackwell publishing.
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Figure 2 Structural maturation of mouse neuromuscular junctions (NMJs). Nerve terminals in postnatal mouse extensor digitorum longus muscles were labeled with zinc iodide-osmium, and acetylcholine receptors (AChRs) were labeled with R-a-bungarotoxin. Arrows point to the preterminal motor axon(s). Several axons are present at birth, but between 1 and 2 weeks after birth, all but one are withdrawn. The surviving axon becomes myelinated 2–3 weeks after birth. As the axon terminal enlarges, the initially uniform granular distribution of AChRs breaks up, leaving areas with low AChR density that come to match the branches of the nerve terminals. NB, newborn.
is about 3000 mm2. How the AChR cluster forms initially and is subsequently maintained are topics of extensive current research. There appear to be at least two components of this process: the aggregation of AChRs within the membrane of the muscle fiber and the enhanced expression of the genes encoding the AChR subunits by the myonuclei closest to the site of nerve contact. AChR aggregation AChRs can exist in a form that is mobile in the plane of the cell membrane. In the case of cultured immature frog muscle precursor cells, such mobile AChRs aggregate at sites of contact with motor axons, suggesting that the nerve induces
aggregation of AChRs in the muscle cell. Much evidence suggests that it is a protein, agrin, which is made by and released from motor axons, that triggers AChR aggregation. Agrin function is associated with its activation of a muscle specific kinase (MuSK), which triggers downstream signaling cascades in the muscle. A second key player in the process of agrininduced AChR aggregation is the 43 kDa protein rapsyn. Rapsyn links AChRs to the plasma membrane and to components of the membrane skeleton. In the absence of rapsyn, no aggregation of AChRs occurs. There is also much evidence that AChRs can selfaggregate, and on suitable substrates they can form clusters similar to those at mature NMJs. Some recent
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studies have shown that in some species and muscles, AChR clusters form in the region of presumptive NMJs before nerve-muscle contacts are made. In these cases, neuromuscular contacts may form selectively on the presumptive postsynaptic sites. On balance, it seems likely that while the muscle fiber surface has an inherent tendency to form AChR clusters, agrin released from the nerve can either trigger clustering de novo or stabilize preexisting clusters. Upregulation of postsynaptic gene expression The second process that contributes to AChR accumulation at the mature NMJ is the enhanced expression of the genes that encode the AChR subunits. This process is discussed below. Within a few days of nerve contact, acetylcholinesterase (AChE), the enzyme that terminates AChaction, accumulates in the synaptic basal lamina that forms between the nerve and muscle cells. As for AChRs, this accumulation is associated with upregulation of the genes encoding AChE. Schwann Cells: The Motor Neurons’ Companion
Throughout most of their life, motor axons are closely accompanied by Schwann cells. There is increasing evidence of a mutual dependency of motor axons and their companion Schwann cells during NMJ development. Thus, if the motor axons are cut at birth, the Schwann cells associated with their degenerating distal portion die. This can be prevented by addition of neuregulin, a growth factor normally produced by the motor neuron. Consistent with a role for neuregulin in promoting Schwann cell survival is a reduction of Schwann cell numbers in mice in which neuregulin expression is reduced by genetic manipulation.
NMJ Maturation: Presynaptic At birth, both the pre- and postsynaptic components of the NMJ of rats and mice are immature in structure, function, and molecular makeup. Nonetheless, neuromuscular transmission is adequate for the simple movements of the neonate. The adult form of the NMJ arises in concert with increasing motor activity during the next 3–4 weeks as the result of a program of coordinated events affecting the presynaptic nerve terminal and the postsynaptic surface of the muscle fiber. Myelination of the Axon
In rats and mice, myelination of the peripheral nerves begins a day or two after birth. Within a further week or so, the extramuscular parts of the nerves are well myelinated. By contrast, although the fine
intramuscular branches leading to individual muscle fibers remain in close contact with Schwann cells, they remain unmyelinated until the elimination of supernumerary innervation is complete. Synapse Elimination
During the first 2 weeks or so after birth, all but one of the axons that initially innervate each muscle fiber withdraws, leaving a sole survivor (Figure 1). A similar process occurs at most of the vertebrate NMJs that have been investigated. In humans, synapse elimination is complete by about 14 weeks of gestation. This important process has been extensively investigated, both at the NMJ and in the CNS. A very different pattern of muscle innervation occurs in the muscle of fish, some muscles in other lower vertebrates, and in a few muscles of mammals. In these muscles, the mature muscle fibers are innervated at multiple sites by numerous axons. Although the development of the innervation pattern in these multiply innervated muscles has not been studied in detail, it is clear that the immature nerve–muscle contacts are not restricted to a single site and that the local competition that occurs in most mammalian muscles does not occur. A common feature of these muscle fibers is that instead of generating action potentials, the contraction is triggered by the summed effects of the local depolarizations at the multiple sites of innervation. Maturation of the Nerve Terminal
Once synapse elimination is complete, the terminal of the sole surviving motor axon expands significantly. This involves a broadening of the regions of contact with the muscle and an overall increase in the area of synaptic contact (Figure 2(a)). As the nerve terminal expands, it retains its close contact with the terminal Schwann cells, which increase in number as a result of continuing cell divisions. The factors that limit or determine the size of the mature terminal are poorly understood. Zones of postsynaptic specialization can be induced in a variety of experimental conditions and may adopt a size and appearance remarkably similar to those at mature NMJs. This suggests that factors within the muscle play an important role in determining NMJ size and its well-established correlation with muscle fiber caliber. This view has found recent support from the discovery that a class of mutations of the gene encoding Dok-7, a postsynaptic protein that regulates MuSK activity, causes a reduction in human NMJ size without significant alteration in muscle fiber size or the local density of AChRs. It remains to be seen whether neuromuscular synaptic size–strength homoestasis in vertebrates is regulated by mechanisms
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similar to those recently shown to operate in Drosophila larvae. Transmitter release While growing motor axons may be able to release ACh in response to depolarization (see above), evidence from in vitro studies indicates that contact of the nerve terminal with muscle cells greatly increases the efficacy of quantal release. The structural basis of this enhanced release is unclear since active zones, the sites of nerve-induced quantal release at mature NMJs, have not been described until the NMJs acquire their mature form. Even at immature NMJs, stimulation of any of the several axons innervating each muscle fiber is often adequate to trigger muscle contraction. However, the quantal content (the number of ACh quanta released by a single nerve impulse) is very low, initially about 5% of the adult value. During the 2–3 week period of synapse elimination, quantal release increases at some of the terminals at each NMJ while it declines at others. In general, greater quantal release is associated with a greater chance of a terminal’s surviving the competitive process. Since transmitter release correlates strongly with synaptic size, and it is the smallest synapses that are ultimately eliminated, this association is perhaps not surprising. After synapse elimination is complete, the quantal content of the sole surviving input increases progressively, reaching its adult value at about 2 months of age. Part of this increase reflects the increasing size of the nerve terminal. In addition, there is an increase in the quantal release per unit area of membrane. This may be related to the maturation of the population of active zones. Ca2+ Channel Switch
The changes in the effectiveness of quantal release are accompanied by a change in the type of calcium channels that trigger release. At adult mammalian NMJs, release is mediated by P/Q-type channels, as can be shown using selective blockers. In contrast, at the NMJs of newborn mice, both P/Q-type and N-type channels contribute to release. The contribution of the N-type channels is lost within 1–2 weeks after birth.
