Isolated primary osteocytes express functional gap junctions in vitro

The differentiation of the neuromuscular junction is a multistep process requiring coordinated interactions between nerve terminals and muscle. Although innervation is not needed for muscle production, the formation of nerve-muscle contacts, intramuscular nerve branching, and neuronal survival require reciprocal signals from nerve and muscle to regulate the formation of synapses. Following the production of muscle fibers, clusters of acetylcholine receptors (AChRs) are concentrated in the central regions of the myofibers via a process termed “prepatterning”. The postsynaptic protein MuSK is essential for this process activating possibly its own expression, in addition to the expression of AChR. AChR complexes (aggregated and stabilized by rapsyn) are thus prepatterned independently of neuronal signals in developing myofibers. ACh released by branching motor nerves causes AChR-induced postsynaptic potentials and positively regulates the localization and stabilization of developing synaptic contacts. These “active” contact sites may prevent AChRs clustering in non-contacted regions and counteract the establishment of additional contacts. ACh-induced signals also cause the dispersion of non-synaptic AChR clusters and possibly the removal of excess AChR. A further neuronal factor, agrin, stabilizes the accumulation of AChR at synaptic sites. Agrin released from the branching motor nerve may form a structural link specifically to the ACh-activated endplates, thereby enhancing MuSK kinase activity and AChR accumulation and preventing dispersion of postsynaptic specializations. The successful stabilization of prepatterned AChR clusters by agrin and the generation of singly innervated myofibers appear to require AChR-mediated postsynaptic potentials indicating that the differentiation of the nerve terminal proceeds only after postsynaptic specializations have formed.     

Nicotine decreases agrin signaling and acetylcholine receptor clustering in C2C12 myotube culture

The clustering of acetylcholine receptors (AChRs) in skeletal muscle fibers is a critical event in neuromuscular synaptogenesis. AChRs in concert with other molecules form postsynaptic scaffolds in response to agrin released from motor neurons as motor neurons near skeletal muscle fibers in development. Agrin drives an intracellular signaling pathway that precedes AChR clustering and includes the tyrosine phosphorylation of AChRs. In C2C12 myotube culture, agrin application stimulates the agrin signaling pathway and AChR clustering. Previous studies have determined that the frequency of spontaneous AChR clustering is decreased and AChRs are partially inactivated when bound by the acetylcholine agonist nicotine. We hypothesized that nicotine interferes with AChR clustering and consequent postsynaptic scaffold formation. In the present study, C2C12 myoblasts were cultured with growth medium to stimulate proliferation and then differentiation medium to stimulate fusion into myotubes. They were bathed in a physiologically relevant concentration of nicotine and then subject to agrin treatment after myotube formation. Our results demonstrate that nicotine decreases agrin-induced tyrosine phosphorylation of AChRs and decreases the frequency of spontaneous as well as agrin-induced AChR clustering. We conclude that nicotine interferes with postsynaptic scaffold formation by preventing the tyrosine phosphorylation of AChRs, an agrin signaling event that precedes AChR clustering.     

The signaling pathways mediated by P2Y nucleotide receptors in the formation and maintenance of the skeletal neuromuscular junction

The motor neuron, the Schwann cell and the muscle cell are highly specialized at the vertebrate skeletal neuromuscular junction (NMJ). The muscle cell surface contains a high local density of acetylcholine (ACh) receptors (AChRs), acetylcholinesterase (AChE) and their interacting macromolecules at the NMJ, forming the postsynaptic specializations. During the early stages of development, the incoming nerve terminal induces the formation of these postsynaptic specializations; the nerve secretes agrin and neuregulin (NRG), which are known to aggregate existing AChRs and to increase the expression of AChR at the synaptic region, respectively. In addition, adenosine 5'-triphosphate (ATP) is stored at the motor nerve terminals and is coreleased with ACh during muscle contraction. Recent evidence suggests that ATP can play a role in forming and maintaining the postsynaptic specializations by activating its corresponding receptors. In particular, one of the nucleotide receptor subtypes, the P2Y(1) receptor, is specifically localized at the NMJs. The gene expression of AChR and AChE is upregulated after the activation of P2Y(1) receptors. Thus, the synaptic ATP together with agrin and NRG can act as a synapse-organizing factor to induce the expression of postsynaptic functional effectors.     

