The neuromuscular junction comprises three types of cells: a motor neuron, a muscle fiber, and Schwann cells. All three types are highly differentiated in the region of the synapse.
The process of synapse formation is initiated when a motor axon, guided by the multiple factors described in Chapter 54, reaches a developing skeletal muscle and approaches an immature muscle fiber. Contact is made and the process of synaptic differentiation begins. As the growth cone begins its transformation into a nerve terminal, the portion of the muscle surface opposite the nerve terminal begins to acquire its own specializations. As development proceeds, synaptic components are added and structural signs of synaptic differentiation become apparent in the pre- and postsynaptic cells and in the synaptic cleft. Eventually the neuromuscular junction acquires its mature and complex form (Figure 55–7A, B).
The neuromuscular junction develops in sequential stages.
A. A growth cone approaches a newly fused myotube (1) and forms a morphologically unspecialized but functional contact (2). The nerve terminal accumulates synaptic vesicles and a basal lamina forms in the synaptic cleft (3). As the muscle matures, multiple axons converge on a single site (4). Finally, all axons but one are eliminated and the surviving terminal matures (5). (Adapted, with permission, from Hall and Sanes 1993.)
B. At the mature neuromuscular junction, pre- and postsynaptic membranes are separated by a synaptic cleft that contains basal lamina and extracellular matrix proteins. Vesicles are clustered at presynaptic release sites, transmitter receptors are clustered in the postsynaptic membrane, and nerve terminals are coated by Schwann cell processes. (Image reproduced, with permission, courtesy of T. Gillingwater.)
Three general features of neuromuscular junction development have provided clues about the molecular mechanisms that underlie synapse formation. First, nerve and muscle organize each other's differentiation. In principle, the precise apposition of pre- and postsynaptic specializations might be explained by independent programming of nerve and muscle properties. However, in muscle cells cultured alone acetylcholine (ACh) receptors are generally distributed uniformly on the surface, although some are clustered as in mature postsynaptic membranes. Yet when motor neurons are added to the cultures they extend neurites that contact the muscle cells more or less randomly, instead of seeking out the ACh receptor clusters. New receptor clusters appear precisely at the points of contact with the presynaptic neurites while preexisting uninnervated clusters eventually disperse (Figure 55–8). Thus factors on or released by motor axons exert a profound influence on the synaptic organization of the muscle cell. Likewise, muscles signal retrogradely to motor nerve terminals. When motor neurons in culture extend neurites, they assemble and transport synaptic vesicles, some of which form aggregates similar to those found in nerve terminals. When the neurites contact muscle cells, new vesicle clusters form opposite the muscle membrane, and most of the preexisting clusters disperse.
Nerve and muscle cells express synaptic components, but synaptic organization requires cell interactions.
Acetylcholine receptors (AChR) are synthesized by muscle cells cultured without neurons. Many receptors are diffusely distributed, but some form high-density aggregates similar to those found in the postsynaptic membrane of the neuromuscular junction. When neurons first contact muscle they do not restrict themselves to the receptor-rich aggregates. Instead, new receptor aggregates form at sites of neurite-muscle contact, and many of the preexisting clusters disperse. Similarly, motor axons contain synaptic vesicles that cluster at sites of neurite contact with muscle cells. (Adapted, with permission, from Anderson and Cohen 1977; Lupa, Gordon, and Hall 1990.)
Second, as these studies showed, motor neurons and muscle cells can synthesize and arrange most synaptic components without each other's help. Uninnervated myotubes can synthesize functional ACh receptors and gather them into high-density aggregates. Likewise, motor axons can form synaptic vesicles and cluster them into varicosities in the absence of muscle. In fact, vesicles in growth cones can synthesize and release ACh in response to electrical stimulation, before the growth cone has reached its target cells. Thus the developmental signals that pass between nerve and muscle do not induce wholesale changes in cell properties; rather they assure that components of the pre- and postsynaptic machinery are organized at the correct time and in the right places. It is useful therefore to think of the intercellular signals that control synaptogenesis as organizers rather than inducers.