NMJ Maturation: Postsynaptic Maturation of the nerve terminal is paralleled by numerous changes in the postsynaptic region of the muscle fiber that contribute to an increase in the speed and efficacy of neuromuscular transmission. This occurs in the context of a substantial increase in muscle fiber diameter. Immature muscle fibers typically have a diameter of 8–10 mm and relatively high electrical input resistance. As a result, relatively little
ACh-induced current is required to depolarize them to the action potential threshold. This helps ensure effective neuromuscular transmission even when ACh output from the nerve is still low. As the muscle grows, the diameter of the fibers also grows, and their input resistance falls. A variety of changes in the postsynaptic region help to ensure and enhance the efficacy of transmission in spite of this. Remodeling of the Postsynaptic Zone
As synapse elimination proceeds, important changes take place in the distribution of key synaptic molecules and in the conformation of the postsynaptic membrane. Redistribution of AChRs and AChE At birth, the AChRs and AChE occupy more or less uniformly the oval plaque that defines the NMJ. With time, holes in this plaque appear where the density of postsynaptic molecules is relatively low. As synapse elimination is completed and the one surviving terminal begins to expand, the regions with a high density of postsynaptic molecules become closely associated with the nerve terminals, mirroring their pretzel-like shape (Figure 2(b)). In the case of AChRs, this redistribution is associated with a substantial increase in the local density, rising to about 10 000 mm2. A number of molecules associated with the AChRs, including rapsyn, utrophin, and syntrophin, undergo a similar redistribution. This suggests that there are close links between them and that these molecules may help to stabilize the AChR cluster by cross-linking to the cytoskeleton. Appearance of voltage-gated sodium channels channels and postsynaptic folds At the mature NMJ, the postsynaptic membrane is highly folded, with the folds extending into the muscle fiber. The voltagegated sodium channels that account for the action potential, of a type termed NaV1, are highly concentrated in the depths of these folds and in a perijunctional zone a few micrometers wide (Figure 3(a)). During development in rats, these channels are first detectable around the time of birth, about a week after the AChRs. At this time, the NaV1 channels are present in highest density in a diffuse band some 200–300 mm2 wide, centered on the AChR cluster but extending well beyond it and lacking its distinct boundary (Figure 3(b)). Thus, the two types of ion channel that are central to the postsynaptic response to the nerve have very different developmental patterns of expression. Formation of the folds The postsynaptic folds begin to develop soon after birth (Figure 4). The factors that
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Figure 3 Voltage-gated sodium channels (NaV1) at the mammalian neuromuscular junction (NMJ). (a) Adult rat. NaV1s, shown in red, occupy the depths of the folded postsynaptic membrane and in the light microscope (left panel) appear as a red fringe surrounding the acetylcholine receptors (AChRs), shown in green. (b) Newborn rat. AChRs are concentrated in discrete spots at the immature NMJs while the NaV1s, which are first detected at this age, have a much more diffuse distribution. (a) Reproduced from Slater CR (2003) Structural determinants of the reliability of synaptic transmission at the vertebrate neuromuscular junction. Journal of Neurocytology 32: 505–522, with the permission from Springer.
control their distribution and growth are poorly understood. It has been suggested that the opening of the fold represents a site of reduced nerve-muscle adhesion, possibly related to the presence of the active zones in the nerve terminal. However, folds also form in a variety of situations in which AChRs form clusters in the absence of the nerve. These include both denervated and regenerating muscle and muscle exposed to exogenously applied agrin, where AChR clusters associated with folds form at sites away from the NMJ. From an early stage in fold formation during normal development, while AChRs are concentrated at the crests of the folds, nearest the nerve, the NaV1 channels occupy the depths of the folds. This is likely to result from a high concentration of NaV1 channels in the new membrane added during the process of fold formation combined with a barrier to diffusion of NaV1 channels into the region of high AChR density. Molecular Differentiation of the Postsynaptic Zone
The remodeling of the postsynaptic region is accompanied by changes in the patterns of isoforms of
the ion channels expressed within it. These come about as a result of the combined effects of increasing muscle activity and signaling molecules released from the nerve on gene transcription in the subsynaptic myonuclei. Changes in AChR expression After birth, the density of AChRs away from the NMJ declines so that in the adult it is generally undetectable. This is the result of suppression of AChR subunit gene transcription by muscle activity, mediated by the binding of myogenic regulatory factors to an E-box sequence in the genes of several AChR subunits. It raises a central question: How is it that AChRs remain at the NMJ itself? In brief, the answer is that agrin released from the nerve and bound to the synaptic basal lamina, through its activation of MuSK, induces activity-resistant transcription of the genes encoding both AChR subunits and a number of other postsynaptic proteins, by the myonuclei in the vicinity of the NMJ (see below). In addition to the activity-mediated suppression of AChR expression away from the NMJ, there is a change in the type of AChRs expressed at the NMJ
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bound ACh to charge entry. However, the slower kinetics of the currents mediated by fetal AChRs mean that immature NMJs are poorly suited to transmit the high-frequency repetitive activity that occurs in the mature animal. It is thus relevant that the changes in AChR properties occur at about the same time as the speeding up of the firing patterns of the motor neuron (see below) and the myelination of the most distal axons that allows those patterns to reach the NMJ.