Lithium inhibits a late step in agrin‐induced AChR aggregation

Agrin activates an intracellular signaling pathway to induce the formation of postsynaptic specializations on muscle fibers. In myotubes in culture, this pathway has been shown to include autophosphorylation of the muscle‐specific kinase MuSK, activation of Src‐family kinases, tyrosine phosphorylation of the acetylcholine receptor (AChR) β subunit, a decrease in receptor detergent extractability, and the accumulation of AChRs into high‐density aggregates. Here we report that treating chick myotubes with lithium prevented any detectable agrin‐induced change in AChR distribution without affecting the number of AChRs or the agrin‐induced change in AChR tyrosine phosphorylation and detergent extractability. Lithium treatment also increased the rate at which AChR aggregates disappeared when agrin was removed. The effects of lithium developed slowly over the course of approximately 12 h. Thus, sensitivity to lithium identifies a late step in the agrin signaling pathway, after agrin‐induced MuSK and AChR phosphorylation, that is necessary for the recruitment of AChRs into visible aggregates. © 2002 Wiley Periodicals, Inc. J Neurobiol 54: 346–357, 2003     

A role for acetylcholine receptors in their own aggregation on muscle cells

Both neurotrophic factors and activity regulate synaptogenesis. At neuromuscular synapses, the neural factor agrin released from motor neuron terminals stimulates postsynaptic specialization by way of the muscle specific kinase MuSK. In addition, activity through acetylcholine receptors (AChRs) has been implicated in the stabilization of pre- and postsynaptic contacts on muscle at various stages of development. We show here that activation of AChRs with specific concentrations of nicotine is sufficient to induce AChR aggregation and that this induction requires the function of L-type calcium channels (L-CaChs). Furthermore, AChR function is required for agrin induced AChR aggregation in C2 muscle cells. The same concentrations of nicotine did not induce observable tyrosine phosphorylation on either MuSK or the AChR beta subunit, suggesting significant differences between the mechanisms of agrin and activity induced aggregation. The AChR/L-CaCh pathway provides a mechanism by which neuromuscular signal transmission can act in concert with the agrin-MuSK signaling cascade to regulate NMJ formation.     

HGF induction of postsynaptic specializations at the neuromuscular junction

A critical event in the formation of vertebrate neuromuscular junctions (NMJs) is the postsynaptic clustering of acetylcholine receptors (AChRs) in muscle. AChR clustering is triggered by the activation of MuSK, a muscle-specific tyrosine kinase that is part of the functional receptor for agrin, a nerve-derived heparan sulfate proteoglycan (HSPG). At the NMJ, heparan sulfate (HS)-binding growth factors and their receptors are also localized but their involvement in postsynaptic signaling is poorly understood. In this study we found that hepatocyte growth factor (HGF), an HS-binding growth factor, surrounded muscle fibers and was localized at NMJs in rat muscle sections. In cultured Xenopus muscle cells, HGF was enriched at spontaneously occurring AChR clusters (hot spots), where HSPGs were also concentrated, and, following stimulation of muscle cells by agrin or cocultured neurons, HGF associated with newly formed AChR clusters. HGF presented locally to cultured muscle cells by latex beads induced new AChR clusters and dispersed AChR hot spots, and HGF beads also clustered phosphotyrosine, activated c-Met, and proteins of dystrophin complex; clustering of AChRs and associated proteins by HGF beads required actin polymerization. Lastly, although bath-applied HGF alone did not induce new AChR clusters, addition of HGF potentiated agrin-dependent AChR clustering in muscle. Our findings suggest that HGF promotes AChR clustering and synaptogenic signaling in muscle during NMJ development.     