A third key feature of neuromuscular junction development is that new synaptic components are added in several distinct steps. The newly formed synapse is not simply a prototype of a fully developed synapse. Although nerve and muscle membrane form close contacts at early stages of synaptogenesis, only later does the synaptic cleft widen and the basal lamina appear. Similarly, ACh receptors accumulate in the postsynaptic membrane before acetylcholinesterase accumulates in the synaptic cleft, and the postsynaptic membrane acquires junctional folds only after the nerve terminal has matured. Several different axons innervate each myotube around the time of birth, but during early postnatal life all but one axon withdraws.
This elaborate sequence is unlikely to be orchestrated by the simple act of contact between nerve and muscle. More probably, multiple signals pass between the cells—the nerve sends a signal to the muscle that triggers the first steps in postsynaptic differentiation, at which point the muscle sends a signal that triggers the initial steps of nerve terminal differentiation. The nerve then sends further signals to the muscle, and this interaction continues.
We now consider retrograde (from muscle to nerve) and anterograde (from nerve to muscle) organizers in more detail.
Differentiation of Motor Nerve Terminals Is Organized by Muscle Fibers
Soon after the growth cone of a motor axon contacts a developing myotube, a rudimentary form of neurotransmission begins. The axon releases ACh in vesicular packets, the transmitter binds to receptors, and the muscle responds with depolarization and weak contraction.
The onset of transmission at the new synapse reflects the intrinsic capabilities of each synaptic partner. Nevertheless, these intrinsic capabilities cannot readily explain the marked increase in the rate of transmitter release that occurs after nerve-muscle contact is made, nor can they explain the accumulation of synaptic vesicles and the assembly of active zones in the small portion of the motor axon that contacts the muscle surface. These developmental steps require signals from muscle to nerve.
A clue to the source of these signals came from studies on the reinnervation of adult muscle. Although axotomy leaves muscle fibers denervated and leads to insertion of ACh receptors in nonsynaptic regions, the postsynaptic apparatus remains largely intact. It is still recognizable by its synaptic nuclei, junctional folds, and the ACh receptors, which remain far more densely packed in synaptic areas than in extrasynaptic areas of the cell. Damaged peripheral axons regenerate readily (unlike those in the central nervous system) and form new neuromuscular junctions that look and perform much like the original ones.
A century ago, Fernando Tello y Muñóz, a student of Santiago Ramón y Cajal, noted that the new junctions form at preexisting synaptic sites on the de-nervated muscle fibers even though the postsynaptic specializations occupy only 0.1% of the muscle fiber surface (Figure 55–9A). Later, electron microscopy showed that specialization in the axon occurs only in the terminals that contact the muscle. For example, active zones form directly opposite the mouths of the postsynaptic junctional folds. These findings imply that motor axons recognize signals associated with the postsynaptic apparatus.
Synaptic portions of basal lamina contain proteins that organize developing nerve terminals.
A. After nerve damage motor axons regenerate and form new neuromuscular junctions. Nearly all of the new synapses form at the original synaptic sites. (Micrograph reproduced, with permission, from Glicksman and Sanes 1983.)
B. A strong preference for innervation at original synaptic sites persists even after the muscle fibers have been removed, leaving behind basal lamina "ghosts." Regenerated axons develop synaptic specialization on contact with the original synaptic sites on the basal lamina. (Micrograph reproduced, with permission, from Glicksman and Sanes 1983.)
C. Following denervation of a skeletal muscle fiber and elimination of mature muscle fibers, muscle satellite cells proliferate and differentiate to form new myofibers. The expression of ACh receptors on the regenerated myofiber surface is concentrated in the synaptic areas of basal lamina, even when reinnervation is prevented. (Micrograph reproduced, with permission, from Burden, Sargent, and McMahan 1979.)