Figure 4 Ultrastructural maturation of mouse neuromuscular junctions (NMJs); development of postsynaptic folds at NMJs in postnatal mouse extensor digitorum longus muscles. (a) In the newborn, few folds are present. Solid arrows, subjunctional cytoplasm rich in coated vesicle and coated pits; open arrows, multivesicular bodies. (b) At 2 weeks, folds are present but less well developed than in adult. (c) In the adult, folds are well formed and closely packed. Solid arrow, junctional folds. a, axon terminal; star, Schwann cell cytoplasm; asterisk, thickened postsynaptic membranes, reflecting accumulation of AChRs and associated protiens. Scale bars ¼ 1 mm. Reproduced from MatthewsBellinger JA and Salpeter MM (1983) Fine structural distribution of acetylcholine receptors at developing mouse neuromuscular junctions. Journal of Neuroscience 3: 644–657. Copyright 1983 by the Society for Neuroscience, with permission.
itself. At birth the predominant fetal form of the AChR has a subunit composition of a2bdg. During the first 2 weeks after birth, the g subunit is replaced by an e subunit to produce the adult form. Expression of the e subunit is not suppressed by muscle activity. The two forms of AChR differ in their channel properties: The immature form has a longer mean open time (typically 7–8 ms) and a lower conductance (40–50 pS) than the adult form (1–2 ms; 50–60 pS). As a result, a single opening of average duration of a fetal AChR channel allows 3–4 times as much charge to enter the cell as an adult channel. This makes the fetal channels more efficient at converting
Changes in NaV1 expression An analogous change in isoform expression occurs for the NaV1 channels. The first NaV1 channels to appear are of an immature form, designated NaV1.5. These differ from the adult form (NaV1.4) in that they open at morenegative membrane potentials. As a result, less depolarization is required to trigger an action potential in an immature muscle than in a mature one. Expression of the gene encoding NaV1.5, like that of the g-AChR subunit gene, is suppressed by muscle activity, both at the NMJ and away from it. As a result, expression of NaV1.5 declines to an undetectable level during the first 2–3 weeks after birth. Expression of the gene encoding NaV1.4 is not sensitive to activity. Its expression first becomes detectable at birth and increases during the next few weeks, both away from the NMJ and at a higher level at the NMJ. The factors that control the onset of NaV1.4 expression and its enhancement at the NMJ are not known. mRNA accumulation The developmental increases in the concentration of AChR and NaV1 at the NMJ are accompanied by localized increases in the levels of the mRNAs that encode them. At the mature NMJ, there is an increase in the concentration of mRNA encoding a number of critical postsynaptic proteins, including AChR subunits, AChE, and NaV1.4, as well as supporting proteins including rapsyn and utrophin. These increases are apparent soon after birth and are probably induced by agrin acting via MuSK. Accumulation of Myonuclei
The upregulation of expression at the NMJ of the genes encoding important components of the postsynaptic membrane is now believed to be induced by agrin acting via MuSK. At the NMJ, agrin is fixed in place by association with the synaptic basal lamina. This ensures that the effects of agrin are confined to the vicinity of the NMJ. The effective sphere of influence of this immobilized agrin is not more than about 100 mm, so myonuclei further than this from the NMJ do not have a synaptic profile of gene expression. At many NMJs, there is an accumulation of 5–10 myonuclei within this sphere of influence.
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This amplifies the effect of the agrin–MuSK signaling system. The clustering of myonuclei begins soon after birth and is complete by about 1 month of age. The events leading to myonuclear accumulation are not yet understood.
Mammalian Motor Unit Maturation The formation and maturation of the mammalian NMJ occurs in the context of the development of the motor unit as a whole. The maturation of the motor neuron, and in particular its pattern of firing, has an effect on the development of the NMJ itself. In turn, the increasing ability of the NMJ to transmit faithfully the patterns of activity arising in the motor neuron to the muscle fibers plays an important part in the acquisition of mature muscle properties. It may also lead to retrograde regulation of motor neuron properties. Firing Patterns
In adult mammals, motor neurons vary considerably in their functional properties. Most notably, some fire action potentials in long trains at a frequency of 10–20 Hz while others fire in short bursts of 5–10 action potentials at frequencies of up to 100 Hz. Motor neurons in neonatal rats and mice fire at a uniformly low frequency and are often synchronized as a result of electrical coupling by gap junctions. The first evidence of faster, more adultlike firing patterns, and of differences between the firing patterns of different motor neurons, is seen about 2 weeks after birth, by which time electrical coupling is lost. The onset of these more adult firing properties coincides with the time of myelination of the most distal intramuscular nerve branches. It is likely that before this happens, these small branches would be unable to fire at high frequencies and could therefore not transmit the features of activity that distinguish different motor neurons in the adult. Efficacy of Neuromuscular Transmission
During the early stage of polyaxonal innervation, more than one input to the muscle fiber is often capable of triggering contraction. As the competitive process unfolds, some inputs, and ultimately only one, come to have a much stronger impact on the muscle fiber than others. There is evidence that each branch of a motor axon competes locally at a given NMJ and that axons with the strongest input to a given NMJ are more likely to survive the competitive process than weaker ones. However, the evidence on this point is not clear cut, and neither of these findings fully explains the final outcome of the competition, in which a single motor axon, with appropriately matched properties, innervates each muscle fiber.
As the NMJ matures, both the pre- and postsynaptic components increase in size. For the muscle fiber, this results in a decrease in input resistance and therefore the need for a greater synaptic current to reach the action potential threshold reliably. This is achieved by the parallel increase in the size of the motor nerve terminal and with it, the quantal content. This, together with the folding of the postsynaptic membrane, results in the great reliability of neuromuscular transmission in the adult. The nature of the feedback mechanisms that regulate synaptic size and strength is intriguing, however, because one hallmark of the normal adult neuromuscular junction is a three- to fivefold excess of transmitter release over that required to trigger an action potential in the muscle fiber. This high safety factor ensures that every action potential entering a motor nerve terminal will normally trigger an action potential in all the muscle fiber it innervates. Development of Muscle Fiber Homogeneity
An important consequence of the process of synapse elimination is that each motor neuron ends up innervating muscle fibers with very similar properties. Clear signs of increasing functional homogeneity of the muscle fibers within motor units are seen in mice 1–2 weeks after birth, as synapse elimination nears completion but before the distinctive patterns of activity of different motor units are well developed. It therefore seems unlikely that differences in activity patterns between motor neurons play a decisive role either in selecting which input survives at a given NMJ or in matching the properties of motor neurons to the muscle fibers they innervate. A possible alternative is that the matching of nerve and muscle cells is achieved by a molecular recognition system that involves activity-dependent expression of surface and/or diffusible molecules that interact so that during the process of synapse elimination, the most compatible nerve-muscle pairs survive at each developing NMJ. Such a mechanism could depend on activity as a driving force without the pattern of activity determining the specific outcome of the competition. As yet, however, no likely candidates for such a molecular recognition scheme have been identified. It is also not yet clear how the properties of a mature muscle fiber are determined. There is good evidence for at least two very different, though not mutually exclusive, mechanisms. On the one hand, there is evidence that myoblasts are predetermined to make fast and slow muscle fibers even before myotube formation occurs. If correct, this mean that myoblasts of the same predisposition may fuse more or less selectively to make myotubes containing nuclei with intrinsically similar properties. On the other
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hand, there is very strong evidence, particularly in mammals, that the properties of adult muscle fibers are sensitive to the patterns of activity they experience. This is consistent with the idea that the properties of a muscle fiber are modified after innervation by the pattern of activity of the motor neuron that innervates it. Both schemes raise important questions about how the muscle fibers innervated by a single motor neuron come to have the same properties.