Distribution of alpha-dystroglycan during embryonic nerve-muscle synaptogenesis

The distribution of alpha-dystroglycan (alpha DG) relative to acetylcholine receptors (AChRs) and neural agrin was examined by immunofluorescent staining with mAb IIH6 in cultures of nerve and muscle cells derived from Xenopus embryos. In Western blots probed with mAb IIH6, alpha DG was evident in membrane extracts of Xenopus muscle but not brain. alpha DG immunofluorescence was present at virtually all synaptic clusters of AChRs and neural agrin. Even microclusters of AChRs and agrin at synapses no older than 1-2 h (the earliest examined) had alpha DG associated with them. alpha DG was also colocalized at the submicrometer level with AChRs at nonsynaptic clusters that have little or no agrin. The number of large (> 4 microns) nonsynaptic clusters of alpha DG, like the number of large nonsynaptic clusters of AChRs, was much lower on innervated than on noninnervated cells. When mAb IIH6 was included in the culture medium, the large nonsynaptic clusters appeared fragmented and less compact, but the accumulation of agrin and AChRs along nerve-muscle contacts was not prevented. It is concluded that during nerve-muscle synaptogenesis, alpha DG undergoes the same nerve- induced changes in distribution as AChRs. We propose a diffusion trap model in which the alpha DG-transmembrane complex participates in the anchoring and recruitment of AChRs and alpha DG during the formation of synaptic as well as nonsynaptic AChR clusters.     

Involvement of p120 catenin in myopodial assembly and nerve–muscle synapse formation

At developing neuromuscular junctions (NMJs), muscles initially contact motor axons by microprocesses, or myopodia, which are induced by nerves and nerve‐secreted agrin, but it is unclear how myopodia are assembled and how they influence synaptic differentiation at the NMJ. Here, we report that treatment of cultured muscle cells with agrin transiently depleted p120 catenin (p120ctn) from cadherin junctions in situ, and increased the tyrosine phosphorylation and decreased the cadherin‐association of p120ctn in cell extracts. Whereas ectopic expression of wild‐type p120ctn in muscle generated myopodia in the absence of agrin, expression of a specific dominant‐negative mutant form of p120ctn, which blocks filopodial assembly in nonmuscle cells, suppressed nerve‐ and agrin‐induction of myopodia. Significantly, approaching neurites triggered reduced acetylcholine receptor (AChR) clustering along the edges of muscle cells expressing mutant p120ctn than of control cells, although the ability of the mutant cells to cluster AChRs was itself normal. Our results indicate a novel role of p120ctn in agrin‐induced myopodial assembly and suggest that myopodia increase muscle–nerve contacts and muscle's access to neural agrin to promote NMJ formation. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Phosphoinositide 3-kinase acts through RAC and Cdc42 during agrin-induced acetylcholine receptor clustering

The formation of the neuromuscular junction (NMJ) is regulated by the nerve-derived heparan sulfate proteoglycan agrin and the muscle-specific kinase MuSK. Agrin induces a signal transduction pathway via MuSK, which promotes the reorganization of the postsynaptic muscle membrane. Activation of MuSK leads to the phosphorylation and redistribution of acetylcholine receptors (AChRs) and other postsynaptic proteins to synaptic sites. The accumulation of high densities of AChRs at postsynaptic regions represents a hallmark of NMJ formation and is required for proper NMJ function. Here we show that phosphoinositide 3-kinase (PI3-K) represents a component of the agrin/MuSK signaling pathway. Muscle cells treated with specific PI3-K inhibitors are unable to form full-size AChR clusters in response to agrin and AChR phosphorylation is reduced. Moreover, agrin-induced activation of Rac and Cdc42 is impaired in the presence of PI3-K inhibitors. PI3-K is localized to the postsynaptic muscle membrane consistent with a role during agrin/MuSK signaling. These results put PI3-K downstream of MuSK as regulator of AChR phosphorylation and clustering. Its role during agrin-stimulated Rac and Cdc42 activation suggests a critical function during cytoskeletal reorganizations, which lead to the redistribution of actin-anchored AChRs.     

Nitric oxide synthase activity is required for postsynaptic differentiation of the embryonic neuromuscular junction

Agrin, a synapse-organizing protein externalized by motor axons at the neuromuscular junction (NMJ), initiates a signaling cascade in muscle cells leading to aggregation of postsynaptic proteins, including acetylcholine receptors (AChRs). We examined whether nitric oxide synthase (NOS) activity is required for agrin-induced aggregation of postsynaptic AChRs at the embryonic NMJ in vivo and in cultured muscle cells. Inhibition of NOS reduced AChR aggregation at embryonic Xenopus NMJs by 50-90%, whereas overexpression of NOS increased AChR aggregate area 2- to 3-fold at these synapses. NOS inhibitors completely blocked agrin-induced AChR aggregation in cultured embryonic muscle cells. Application of NO donors to muscle cells induced AChR clustering in the absence of agrin. Our results indicate that NOS activity is necessary for postsynaptic differentiation of embryonic NMJs and that NOS is a likely participant in the agrin-MuSK signaling pathway of skeletal muscle cells.     