When regenerating axons reach a muscle fiber they encounter the basal lamina of the synaptic cleft. To explore the significance of this association, muscles were damaged in vivo in a way that killed the muscle fibers but left their basal lamina intact. The necrotic fibers were phagocytized, leaving behind basal lamina sheaths on which synaptic sites were readily recognizable. At the same time that the muscle was damaged the nerve was cut and allowed to regenerate. Under these conditions motor axons reinnervated the empty basal lamina sheaths, contacting synaptic sites as precisely as they would have if muscle fibers were present. Moreover, nerve terminals developed at these sites and active zones even formed opposite struts of basal lamina that once lined junctional folds. These observations implied that components of the basal lamina organize presynaptic specialization (Figure 55–9B).
Several such molecular organizers have now been identified. Among the best studied are isoforms of the protein laminin. Laminins are major components of all basal laminae and promote axon outgrowth in many neuronal types (see Chapter 54). They are heterotrimers of α, β, and γ chains, comprising a family of at least five α, four β, and three γ chains. Muscle fibers synthesize multiple laminin isoforms and incorporate them into the basal lamina. Laminin-211, a heterotrimer containing the α2, β1, and γ1 chains, is the major laminin in the basal lamina, and its absence leads to severe muscular dystrophy. In the synaptic cleft, however, isoforms bearing the β2 chain predominate (Figure 55–10A).
Different laminin isoforms are localized at synaptic and extrasynaptic areas of the basal lamina.
A. Different laminin isoforms are found in synaptic (brown) and extrasynaptic (green) areas of basal lamina. Isoforms, containing the β2 chain, are concentrated in the synaptic areas.
B. Maturation of neuromuscular junctions is impaired in mice lacking β2 laminins. These mutants have few active zones, and the synaptic cleft is invaded by Schwann cell processes (blue). (Micrograph reproduced, with permission, from Noakes et al. 1995.)
In vitro motor axons that encounter a deposit of β2-containing laminin stop growing, accumulate synaptic vesicles, and acquire the ability to release neurotransmitter. Conversely, the development of nerve terminals and Schwann cells is perturbed in mutant mice that lack the β2 laminin (Figure 55–10B). These laminins appear to act by binding to voltage-sensitive calcium channels that reside in the axon terminal membrane, where they couple activity to transmitter release. Laminins act on the extracellular domain of the channels whereas the intracellular segment recruits or stabilizes other components of the release apparatus.
Because presynaptic specialization proceeds to some extent in the absence of laminins, additional retrograde organizers of axonal specialization must exist. Among these are members of the fibroblast growth factor and collagen IV families, both produced by muscle cells. Thus target-derived proteins from multiple families collaborate to organize the presynaptic nerve terminal.
Differentiation of the Postsynaptic Muscle Membrane Is Organized by the Motor Nerve
Soon after myoblasts fuse to form myotubes, the genes that encode ACh receptor subunits are activated. Receptor subunits are synthesized, assembled into pentamers in the endoplasmic reticulum, and inserted into the plasma membrane. As noted above, some receptors spontaneously form aggregates, but the majority are distributed throughout the membrane at a low density, approximately 1,000 per μm2.
Once synapse formation is complete, however, the distribution of the receptors changes drastically. The receptors become concentrated at the synaptic sites of the membrane (to a density up to 10,000 per μm2) and depleted in the nonsynaptic membrane (reduced to 10 per μm2 or less). This thousand-fold difference in ACh receptor density occurs a few tens of micrometers from the edge of the nerve terminal.
Appreciation of the critical role of the nerve in the redistribution of ACh receptors inspired a search for factors that might promote their clustering. This quest led to the discovery of a proteoglycan, agrin. Agrin is synthesized by motor neurons, transported down the axon, released from nerve terminals, and incorporated into the synaptic cleft (Figure 55–11A, B). Some agrin isoforms are also made by muscle cells, but the neuronal isoforms are about a thousand-fold more active in aggregating ACh receptors.