Conclusions The many changes in the structural, functional, and molecular properties of the NMJ that occur during its maturation adapt it for reliable high-frequency activation of mature muscle fibers. These changes are matched to complementary changes in the nerve and the muscle. Their overall effect is the conversion of an immature system that is good at generating slow muscle contractions in response to low-frequency activity in the nerve to a much faster system, adapted to the needs of a freely moving and increasingly independent animal. The events that give rise to the mature NMJ are part of a coherent developmental program that defines the patterns of expression of a number of proteins, such as ion channels, that play central roles in neuromuscular transmission. In addition, it determines the size and conformation of the NMJ. Both aspects of the program have important consequences for the efficacy and reliability of the mature NMJ. See also: Neuromuscular Junction: Neuronal Regulation of Gene Transcription at the Vertebrate; Neuromuscular Junction: Synapse Elimination; Neuromuscular Junction Plasticity in Mammals and Botulinum Toxins; Neuromuscular Junction (NMJ): Acetylcholinesterases; Neuromuscular Junction (NMJ): Postsynaptic Basal Lamina; Neuromuscular Connections: Vertebrate Patterns of; Presynaptic Events in Neuromuscular Transmission; Schwann Cells and Plasticity of the Neuromuscular Junction.
Further Reading Banks GB, Fuhrer C, Adams ME, et al. (2003) The postsynaptic submembrane machinery at the neuromuscular junction: Requirement for rapsyn and the utrophin/dystrophin-associated complex. Journal of Neurocytology 32: 709–726. Bellinger JA and Salpeter MM (1983) Fine structural distribution of acetylcholine receptors at developing mouse neuromuscular junctions. Journal of Neuroscience 3: 644–657. Bennett MR and Pettigrew AG (1974) The formation of synapses in striated muscle during development. Journal of Physiology 241: 515–545. Bewick GS, Reid B, Jawaid S, et al. (2004) Postnatal emergence of mature release properties in terminals of rat fast- and slow-twitch muscles. European Journal of Neuroscience 19: 2967–2976. Jansen JK and Fladby T (1990) The perinatal reorganization of the innervation of skeletal muscle in mammals. Progress in Neurobiology 34: 39–90. Landmesser LT (2001) The acquisition of motoneuron subtype identity and motor circuit formation. International Journal of Developmental Neuroscience 19: 175–182. Lichtman JW and Sanes JR (2003) Watching the neuromuscular junction. Journal of Neurocytology 32: 767–775. Marques MJ, Conchello JA, and Lichtman JW (2000) From plaque to pretzel: Fold formation and acetylcholine receptor loss at the developing neuromuscular junction. Journal of Neuroscience 20: 3663–3675. Matthews-Bellinger JA and Salpeter MM (1983) Fine structural distribution of acetylcholine receptors at developing mouse neuromuscular junctions. Journal of Neuroscience 3: 644–657. McMahan UJ (1990) The agrin hypothesis. Cold Spring Harbor Symposia on Quantitative Biology 55: 407–418. Sanes JR and Lichtman JW (1999) Development of the vertebrate neuromuscular junction. Annual Review of Neuroscience 22: 389–442. Sheard PW and Duxson MJ (1996) Composition of newly forming motor units in prenatal rat intercostal muscle. Developmental Dynamics 205: 196–212. Slater CR (1982) Postnatal maturation of nerve-muscle junctions in hindlimb muscles of the mouse. Developmental Biology 94: 11–22. Slater CR (2003) Structural determinants of the reliability of synaptic transmission at the vertebrate neuromuscular junction. Journal of Neurocytology 32: 505–522. Urbano FJ, Rosato-Siri MD, and Uchitel OD (2002) Calcium channels involved in neurotransmitter release at adult, neonatal and P/Q-type deficient neuromuscular junctions. Molecular Membrane Biology 19: 293–300.
Neuromuscular Junction (NMJ): Mammalian Development C R Slater, University of Newcastle, Newcastle upon Tyne, UK ã 2009 Elsevier Ltd. All rights reserved.
Introduction The neuromuscular junction (NMJ) is an example of a highly specialized, asymmetric, cell-cell junction. The speed with which fast chemical transmission occurs requires that the specializations of the postsynaptic muscle cell surface that allow it to respond to the transmitter released from the nerve are less than a micrometer from the presynaptic release sites. An important question concerning the development of the NMJ is therefore, what are the mechanisms that account for the alignment and proximity of the distinctive specializations of its pre- and postsynaptic components? A second important question concerns the specificity of the mature nerve-muscle contacts. What part does the process of NMJ formation play in achieving functional matching of individual motor neurons to the muscle fibers they innervate? This article gives an overview of the way mammalian NMJs develop. Most of the information has come from rats and mice, in which the most detailed studies of NMJ formation in vertebrates have been made. The article starts with the origin of motor neurons and skeletal muscle fibers and then considers how the NMJ itself is formed.
Development of Motor Neurons Birth
Motor neurons are among the earliest nerve cells to be born, that is, to complete their final round of DNA synthesis. Soon after cell birth, the motor neurons begin to extend an axon that leaves the spinal cord in the nascent ventral roots. In mammals, the motor neurons that innervate an individual muscle are usually grouped into a longitudinally oriented column that extends over two to three spinal segments. Axon Outgrowth and Motor Neuron Identity
The immature motor axons leave the spinal cord even before their target muscles have formed. As they grow, the axons select paths that lead to the muscles they are destined to innervate. The ability of an immature motor neuron to make such decisions indicates that it has some knowledge of its identity, and that different motor neurons therefore have different identities. In adults, motor neurons that innervate
slowly contracting nonfatigable muscle fibers tend to be rich in oxidative enzymes that can be visualized by appropriate histochemical techniques. Well before the first NMJs are formed, embryonic motor neurons already differ in their oxidative enzyme profiles. This supports the idea that the motor neurons that innervate a single muscle differ in their properties and that those differences arise before any interaction with the muscle occurs. Once contact with the appropriate premuscle mass has been established, but not before, the axons branch extensively. In rats and mice, functional contacts with newly formed limb muscles are first present around embryonic day 14 (E14), a week before birth. A similar stage occurs in humans at about week 9 of gestation. Release of Acetylcholine from Growth Cones
The terminals of cultured motor neurons can release acetylcholine (ACh), the chemical transmitter at neuromuscular junctions, even before they make contact with muscle. This suggests that motor neurons in vivo also synthesize ACh and have the necessary specializations for its activity-dependent release at an early stage of their development.