Neural agrin increases postsynaptic ACh receptor packing by elevating rapsyn protein at the mouse neuromuscular synapse

Fluorescence resonance energy transfer (FRET) experiments at neuromuscular junctions in the mouse tibialis anterior muscle show that postsynaptic acetylcholine receptors (AChRs) become more tightly packed during the first month of postnatal development. Here, we report that the packing of AChRs into postsynaptic aggregates was reduced in 4-week postnatal mice that had reduced amounts of the AChR-associated protein, rapsyn, in the postsynaptic membrane (rapsyn(+/-) mice). We hypothesize that nerve-derived agrin increases postsynaptic expression and targeting of rapsyn, which then drives the developmental increase in AChR packing. Neural agrin treatment elevated the expression of rapsyn in C2 myotubes by a mechanism that involved slowing of rapsyn protein degradation. Similarly, exposure of synapses in postnatal muscle to exogenous agrin increased rapsyn protein levels and elevated the intensity of anti-rapsyn immunofluorescence, relative to AChR, in the postsynaptic membrane. This increase in the rapsyn-to-AChR immunofluorescence ratio was associated with tighter postsynaptic AChR packing and slowed AChR turnover. Acute blockade of synaptic AChRs with alpha-bungarotoxin lowered the rapsyn-to-AChR immunofluorescence ratio, suggesting that AChR signaling also helps regulate the assembly of extra rapsyn in the postsynaptic membrane. The results suggest that at the postnatal neuromuscular synapse agrin signaling elevates the expression and targeting of rapsyn to the postsynaptic membrane, thereby packing more AChRs into stable, functionally-important AChR aggregates.     

Regulation of agrin-induced acetylcholine receptor aggregation by Ca++ and phorbol ester

Agrin, a protein extracted from the electric organ of Torpedo californica, induces the formation of specializations on cultured chick myotubes that resemble the postsynaptic apparatus at the neuromuscular junction. The aim of the studies reported here was to characterize the effects of agrin on the distribution of acetylcholine receptors (AChRs) and cholinesterase as a step toward determining agrin's mechanism of action. When agrin was added to the medium bathing chick myotubes small (less than 4 micron 2) aggregates of AChRs began to appear within 2 h and increased rapidly in number until 4 h. Over the next 12-20 h the number of aggregates per myotube decreased as the mean size of each aggregate increased to approximately 15 micron 2. The accumulation of AChRs into agrin-induced aggregates occurred primarily by lateral migration of AChRs already in the myotube plasma membrane at the time agrin was added to the cultures. Aggregates of AChRs and cholinesterase remained as long as agrin was present in the medium; if agrin was removed the number of aggregates declined slowly. The formation and maintenance of agrin-induced AChR aggregates required Ca++, Co++ and Mn++ inhibited agrin-induced AChR aggregation and increased the rate of aggregate dispersal. Mg++ and Sr++ could not substitute for Ca++. Agrin-induced receptor aggregation also was inhibited by phorbol 12-myristate 13-acetate, an activator of protein kinase C, and by inhibitors of energy metabolism. The similarities between agrin's effects on cultured myotubes and events that occur during formation of neuromuscular junctions support the hypothesis that axon terminals release molecules similar to agrin that induce the differentiation of the postsynaptic apparatus.     

L-type calcium channels mediate acetylcholine receptor aggregation on cultured muscle

Agrin activation of muscle specific kinase (MuSK) initiates postsynaptic development on skeletal muscle that includes the aggregation of acetylcholine receptors (AChRs; Glass et al. [1996]: Cell 85: 513-523; Gautam et al. [1996]: Cell 85: 525-535). Although the agrin/MuSK signaling pathway remains largely unknown, changes in intracellular calcium levels are required for agrin-induced AChR aggregation (Megeath and Fallon [1998]: J Neurosci 18: 672-678). Here, we show that L-type calcium channels (L-CaChs) are required for full agrin-induced aggregation of AChRs and sufficient to induce agrin-independent AChR aggregation. Blockade of L-CaChs in muscle cultures inhibited agrin-induced AChR aggregation but not tyrosine phosphorylation of MuSK or AChR beta subunits. Activation of L-CaChs in the absence of agrin induced AChR aggregation but not tyrosine phosphorylation of MuSK or AChR beta subunits. Agrin responsiveness was significantly reduced in primary muscle cultures from the muscular dysgenesis mouse, a natural mutant, which does not express the L-CaCh. Our results establish a novel role for L-CaChs as important sources of the intracellular calcium necessary for the aggregation of AChRs.     

