Synaptic vesicle distribution and release at rat diaphragm neuromuscular junctions

Synaptic vesicle release at the neuromuscular junction (NMJ) is highly reliable and is vital to the success of synaptic transmission. We examined synaptic vesicle number, distribution, and release at individual type-identified rat diaphragm NMJ. Three-dimensional reconstructions of electron microscopy images were used to obtain novel measurements of active zone distribution and the number of docked synaptic vesicles. Diaphragm muscle-phrenic nerve preparations were used to perform electrophysiological measurements of the decline in quantal content (QC) during repetitive phrenic nerve stimulation. The number of synaptic vesicles available for release vastly exceeds those released with a single stimulus, thus reflecting a relatively low probability of release for individual docked vesicles and at each active zone. There are two components that describe the decline in QC resulting from repetitive stimulation: a rapid phase (<0.5 s) and a delayed phase (<2.5 s). Differences in the initial rapid decline in QC were evident across type-identified presynaptic terminals (fiber type classification based on myosin heavy chain composition). At terminals innervating type IIx and/or IIb fibers, the initial decline in QC during repetitive stimulation matched the predicted depletion of docked synaptic vesicles. In contrast, at terminals innervating type I or IIa fibers, a faster than predicted decline in QC with repetitive stimulation suggests that a decrease in the probability of release at these terminals plays a role in addition to depletion of docked vesicles. Differences in QC decline likely reflect fiber-type specific differences in activation history and correspond with well-described differences in neuromuscular transmission across muscle fiber types.     

Morphological variability is greater at developing than mature mouse neuromuscular junctions

The neuromuscular junction (NMJ) is the highly specialised peripheral synapse formed between lower motor neuron terminals and muscle fibres. Post-synaptic acetylcholine receptors (AChRs), which are found in high density in the muscle membrane, bind to acetylcholine released into the synaptic cleft of the NMJ, thereby enabling the conversion of motor action potentials to muscle contractions. NMJs have been studied for many years as a general model for synapse formation, development and function, and are known to be early sites of pathological changes in many neuromuscular diseases. However, information is limited on the diversity of NMJs in different muscles, how synaptic morphology changes during development, and the relevance of these parameters to neuropathology. Here, this crucial gap was addressed using a robust and standardised semi-automated workflow called NMJ-morph to quantify features of pre- and post-synaptic NMJ architecture in an unbiased manner. Five wholemount muscles from wild-type mice were dissected and compared at immature (post-natal day, P7) and early adult (P31−32) timepoints. The inter-muscular variability was greater in mature post-synaptic AChR morphology than that of the pre-synaptic motor neuron terminal. Moreover, the developing NMJ showed greater differences across muscles than the mature synapse, perhaps due to the observed distinctions in synaptic growth between muscles. Nevertheless, the amount of nerve to muscle contact was consistent, suggesting that pathological denervation can be reliably compared across different muscles in mouse models of neurodegeneration. Additionally, mature post-synaptic endplate diameters correlated with fibre type, independently of muscle fibre diameter. Altogether, this work provides detailed information on healthy pre- and post-synaptic NMJ morphology from five anatomically and functionally distinct mouse muscles, delivering useful reference data for future comparison with neuromuscular disease models.     

Phrenic motor unit recruitment during ventilatory and non-ventilatory behaviors

Phrenic motoneurons are located in the cervical spinal cord and innervate the diaphragm muscle, the main inspiratory muscle in mammals. Similar to other skeletal muscles, phrenic motoneurons and diaphragm muscle fibers form motor units which are the final element of neuromotor control. In addition to their role in sustaining ventilation, phrenic motor units are active in other non-ventilatory behaviors important for airway clearance such as coughing or sneezing. Diaphragm muscle fibers comprise all fiber types and are commonly classified based on expression of contractile proteins including myosin heavy chain isoforms. Although there are differences in contractile and fatigue properties across motor units, there is a matching of properties for the motor neuron and muscle fibers within a motor unit. Motor units are generally recruited in order such that fatigue-resistant motor units are recruited earlier and more often than more fatigable motor units. Thus, in sustaining ventilation, fatigue-resistant motor units are likely required. Based on a series of studies in cats, hamsters and rats, an orderly model of motor unit recruitment was proposed that takes into consideration the maximum forces generated by single type-identified diaphragm muscle fibers as well as the proportion of the different motor unit types. Using this model, eupnea can be accomplished by activation of only slow-twitch diaphragm motor units and only a subset of fast-twitch, fatigue-resistant units. Activation of fast-twitch fatigable motor units only becomes necessary when accomplishing tasks that require greater force generation by the diaphragm muscle, e.g., sneezing and coughing.     