Development of Skeletal Muscle Fibers Myotube Formation
Most vertebrate skeletal muscle fibers arise during development from the fusion of many mononucleated postmitotic myoblasts. These spindle-shaped cells line up and fuse to form the primitive myotubes. As the myotubes grow and mature into muscle fibers, they incorporate additional myoblasts. In adult muscle fibers, there is typically one nucleus for every 10 mm of length. Thus, a single fiber in a large human muscle, such as vastus lateralis, which has fibers up to 20 cm long, has up to 20 000 nuclei. Kinetics of Primary and Secondary Myotube Formation
In mammalian muscles, myotube formation occurs in two phases. The first involves the formation of an initial cohort of relatively few primary myotubes. As the muscle elongates, further myoblasts line up along the primary myotubes and eventually fuse to form secondary myotubes. These form initially roughly midway between the ends of the primary myotubes and then grow rapidly in length as they add new myoblasts. The two populations of myotubes, and the mature muscle fibers that form from them, have
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somewhat different functional properties. The great majority of fibers in the adult muscles are derived from secondary myotubes. Origin of Muscle Fiber Types
Most adult mammalian muscles contain fibers of different functional types. Some are specialized for relatively slow, sustained contraction whereas others are specialized for fast, high-powered contractions. At birth, the muscle fibers in rats and mice contract uniformly slowly. Differentiation into faster and slower contracting fibers begins soon after birth and is well established 2–3 weeks later. Most of the muscle fibers that develop from primary myotubes end up as slow in the adult, while secondary myotubes give rise to both fast and slow fibers. These distinctive properties arise from the pattern of genes expressed by the fibers. Most of the nuclei in each fiber express the same set of genes, raising the as yet unanswered question of how that homogeneity of gene expression comes about.
Polyaxonal Innervation
A distinctive feature of the early motor innervation of vertebrates is that several motor neurons initially innervate each muscle fiber. This innervation occurs at a single postsynaptic site that is thus contacted by the terminal axons of several motor neurons (Figure 1). Myotubes cultured in vitro can acquire polyneuronal innervation, but such multiple inputs are normally distributed over the myotube surface rather than focused on a single site. The forces that initially restrict the immature nerve terminals to this single site in vivo are not known. AChR Accumulation
A high density of AChRs is a hallmark of the postsynaptic membrane of the vertebrate NMJ. A distinct cluster of AChRs, detectable after labeling with fluorescent conjugates of the snake toxin a-bungarotoxin, is present from a very early stage of NMJ formation (Figure 2). Within the immature cluster, AChRs are often gathered into microclusters less than 1 mm across. The mean density of AChRs within the plaque
Early Appearance of Delocalized Postsynaptic Properties
Early Development of the NMJ Initial Nerve–Muscle Encounters
Muscle fibers become innervated very soon after they first form. In rats and mice, signs of functional innervation can be detected within a day or two of the earliest myotube formation at about E12–14, depending on the muscle. The early nerve-muscle contacts lack many of the structural features of mature NMJs but are characterized by a high density of AChRs in the muscle fiber membrane. Whether the AChR clusters form before or after nerve contact is a matter of continuing debate (see below). Indeed, there is evidence that NMJs in different muscles in the same species form by different sequences of events.
Axon Muscle fiber
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Even before they are innervated, immature muscle fibers begin to express the receptor proteins that allow them to respond to ACh (acetylcholine receptors, or AChRs). AChRs of the fetal form, containing a2bdg subunits, are initially present over the whole fiber surface at a density of about 500 mm2. Other molecules that play roles in AChR localization and gene expression, such as rapsyn and muscle specific kinase (MuSK; see ‘AChR aggregation’ below), have a similar pattern of expression. As the muscle fibers become increasingly active, their activity suppresses the expression of all these proteins away from the NMJ.
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Figure 1 Development of muscle innervation. (a) Comparison of polyneuronal innervation of muscles in newborn rats or mice (left) with the adult state (right). (b) Changes in the number of axon terminals innervating neuromuscular junctions in rat diaphragm with age. (b) Reproduced from Bennett MR and Pettigrew AG (1974) The formation of synapses in striated muscle during development. Journal of Physiology 241: 515–545, with permission from Blackwell publishing.
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Figure 2 Structural maturation of mouse neuromuscular junctions (NMJs). Nerve terminals in postnatal mouse extensor digitorum longus muscles were labeled with zinc iodide-osmium, and acetylcholine receptors (AChRs) were labeled with R-a-bungarotoxin. Arrows point to the preterminal motor axon(s). Several axons are present at birth, but between 1 and 2 weeks after birth, all but one are withdrawn. The surviving axon becomes myelinated 2–3 weeks after birth. As the axon terminal enlarges, the initially uniform granular distribution of AChRs breaks up, leaving areas with low AChR density that come to match the branches of the nerve terminals. NB, newborn.