Agrin induces aggregation of ACh receptors at synaptic sites.
A. Agrin is a large (~400 kDa) extracellular matrix proteoglycan. Alternative splicing includes a "z" exon that confers the ability to cluster ACh receptors. When released by a nerve terminal, agrin binds Lrp4 on the muscle membrane, activating the membrane-associated receptor tyrosine kinase MuSK and triggering an intracellular cascade that results in ACh receptor clustering. Clustering is mediated by rapsyn, a cytoplasmic ACh receptor-associated protein. (Adapted, with permission, from DeChiara et al. 1996.)
B. Few ACh receptor clusters form on myofibers grown in culture under control conditions, but addition of agrin induces ACh receptor clustering. (Adapted, with permission, from Misgeld et al. 2005.)
C. Muscles from wild type neonatal mice and from three mutant types. Muscles were labeled for ACh receptors (green) and motor axons (brown). In wild type mice ACh receptor clusters have formed under each nerve terminal by birth, whereas in agrin mutants most clusters have dispersed. ACh receptors are also absent in MuSK mutant mice. When the genes for agrin and ChAT (choline acetyltransferase) are mutated, clusters of ACh receptors remain, indicating that agrin works by counteracting receptor dispersion mediated by ACh. All three mutant conditions also show axonal abnormalities, reflecting the inability of the muscle to supply retrograde factors. (ACh, acetylcholine; MuSK, muscle-specific trk-related receptor with a kringle domain.) (Adapted, with permission, from Gautam et al. 1996.)
The phenotype of mutant mice lacking agrin shows that agrin has a central role in the organization of ACh receptors. Agrin mutants have grossly perturbed neuromuscular junctions and die at birth. The number, size, and density of ACh receptor aggregates are severely reduced in these mice (Figure 55–11C). Other components of the postsynaptic apparatus—including cytoskeletal, membrane, and basal lamina proteins—are also reduced. Interestingly, the differentiation of presynaptic elements is also perturbed. However, the defects in the presynaptic element do not result directly from lack of agrin in the motor neuron, but rather indirectly from the failure of the disorganized postsynaptic apparatus to generate signals for presynaptic specialization.
How does agrin work? Agrin's major receptor is a complex of a muscle-specific tyrosine kinase called MuSK (muscle-specific trk-related receptor with a kringle domain) and a coreceptor subunit called LRP4 (Figure 55–11A). MuSK is normally concentrated at synaptic sites in the muscle membrane, and muscles of mutant mice lacking MuSK do not have ACh receptor clusters (Figure 55–11C). Myotubes generated in vitro from these mutants express normal levels of ACh receptors, but these receptors cannot be clustered by agrin. MuSK therefore appears to be a critical component of the receptor for agrin. LRP4 functions together with MuSK, an adaptor protein Dok-7, and a cytoplasmic protein rapsyn. LRP4 forms a complex with MuSK and binds agrin efficiently. Dok-7 binds MuSK and signals to rapsyn, which is also necessary for ACh receptor clustering. Rapsyn is co-localized with ACh receptors in vivo, is present at ACh receptor clusters soon after they form, and can induce the aggregation of ACh receptors in vitro. In mice lacking rapsyn muscles form normally and ACh receptors accumulate in normal numbers but fail to aggregate at the synaptic sites on the membrane.
Thus an extracellular protein (agrin), transmembrane proteins (MuSK and LRP4), an adaptor protein (Dok-7), and a cytoskeletal protein (rapsyn) form a chain that links commands from the motor axon to ACh receptor clustering in the muscle membrane.