Agrin-induced reorganization of extracellular matrix components on cultured myotubes: relationship to AChR aggregation

Agrin, an extracellular matrix-associated protein extracted from synapse-rich tissues, induces the accumulation of acetylcholine receptors (AChRs) and other synaptic components into discrete patches on cultured myotubes. The appearance of agrin-like molecules at neuromuscular junctions suggests that it may direct synaptic organization in vivo. In the present study we examined the role of extracellular matrix components in agrin-induced differentiation. We used immunohistochemical techniques to visualize the spatial and temporal distribution of laminin, a heparan sulfate proteoglycan (HSPG), fibronectin, and type IV collagen on cultured chick myotubes during agrin-induced aggregation of AChRs. Myotubes displayed significant amounts of laminin and HSPG, lesser amounts of type IV collagen, and little, if any, fibronectin. Agrin treatment caused cell surface laminin and HSPG to patch, while collagen and fibronectin distributions were generally unaffected. Many of the agrin-induced laminin and HSPG patches colocalized with AChR patches, raising the possibility of a causal relationship between matrix patching and AChR accumulations. However, patching of AChRs (complete within a few hours) preceded that of laminin or HSPG (not complete until 15-20 h), making it unlikely that matrix accumulations initiate AChR patching at agrin-induced sites. Conversely, when AChR patching was blocked by treatment with anti-AChR antibody mAb 35, agrin was still able to effect patching of laminin and HSPG. Taken together, these findings suggest that agrin-induced accumulations of AChR and laminin/HSPG are not mechanistically linked.     

Local induction of acetylcholine receptor clustering in myotube cultures using microfluidic application of agrin

During neuromuscular synaptogenesis, the exchange of spatially localized signals between nerve and muscle initiates the coordinated focal accumulation of the acetylcholine (ACh) release machinery and the ACh receptors (AChRs). One of the key first steps is the release of the proteoglycan agrin focalized at the axon tip, which induces the clustering of AChRs on the postsynaptic membrane at the neuromuscular junction. The lack of a suitable method for focal application of agrin in myotube cultures has limited the majority of in vitro studies to the application of agrin baths. We used a microfluidic device and surface microengineering to focally stimulate muscle cells with agrin at a small portion of their membrane and at a time and position chosen by the user. The device is used to verify the hypothesis that focal application of agrin to the muscle cell membrane induces local aggregation of AChRs in differentiated C2C12 myotubes.     

Agrin elicits membrane lipid condensation at sites of acetylcholine receptor clusters in C2C12 myotubes

The formation of the neuromuscular junction is characterized by the progressive accumulation of nicotinic acetylcholine receptors (AChRs) in the postsynaptic membrane facing the nerve terminal, induced predominantly through the agrin/muscle-specific kinase (MuSK) signaling cascade. However, the cellular mechanisms linking MuSK activation to AChR clustering are still poorly understood. Here, we investigate whether lipid rafts are involved in agrin-elicited AChR clustering in a mouse C2C12 cell line. We observed that in C2C12 myotubes, both AChR clustering and cluster stability were dependent on cholesterol, because depletion by methyl-beta-cyclodextrin inhibited cluster formation or dispersed established clusters. Importantly, AChR clusters resided in ordered membrane domains, a biophysical property of rafts, as probed by Laurdan two-photon fluorescence microscopy. We isolated detergent-resistant membranes (DRMs) by three different biochemical procedures, all of which generate membranes with similar cholesterol/GM1 ganglioside contents, and these were enriched in several postsynaptic components, notably AChR, syntrophin, and raft markers flotillin-2 and caveolin-3. Agrin did not recruit AChRs into DRMs, suggesting that they are present in rafts independently of agrin activation. Consequently, in C2C12 myotubes, agrin likely triggers AChR clustering or maintains clusters through the coalescence of lipid rafts. These data led us to propose a model in which lipid rafts play a pivotal role in the assembly of the postsynaptic membrane at the neuromuscular junction upon agrin signaling.     