Three-dimensional neuron–muscle constructs with neuromuscular junctions

This paper describes a fabrication method of muscle tissue constructs driven by neurotransmitters released from activated motor neurons. The constructs consist of three-dimensional (3D) free-standing skeletal muscle fibers co-cultured with motor neurons. We differentiated mouse neural stem cells (mNSCs) cultured on the skeletal muscle fibers into neurons that extend their processes into the muscle fibers. We found that acetylcholine receptors (AChRs) were formed at the connection between the muscle fibers and the neurons. The neuron–muscle constructs consist of highly aligned, long and matured muscle fibers that facilitate wide contractions of muscle fibers in a single direction. The contractions of the neuron–muscle construct were observed after glutamic acid activation of the neurons. The contraction was stopped by treatment with curare, an neuromuscular junction (NMJ) antagonist. These results indicate that our method succeeded in the formation of NMJs in the neuron–muscle constructs. The neuron–muscle construct system can potentially be used in pharmacokinetic assays related to NMJ disease therapies and in soft-robotic actuators.     

Morphology of diaphragm neuromuscular junctions on different fibre types

Summary We hypothesize that the morphology of the neuromuscular junction on different muscle fibre types varies, reflecting differences in activation history. In the rat diaphragm muscle, we used a three-colour fluorescent immunocytochemical technique to simultaneously visualize (1) innervating axons and presynaptic nerve terminals, (2) motor endplates and (3) myosin heavy chain isoform expression (muscle fibre type). Laser-scanning confocal microscopy was then used to optically section the triple-labelled muscle fibres, and create three-dimensional views of the neuromuscular junction. Type I fibres were innervated by the smallest axons, and type IIa, IIx and IIb fibres by progressively larger axons. Absolute planar areas of nerve terminals and endplates progressively increased from type I, IIa, IIx to IIb fibres. When normalized for fibre diameter planar areas of nerve terminals were largest on type I fibres, with no difference among type II fibres. The normalized planar area of endplates were larger for type I and IIb fibres, compared to type IIa and IIx fibres. The three-dimensional surface area of endplates was largest on type I fibres, with no differences across type II fibres. When normalized for fibre diameter, endplate surface areas increase progressively from type I, IIa, IIx to IIb fibres. The branches increased progressively from type I, IIa, IIx to IIb fibres. Conversely, individual branch length was longest on type I fibres, and shortest on type IIb fibres. The extent of overlap of pre- and postsynaptic elements of the neuromuscular junction decreased progressively on type I, IIa, IIx and IIb fibres. We conclude that these morphological differences at the neuromuscular function of different fibre types reflect differences in activation history and may underlie phenotypic differences in neuromuscular transmission.     

The emerging role of the sympathetic nervous system in skeletal muscle motor innervation and sarcopenia

Examining neural etiologic factors’role in the decline of neuromuscular function with aging is essential to our understanding of the mechanisms underlying sarcopenia, the age-dependent decline in muscle mass, force and power. Innervation of the skeletal muscle by both motor and sympathetic axons has been established, igniting interest in determining how the sympathetic nervous system (SNS) affect skeletal muscle composition and function throughout the lifetime. Selective expression of the heart and neural crest derivative 2 gene in peripheral SNs increases muscle mass and force regulating skeletal muscle sympathetic and motor innervation; improving acetylcholine receptor stability and NMJ transmission; preventing inflammation and myofibrillar protein degradation; increasing autophagy; and probably enhancing protein synthesis. Elucidating the role of central SNs will help to define the coordinated response of the visceral and neuromuscular system to physiological and pathological challenges across ages.This review discusses the following questions: (1) Does the SNS regulate skeletal muscle motor innervation? (2) Does the SNS regulate presynaptic and postsynaptic neuromuscular junction (NMJ) structure and function? (3) Does sympathetic neuron (SN) regulation of NMJ transmission decline with aging? (4) Does maintenance of SNs attenuate aging sarcopenia? and (5) Do central SN group relays influence sympathetic and motor muscle innervation?     

Abnormalities in neuromuscular junction structure and skeletal muscle function in mice lacking the P2X2 nucleotide receptor

ATP is co-released in significant quantities with acetylcholine from motor neurons at skeletal neuromuscular junctions (NMJ). However, the role of this neurotransmitter in muscle function remains unclear. The P2X2 ion channel receptor subunit is expressed during development of the skeletal NMJ, but not in adult muscle fibers, although it is re-expressed during muscle fiber regeneration. Using mice deficient for the P2X2 receptor subunit for ATP (P2X2(-/-)), we demonstrate a role for purinergic signaling in NMJ development. Whereas control NMJs were characterized by precise apposition of pre-synaptic motor nerve terminals and post-synaptic junctional folds rich in acetylcholine receptors (AChRs), NMJs in P2X2(-/-) mice were disorganized: misapposition of nerve terminals and post-synaptic AChR expression localization was common; the density of post-synaptic junctional folds was reduced; and there was increased end-plate fragmentation. These changes in NMJ structure were associated with muscle fiber atrophy. In addition there was an increase in the proportion of fast type muscle fibers. These findings demonstrate a role for P2X2 receptor-mediated signaling in NMJ formation and suggest that purinergic signaling may play an as yet largely unrecognized part in synapse formation.     