Nevertheless, postsynaptic differentiation can occur in the absence of this transduction pathway. This capacity was apparent in early studies on cultured muscle (see Figure 55–8) and is also seen in vivo: ACh receptor clusters form initially but then disperse in agrin mutants (Figure 55–11C). Clustering also occurs in muscles that lack innervation entirely. Conversely, no clustering occurs in mutant animals lacking MuSK, LRP4, Dok-7, or rapsyn. Thus the signaling pathway that initiates postsynaptic differentiation can be activated without agrin, but agrin is required to maintain clustering of ACh receptors.
The role of agrin is perhaps best understood in terms of the requirement that pre- and postsynaptic specializations be perfectly aligned. Acetycholine receptor aggregates persist in uninnervated muscles but disappear in agrin mutant muscles, suggesting that axons sculpt the postsynaptic membrane through the combined action of agrin and a dispersal factor. One major dispersal factor is ACh itself; clustering persists in mutants that lack both agrin and ACh (Figure 55–11C). Thus agrin may render ACh receptors immune to the declustering effects of ACh. Through a combination of positive and negative factors, the motor neuron ensures that the patches of postsynaptic membrane contacted by axon branches are rich in ACh receptors.
The Nerve Regulates Transcription of Acetylcholine Receptor Genes
Along with redistribution of ACh receptor in the plane of the membrane, the motor nerve orchestrates the transcriptional program responsible for expression of ACh receptor genes in muscle. To understand this aspect of transcriptional control, it is important to appreciate the geometry of the muscle.
Individual muscle fibers are often more than a centimeter long and contain hundreds of nuclei along their length. Most nuclei are far from the synapse, but a few are clustered beneath the synaptic membrane, so that their transcribed and translated products do not have far to go to reach the synapse. In newly formed myotubes most nuclei express genes encoding the ACh receptor α-, β-, δ-, and γ-subunits. In adult muscles, however, only synaptic nuclei express ACh receptor genes; nonsynaptic nuclei do not. This change in pattern occurs in three steps.
During early stages of synapse formation the ACh receptor subunit genes are expressed at higher levels in synaptic nuclei than in their nonsynaptic neighbors (Figure 55–12). Signals acting through MuSK are needed for this specialization. Around the time of birth, ACh receptor gene expression shuts down in nonsynaptic nuclei. This change reflects a repressive effect of the nerve, as originally shown by studies of denervated muscle. When muscle fibers are denervated, as happens when the motor nerve is damaged, the density of ACh receptors in the postsynaptic membrane increases markedly, a phenomenon termed denervation supersensitivity.
Clustering of ACh receptors at the neuromuscular junction results from transcriptional regulation and local protein trafficking.
A. Acetylcholine receptors (AChR) are distributed diffusely on the surface of embryonic myotubes.
B. After the muscle is innervated by a motor axon the number of receptors in extrasynaptic regions decreases, whereas receptor density at the synapse increases. This reflects the aggregation of preexisting receptors and enhanced expression of ACh receptor genes in nuclei that lie directly beneath the nerve terminal. In addition, the transcription of receptor genes is repressed in nuclei in extrasynaptic regions. Electrical activity in muscle represses ACh gene expression in nonsynaptic nuclei, leading to a lower density of ACh receptors in these regions. The nuclei at synaptic sites are immune to this repressive effect. Following denervation, ACh receptor gene expression is upregulated in extrasynaptic nuclei, although not to the high level attained by synaptic nuclei. Paralysis mimics the effect of denervation, whereas electrical stimulation of denervated muscle mimics the influence of the nerve and decreases the density of ACh receptors in the extrasynaptic membrane.
This repressive effect of the nerve is mediated by electrical activation of the muscle. Under normal conditions the nerve keeps the muscle electrically active, and active muscle synthesizes fewer ACh receptors than inactive muscle. Indeed, direct stimulation of denervated muscle through implanted electrodes decreases ACh receptor expression, preventing or reversing the effect of denervation (Figure 55–12B). Conversely, when nerve activity is blocked by application of a local anesthetic, the number of ACh receptors throughout the muscle fiber increases, even though the synapse is intact.