Recycling of acetylcholine receptors at ectopic postsynaptic clusters induced by exogenous agrin in living rats

During the development of the neuromuscular junction, motor axons induce the clustering of acetylcholine receptors (AChRs) and increase their metabolic stability in the muscle membrane. Here, we asked whether the synaptic organizer agrin might regulate the metabolic stability and density of AChRs by promoting the recycling of internalized AChRs, which would otherwise be destined for degradation, into synaptic sites. We show that at nerve-free AChR clusters induced by agrin in extrasynaptic membrane, internalized AChRs are driven back into the ectopic synaptic clusters where they intermingle with pre-existing and new receptors. The extent of AChR recycling depended on the strength of the agrin stimulus, but not on the development of junctional folds, another hallmark of mature postsynaptic membranes. In chronically denervated muscles, in which both AChR stability and recycling are significantly decreased by muscle inactivity, agrin maintained the amount of recycled AChRs at agrin-induced clusters at a level similar to that at denervated original endplates. In contrast, AChRs did not recycle at agrin-induced clusters in C2C12 or primary myotubes. Thus, in muscles in vivo, but not in cultured myotubes, neural agrin promotes the recycling of AChRs and thereby increases their metabolic stability.     

Localized acetylcholine receptor clustering dynamics in response to microfluidic focal stimulation with agrin

Agrin is a proteoglycan secreted by the motor neuron's growing axon terminal upon contact with the muscle during embryonic development. It was long thought that agrin's role was to trigger the clustering of acetylcholine receptors (AChRs) to nascent synapse sites. However, agrin-predating, protosynaptic AChR clusters are present well before innervation in the embryo and in myotube cultures, yet no role has been conclusively ascribed to agrin. We used a microfluidic device to focally deliver agrin to protosynaptic AChR clusters in micropatterned myotube cultures. The distribution of AChRs labeled with fluorescent bungarotoxin was imaged at various time points over >24 h. We find that a 4-h focal application of agrin (100 nM) preferentially reduces AChR loss at agrin-exposed clusters by 17% relative to the agrin-deprived clusters on the same myotube. In addition, the focal application increases the addition of AChRs preferentially at the clusters by 10% relative to the agrin-exposed, noncluster areas. Taken together, these findings suggest that a focal agrin stimulus can play a key stabilizing role in the aggregation of AChRs at the early stages of synapse formation. This methodology is generally applicable to various developmental processes and cell types, including neurons and stem cells.     

Formation of complex AChR aggregates in vitro requires α‐dystrobrevin

Efficient function at the neuromuscular junction requires high‐density aggregates of acetylcholine receptors (AChRs) to be precisely aligned with the motor nerve terminal. A collaborative effort between the motor neuron and muscle intrinsic factors drives the formation and maintenance of these AChR aggregates. α‐Dystrobrevin (αDB), a cytoplasmic protein found at the postsynaptic membrane, has been implicated in the regulation of AChR aggregate density and patterning. To investigate the contribution of αDB to the muscle intrinsic program regulating AChR aggregate development, we analyzed the formation of complex, pretzel‐like AChR aggregates on primary muscle cell cultures derived from αDB knockout (αDB‐KO) mice in the absence of nerve or agrin. In myotubes lacking αDB, complex AChR aggregates failed to form, whereas aggregates formed readily in wildtype myotubes. Five major isoforms of αDB are expressed in skeletal muscle: αDB1, αDB1(?), αDB2, αDB2(?), and αDB3. Expression of αDB1 or αDB1(?) in αDB‐KO myotubes restored formation of complex AChR aggregates similar to those in wildtype myotubes. In contrast, individual expression of αDB2, αDB2(?), αDB3, or an αDB1 phosphorylation mutant resulted in the formation of few, if any, complex AChR aggregates. Collectively, these data suggest that αDB is a significant component of the muscle intrinsic program that mediates the formation of complex AChR aggregates and that αDB's tyrosine phosphorylation sites are of particular functional importance to this program. Although the muscle intrinsic program appears to influence synaptogenesis, the formation of complex mature AChR aggregates in αDB‐KO mice (with the motor neuron present) suggests the motor neuron, not the muscle intrinsic program, is the major stimulus driving the maturation of AChRs from plaque to pretzel in vivo. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009