is about 3000 mm2. How the AChR cluster forms initially and is subsequently maintained are topics of extensive current research. There appear to be at least two components of this process: the aggregation of AChRs within the membrane of the muscle fiber and the enhanced expression of the genes encoding the AChR subunits by the myonuclei closest to the site of nerve contact. AChR aggregation AChRs can exist in a form that is mobile in the plane of the cell membrane. In the case of cultured immature frog muscle precursor cells, such mobile AChRs aggregate at sites of contact with motor axons, suggesting that the nerve induces
aggregation of AChRs in the muscle cell. Much evidence suggests that it is a protein, agrin, which is made by and released from motor axons, that triggers AChR aggregation. Agrin function is associated with its activation of a muscle specific kinase (MuSK), which triggers downstream signaling cascades in the muscle. A second key player in the process of agrininduced AChR aggregation is the 43 kDa protein rapsyn. Rapsyn links AChRs to the plasma membrane and to components of the membrane skeleton. In the absence of rapsyn, no aggregation of AChRs occurs. There is also much evidence that AChRs can selfaggregate, and on suitable substrates they can form clusters similar to those at mature NMJs. Some recent
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studies have shown that in some species and muscles, AChR clusters form in the region of presumptive NMJs before nerve-muscle contacts are made. In these cases, neuromuscular contacts may form selectively on the presumptive postsynaptic sites. On balance, it seems likely that while the muscle fiber surface has an inherent tendency to form AChR clusters, agrin released from the nerve can either trigger clustering de novo or stabilize preexisting clusters. Upregulation of postsynaptic gene expression The second process that contributes to AChR accumulation at the mature NMJ is the enhanced expression of the genes that encode the AChR subunits. This process is discussed below. Within a few days of nerve contact, acetylcholinesterase (AChE), the enzyme that terminates AChaction, accumulates in the synaptic basal lamina that forms between the nerve and muscle cells. As for AChRs, this accumulation is associated with upregulation of the genes encoding AChE. Schwann Cells: The Motor Neurons’ Companion
Throughout most of their life, motor axons are closely accompanied by Schwann cells. There is increasing evidence of a mutual dependency of motor axons and their companion Schwann cells during NMJ development. Thus, if the motor axons are cut at birth, the Schwann cells associated with their degenerating distal portion die. This can be prevented by addition of neuregulin, a growth factor normally produced by the motor neuron. Consistent with a role for neuregulin in promoting Schwann cell survival is a reduction of Schwann cell numbers in mice in which neuregulin expression is reduced by genetic manipulation.
NMJ Maturation: Presynaptic At birth, both the pre- and postsynaptic components of the NMJ of rats and mice are immature in structure, function, and molecular makeup. Nonetheless, neuromuscular transmission is adequate for the simple movements of the neonate. The adult form of the NMJ arises in concert with increasing motor activity during the next 3–4 weeks as the result of a program of coordinated events affecting the presynaptic nerve terminal and the postsynaptic surface of the muscle fiber. Myelination of the Axon
In rats and mice, myelination of the peripheral nerves begins a day or two after birth. Within a further week or so, the extramuscular parts of the nerves are well myelinated. By contrast, although the fine
intramuscular branches leading to individual muscle fibers remain in close contact with Schwann cells, they remain unmyelinated until the elimination of supernumerary innervation is complete. Synapse Elimination
During the first 2 weeks or so after birth, all but one of the axons that initially innervate each muscle fiber withdraws, leaving a sole survivor (Figure 1). A similar process occurs at most of the vertebrate NMJs that have been investigated. In humans, synapse elimination is complete by about 14 weeks of gestation. This important process has been extensively investigated, both at the NMJ and in the CNS. A very different pattern of muscle innervation occurs in the muscle of fish, some muscles in other lower vertebrates, and in a few muscles of mammals. In these muscles, the mature muscle fibers are innervated at multiple sites by numerous axons. Although the development of the innervation pattern in these multiply innervated muscles has not been studied in detail, it is clear that the immature nerve–muscle contacts are not restricted to a single site and that the local competition that occurs in most mammalian muscles does not occur. A common feature of these muscle fibers is that instead of generating action potentials, the contraction is triggered by the summed effects of the local depolarizations at the multiple sites of innervation. Maturation of the Nerve Terminal
Once synapse elimination is complete, the terminal of the sole surviving motor axon expands significantly. This involves a broadening of the regions of contact with the muscle and an overall increase in the area of synaptic contact (Figure 2(a)). As the nerve terminal expands, it retains its close contact with the terminal Schwann cells, which increase in number as a result of continuing cell divisions. The factors that limit or determine the size of the mature terminal are poorly understood. Zones of postsynaptic specialization can be induced in a variety of experimental conditions and may adopt a size and appearance remarkably similar to those at mature NMJs. This suggests that factors within the muscle play an important role in determining NMJ size and its well-established correlation with muscle fiber caliber. This view has found recent support from the discovery that a class of mutations of the gene encoding Dok-7, a postsynaptic protein that regulates MuSK activity, causes a reduction in human NMJ size without significant alteration in muscle fiber size or the local density of AChRs. It remains to be seen whether neuromuscular synaptic size–strength homoestasis in vertebrates is regulated by mechanisms
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similar to those recently shown to operate in Drosophila larvae. Transmitter release While growing motor axons may be able to release ACh in response to depolarization (see above), evidence from in vitro studies indicates that contact of the nerve terminal with muscle cells greatly increases the efficacy of quantal release. The structural basis of this enhanced release is unclear since active zones, the sites of nerve-induced quantal release at mature NMJs, have not been described until the NMJs acquire their mature form. Even at immature NMJs, stimulation of any of the several axons innervating each muscle fiber is often adequate to trigger muscle contraction. However, the quantal content (the number of ACh quanta released by a single nerve impulse) is very low, initially about 5% of the adult value. During the 2–3 week period of synapse elimination, quantal release increases at some of the terminals at each NMJ while it declines at others. In general, greater quantal release is associated with a greater chance of a terminal’s surviving the competitive process. Since transmitter release correlates strongly with synaptic size, and it is the smallest synapses that are ultimately eliminated, this association is perhaps not surprising. After synapse elimination is complete, the quantal content of the sole surviving input increases progressively, reaching its adult value at about 2 months of age. Part of this increase reflects the increasing size of the nerve terminal. In addition, there is an increase in the quantal release per unit area of membrane. This may be related to the maturation of the population of active zones. Ca2+ Channel Switch
The changes in the effectiveness of quantal release are accompanied by a change in the type of calcium channels that trigger release. At adult mammalian NMJs, release is mediated by P/Q-type channels, as can be shown using selective blockers. In contrast, at the NMJs of newborn mice, both P/Q-type and N-type channels contribute to release. The contribution of the N-type channels is lost within 1–2 weeks after birth.