In essence, then, the nerve uses ACh to repress expression of ACh receptor genes extrasynaptically. Current that passes through the channel of the receptor leads to an action potential that propagates along the entire muscle fiber. This depolarization opens voltage-dependent Ca2+ channels, leading to an influx of Ca2+, which activates a signal transduction cascade that reaches nonsynaptic nuclei and regulates transcription of ACh receptor genes. Thus the same voltage changes that produce muscle contraction over a period of milliseconds also regulate transcription of ACh receptor genes over a period of days.
The increase in transcription of ACh receptor genes in nuclei beneath the synapse, along with the decrease in nuclei distant from synapses, leads to localization of ACh receptor mRNA and thus preferential synthesis and insertion of ACh receptors near synaptic sites. This local synthesis is reminiscent of that seen at postsynaptic sites on dendritic spines in the brain. Local synthesis in muscle is advantageous since ACh receptors synthesized near the ends of fibers would never reach the synapse without degradation.
We have used the ACh receptor as an example of postsynaptic differentiation, but many components of the postsynaptic apparatus are regulated in similar ways—their aggregation depends on agrin and MuSK, and their transcription is enhanced in synaptic nuclei and repressed in extrasynaptic nuclei by electrical activity. Thus synaptic components have tailor-made regulatory mechanisms, but many of these components are regulated in parallel.
The Neuromuscular Junction Matures in a Series of Steps
The adult neuromuscular junction is dramatically different in its molecular architecture, shape, size, and functional properties from the simple nerve-muscle contact that initiates neurotransmission in the embryo. Maturation of the nerve terminal, the postsynaptic membrane, and the intervening synaptic cleft occurs in a complex series of steps. We illustrate this step-wise synaptic construction with a continued focus on the development of ACh receptors.
As we have seen, ACh receptors aggregate in the plane of the membrane as the neuromuscular junction begins to form, and receptor gene transcription is enhanced in synaptic nuclei. A few days later activity begins to decrease the level of extrasynaptic receptors and the stability of receptors changes. In embryonic muscle ACh receptors are turned over rapidly (with a half-life of approximately 1 day) throughout the membrane, whereas in adult muscle they are relatively stable (with a half-life of approximately 2 weeks). The metabolic stabilization of ACh receptors helps concentrate them at synaptic sites and stabilize the postsynaptic apparatus.
During the first few postnatal days the composure of the ACh receptor changes, shuts off the δ gene and activates the ε gene. As a result, new ACh receptors inserted in the membrane are composed of α-, β-, δ-, and ε-subunits rather than α-, β-, δ-, and γ-subunits. This altered subunit composition tunes the receptor in a way that is suited to its mature function. However, although it occurs at the same time as the metabolic stabilization, the two changes are not causally linked.
These molecular changes in the postsynaptic membrane are accompanied by changes in its shape (Figure 55–13). Soon after birth junctional folds begin to form in the postsynaptic membrane and ACh receptors become concentrated at the crests of the folds, along with rapsyn, whereas other membrane and cytoskeletal proteins are localized in the depths of the folds. The initial aggregate of ACh receptors appears to have a plaque-like appearance. Perforations that undergo fusion and fission eventually transform the dense plaque into a pretzel-shape that follows the branches of the nerve ending. Finally, the postsynaptic membrane enlarges and eventually contains many more ACh receptors than were present in the initial cluster. Each of these changes occurs while the synapse is functional, implying that ongoing activity plays an important role in synaptic maturation.
The postsynaptic membrane at the neuro-muscular junction matures in stages.
During early embryogenesis ACh receptors exist as loose aggregates. Later these aggregates condense into a plaque-like structure. After birth the dense cluster opens up as the nerve develops multiple terminals. These axon branches expand in an intercalary fashion as the muscle grows, and the plaque indents to form a gutter, which then invaginates to form folds. Receptors are concentrated at the crests of the folds. (Adapted, with permission, from Sanes and Lichtman 2001.)