NMJ Maturation: Postsynaptic Maturation of the nerve terminal is paralleled by numerous changes in the postsynaptic region of the muscle fiber that contribute to an increase in the speed and efficacy of neuromuscular transmission. This occurs in the context of a substantial increase in muscle fiber diameter. Immature muscle fibers typically have a diameter of 8–10 mm and relatively high electrical input resistance. As a result, relatively little
ACh-induced current is required to depolarize them to the action potential threshold. This helps ensure effective neuromuscular transmission even when ACh output from the nerve is still low. As the muscle grows, the diameter of the fibers also grows, and their input resistance falls. A variety of changes in the postsynaptic region help to ensure and enhance the efficacy of transmission in spite of this. Remodeling of the Postsynaptic Zone
As synapse elimination proceeds, important changes take place in the distribution of key synaptic molecules and in the conformation of the postsynaptic membrane. Redistribution of AChRs and AChE At birth, the AChRs and AChE occupy more or less uniformly the oval plaque that defines the NMJ. With time, holes in this plaque appear where the density of postsynaptic molecules is relatively low. As synapse elimination is completed and the one surviving terminal begins to expand, the regions with a high density of postsynaptic molecules become closely associated with the nerve terminals, mirroring their pretzel-like shape (Figure 2(b)). In the case of AChRs, this redistribution is associated with a substantial increase in the local density, rising to about 10 000 mm2. A number of molecules associated with the AChRs, including rapsyn, utrophin, and syntrophin, undergo a similar redistribution. This suggests that there are close links between them and that these molecules may help to stabilize the AChR cluster by cross-linking to the cytoskeleton. Appearance of voltage-gated sodium channels channels and postsynaptic folds At the mature NMJ, the postsynaptic membrane is highly folded, with the folds extending into the muscle fiber. The voltagegated sodium channels that account for the action potential, of a type termed NaV1, are highly concentrated in the depths of these folds and in a perijunctional zone a few micrometers wide (Figure 3(a)). During development in rats, these channels are first detectable around the time of birth, about a week after the AChRs. At this time, the NaV1 channels are present in highest density in a diffuse band some 200–300 mm2 wide, centered on the AChR cluster but extending well beyond it and lacking its distinct boundary (Figure 3(b)). Thus, the two types of ion channel that are central to the postsynaptic response to the nerve have very different developmental patterns of expression. Formation of the folds The postsynaptic folds begin to develop soon after birth (Figure 4). The factors that
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Figure 3 Voltage-gated sodium channels (NaV1) at the mammalian neuromuscular junction (NMJ). (a) Adult rat. NaV1s, shown in red, occupy the depths of the folded postsynaptic membrane and in the light microscope (left panel) appear as a red fringe surrounding the acetylcholine receptors (AChRs), shown in green. (b) Newborn rat. AChRs are concentrated in discrete spots at the immature NMJs while the NaV1s, which are first detected at this age, have a much more diffuse distribution. (a) Reproduced from Slater CR (2003) Structural determinants of the reliability of synaptic transmission at the vertebrate neuromuscular junction. Journal of Neurocytology 32: 505–522, with the permission from Springer.
control their distribution and growth are poorly understood. It has been suggested that the opening of the fold represents a site of reduced nerve-muscle adhesion, possibly related to the presence of the active zones in the nerve terminal. However, folds also form in a variety of situations in which AChRs form clusters in the absence of the nerve. These include both denervated and regenerating muscle and muscle exposed to exogenously applied agrin, where AChR clusters associated with folds form at sites away from the NMJ. From an early stage in fold formation during normal development, while AChRs are concentrated at the crests of the folds, nearest the nerve, the NaV1 channels occupy the depths of the folds. This is likely to result from a high concentration of NaV1 channels in the new membrane added during the process of fold formation combined with a barrier to diffusion of NaV1 channels into the region of high AChR density. Molecular Differentiation of the Postsynaptic Zone
The remodeling of the postsynaptic region is accompanied by changes in the patterns of isoforms of
the ion channels expressed within it. These come about as a result of the combined effects of increasing muscle activity and signaling molecules released from the nerve on gene transcription in the subsynaptic myonuclei. Changes in AChR expression After birth, the density of AChRs away from the NMJ declines so that in the adult it is generally undetectable. This is the result of suppression of AChR subunit gene transcription by muscle activity, mediated by the binding of myogenic regulatory factors to an E-box sequence in the genes of several AChR subunits. It raises a central question: How is it that AChRs remain at the NMJ itself? In brief, the answer is that agrin released from the nerve and bound to the synaptic basal lamina, through its activation of MuSK, induces activity-resistant transcription of the genes encoding both AChR subunits and a number of other postsynaptic proteins, by the myonuclei in the vicinity of the NMJ (see below). In addition to the activity-mediated suppression of AChR expression away from the NMJ, there is a change in the type of AChRs expressed at the NMJ
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bound ACh to charge entry. However, the slower kinetics of the currents mediated by fetal AChRs mean that immature NMJs are poorly suited to transmit the high-frequency repetitive activity that occurs in the mature animal. It is thus relevant that the changes in AChR properties occur at about the same time as the speeding up of the firing patterns of the motor neuron (see below) and the myelination of the most distal axons that allows those patterns to reach the NMJ.
Figure 4 Ultrastructural maturation of mouse neuromuscular junctions (NMJs); development of postsynaptic folds at NMJs in postnatal mouse extensor digitorum longus muscles. (a) In the newborn, few folds are present. Solid arrows, subjunctional cytoplasm rich in coated vesicle and coated pits; open arrows, multivesicular bodies. (b) At 2 weeks, folds are present but less well developed than in adult. (c) In the adult, folds are well formed and closely packed. Solid arrow, junctional folds. a, axon terminal; star, Schwann cell cytoplasm; asterisk, thickened postsynaptic membranes, reflecting accumulation of AChRs and associated protiens. Scale bars ¼ 1 mm. Reproduced from MatthewsBellinger JA and Salpeter MM (1983) Fine structural distribution of acetylcholine receptors at developing mouse neuromuscular junctions. Journal of Neuroscience 3: 644–657. Copyright 1983 by the Society for Neuroscience, with permission.
itself. At birth the predominant fetal form of the AChR has a subunit composition of a2bdg. During the first 2 weeks after birth, the g subunit is replaced by an e subunit to produce the adult form. Expression of the e subunit is not suppressed by muscle activity. The two forms of AChR differ in their channel properties: The immature form has a longer mean open time (typically 7–8 ms) and a lower conductance (40–50 pS) than the adult form (1–2 ms; 50–60 pS). As a result, a single opening of average duration of a fetal AChR channel allows 3–4 times as much charge to enter the cell as an adult channel. This makes the fetal channels more efficient at converting
Changes in NaV1 expression An analogous change in isoform expression occurs for the NaV1 channels. The first NaV1 channels to appear are of an immature form, designated NaV1.5. These differ from the adult form (NaV1.4) in that they open at morenegative membrane potentials. As a result, less depolarization is required to trigger an action potential in an immature muscle than in a mature one. Expression of the gene encoding NaV1.5, like that of the g-AChR subunit gene, is suppressed by muscle activity, both at the NMJ and away from it. As a result, expression of NaV1.5 declines to an undetectable level during the first 2–3 weeks after birth. Expression of the gene encoding NaV1.4 is not sensitive to activity. Its expression first becomes detectable at birth and increases during the next few weeks, both away from the NMJ and at a higher level at the NMJ. The factors that control the onset of NaV1.4 expression and its enhancement at the NMJ are not known. mRNA accumulation The developmental increases in the concentration of AChR and NaV1 at the NMJ are accompanied by localized increases in the levels of the mRNAs that encode them. At the mature NMJ, there is an increase in the concentration of mRNA encoding a number of critical postsynaptic proteins, including AChR subunits, AChE, and NaV1.4, as well as supporting proteins including rapsyn and utrophin. These increases are apparent soon after birth and are probably induced by agrin acting via MuSK. Accumulation of Myonuclei
The upregulation of expression at the NMJ of the genes encoding important components of the postsynaptic membrane is now believed to be induced by agrin acting via MuSK. At the NMJ, agrin is fixed in place by association with the synaptic basal lamina. This ensures that the effects of agrin are confined to the vicinity of the NMJ. The effective sphere of influence of this immobilized agrin is not more than about 100 mm, so myonuclei further than this from the NMJ do not have a synaptic profile of gene expression. At many NMJs, there is an accumulation of 5–10 myonuclei within this sphere of influence.
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This amplifies the effect of the agrin–MuSK signaling system. The clustering of myonuclei begins soon after birth and is complete by about 1 month of age. The events leading to myonuclear accumulation are not yet understood.
Mammalian Motor Unit Maturation The formation and maturation of the mammalian NMJ occurs in the context of the development of the motor unit as a whole. The maturation of the motor neuron, and in particular its pattern of firing, has an effect on the development of the NMJ itself. In turn, the increasing ability of the NMJ to transmit faithfully the patterns of activity arising in the motor neuron to the muscle fibers plays an important part in the acquisition of mature muscle properties. It may also lead to retrograde regulation of motor neuron properties. Firing Patterns
In adult mammals, motor neurons vary considerably in their functional properties. Most notably, some fire action potentials in long trains at a frequency of 10–20 Hz while others fire in short bursts of 5–10 action potentials at frequencies of up to 100 Hz. Motor neurons in neonatal rats and mice fire at a uniformly low frequency and are often synchronized as a result of electrical coupling by gap junctions. The first evidence of faster, more adultlike firing patterns, and of differences between the firing patterns of different motor neurons, is seen about 2 weeks after birth, by which time electrical coupling is lost. The onset of these more adult firing properties coincides with the time of myelination of the most distal intramuscular nerve branches. It is likely that before this happens, these small branches would be unable to fire at high frequencies and could therefore not transmit the features of activity that distinguish different motor neurons in the adult. Efficacy of Neuromuscular Transmission
During the early stage of polyaxonal innervation, more than one input to the muscle fiber is often capable of triggering contraction. As the competitive process unfolds, some inputs, and ultimately only one, come to have a much stronger impact on the muscle fiber than others. There is evidence that each branch of a motor axon competes locally at a given NMJ and that axons with the strongest input to a given NMJ are more likely to survive the competitive process than weaker ones. However, the evidence on this point is not clear cut, and neither of these findings fully explains the final outcome of the competition, in which a single motor axon, with appropriately matched properties, innervates each muscle fiber.
As the NMJ matures, both the pre- and postsynaptic components increase in size. For the muscle fiber, this results in a decrease in input resistance and therefore the need for a greater synaptic current to reach the action potential threshold reliably. This is achieved by the parallel increase in the size of the motor nerve terminal and with it, the quantal content. This, together with the folding of the postsynaptic membrane, results in the great reliability of neuromuscular transmission in the adult. The nature of the feedback mechanisms that regulate synaptic size and strength is intriguing, however, because one hallmark of the normal adult neuromuscular junction is a three- to fivefold excess of transmitter release over that required to trigger an action potential in the muscle fiber. This high safety factor ensures that every action potential entering a motor nerve terminal will normally trigger an action potential in all the muscle fiber it innervates. Development of Muscle Fiber Homogeneity
An important consequence of the process of synapse elimination is that each motor neuron ends up innervating muscle fibers with very similar properties. Clear signs of increasing functional homogeneity of the muscle fibers within motor units are seen in mice 1–2 weeks after birth, as synapse elimination nears completion but before the distinctive patterns of activity of different motor units are well developed. It therefore seems unlikely that differences in activity patterns between motor neurons play a decisive role either in selecting which input survives at a given NMJ or in matching the properties of motor neurons to the muscle fibers they innervate. A possible alternative is that the matching of nerve and muscle cells is achieved by a molecular recognition system that involves activity-dependent expression of surface and/or diffusible molecules that interact so that during the process of synapse elimination, the most compatible nerve-muscle pairs survive at each developing NMJ. Such a mechanism could depend on activity as a driving force without the pattern of activity determining the specific outcome of the competition. As yet, however, no likely candidates for such a molecular recognition scheme have been identified. It is also not yet clear how the properties of a mature muscle fiber are determined. There is good evidence for at least two very different, though not mutually exclusive, mechanisms. On the one hand, there is evidence that myoblasts are predetermined to make fast and slow muscle fibers even before myotube formation occurs. If correct, this mean that myoblasts of the same predisposition may fuse more or less selectively to make myotubes containing nuclei with intrinsically similar properties. On the other
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hand, there is very strong evidence, particularly in mammals, that the properties of adult muscle fibers are sensitive to the patterns of activity they experience. This is consistent with the idea that the properties of a muscle fiber are modified after innervation by the pattern of activity of the motor neuron that innervates it. Both schemes raise important questions about how the muscle fibers innervated by a single motor neuron come to have the same properties.
Conclusions The many changes in the structural, functional, and molecular properties of the NMJ that occur during its maturation adapt it for reliable high-frequency activation of mature muscle fibers. These changes are matched to complementary changes in the nerve and the muscle. Their overall effect is the conversion of an immature system that is good at generating slow muscle contractions in response to low-frequency activity in the nerve to a much faster system, adapted to the needs of a freely moving and increasingly independent animal. The events that give rise to the mature NMJ are part of a coherent developmental program that defines the patterns of expression of a number of proteins, such as ion channels, that play central roles in neuromuscular transmission. In addition, it determines the size and conformation of the NMJ. Both aspects of the program have important consequences for the efficacy and reliability of the mature NMJ. See also: Neuromuscular Junction: Neuronal Regulation of Gene Transcription at the Vertebrate; Neuromuscular Junction: Synapse Elimination; Neuromuscular Junction Plasticity in Mammals and Botulinum Toxins; Neuromuscular Junction (NMJ): Acetylcholinesterases; Neuromuscular Junction (NMJ): Postsynaptic Basal Lamina; Neuromuscular Connections: Vertebrate Patterns of; Presynaptic Events in Neuromuscular Transmission; Schwann Cells and Plasticity of the Neuromuscular Junction.
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