Trophic factor expression in phrenic motor neurons

The function of a motor neuron and the muscle fibers it innervates (i.e., a motor unit) determines neuromotor output. Unlike other skeletal muscles, respiratory muscles (e.g., the diaphragm, DIAm) must function from birth onwards in sustaining ventilation. DIAm motor units are capable of both ventilatory and non-ventilatory behaviors, including expulsive behaviors important for airway clearance. There is significant diversity in motor unit properties across different types of motor units in the DIAm. The mechanisms underlying the development and maintenance of motor unit diversity in respiratory muscles (including the DIAm) are not well understood. Recent studies suggest that trophic factor influences contribute to this diversity. Remarkably little is known about the expression of trophic factors and their receptors in phrenic motor neurons. This review will focus on the contribution of trophic factors to the establishment and maintenance of motor unit diversity in the DIAm, during development and in response to injury or disease.     

Synaptic vesicle pools at diaphragm neuromuscular junctions vary with motoneuron soma, not axon terminal, inactivity

Both spinal hemisection (SH) at C2 and tetrodotoxin (TTX) phrenic nerve blockade result in diaphragm muscle paralysis and inactivity of the phrenic axon terminals. However, phrenic motoneuron somata are inactive with SH but remain active with TTX phrenic nerve blockade. Neuromuscular transmission failure with repeated activation decreases following SH and increases following TTX phrenic nerve blockade, suggesting that matching (or mismatching) of somal and synaptic inactivities of phrenic motoneurons differentially regulates synaptic vesicle pools at diaphragm neuromuscular junctions. At individual type-identified rat diaphragm presynaptic terminals, the size of the releasable pool of synaptic vesicles was analyzed by fluorescence confocal microscopy of N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl) pyridinium dibromide (FM4-64) uptake and synaptic vesicle density at active zones was determined using transmission electron microscopy. After 14 days of SH and TTX-induced diaphragm muscle inactivity, neuromuscular junction size was not different at type I or IIa fibers, but increased at type IIx and/or IIb fibers (by 51% in SH and 35% in TTX) compared with control. With SH, synaptic vesicle pool size and density increased at presynaptic terminals innervating type I or IIa fibers (17 and 63%, respectively; P<0.001) and type IIx and/or IIb fibers (41 and 31%, respectively; P<0.001) when compared with controls. Following TTX, synaptic vesicle pool size and density decreased by 64 and 17%, respectively, at presynaptic terminals innervating type I or IIa fibers, and by 50 and 36%, respectively, at type IIx and/or IIb fibers (P<0.001, for all comparisons). Thus, matching motoneuron soma and axon terminal inactivity (SH) increases the size and density of releasable synaptic vesicle pools at adult rat diaphragm neuromuscular junctions. Mismatching motoneuron soma and axon terminal inactivities (TTX) results in converse presynaptic adaptations. Inactivity-induced neuromuscular plasticity reflects specific adaptations in the size and density of synaptic vesicle pools that depend on motoneuron soma rather than axon terminal (or muscle fiber) inactivity.     

Subcellular localization of the glutamate transporters GLAST and GLT at the neuromuscular junction in rodents

The vertebrate neuromuscular junction (NMJ) is known to be a cholinergic synapse at which acetylcholine (ACh) is released from the presynaptic terminal to act on postsynaptic nicotinic ACh receptors. There is now growing evidence that glutamate, which is the main excitatory transmitter in the CNS and at invertebrate NMJs, may have a signaling function together with ACh also at the vertebrate NMJ. In the CNS, the extracellular concentration of glutamate is kept at a subtoxic level by Na(+)-driven high-affinity glutamate transporters located in plasma membranes of astrocytes and neurons. The glutamate transporters are also pivotal for shaping glutamate receptor responses at synapses. In order to throw further light on the potential role of glutamate as a cotransmitter at the NMJ we used high-resolution immunocytochemical methods to investigate the localization of the plasma membrane glutamate transporters GLAST (glutamate aspartate transporter) and GLT (glutamate transporter 1) in rat and mice NMJ regions. Confocal laser-scanning immunocytochemistry showed that GLT is restricted to the NMJ in rat and mouse skeletal muscle. Lack of labeling signal in knock-out mice confirmed that the immunoreactivity observed at the NMJ was specific for GLT. GLAST was also localized at the NMJ in rat but not detected in mouse NMJ (while abundant in mouse brain). Post-embedding electron microscopic immunocytochemistry and quantitative analyses in rat showed that GLAST and GLT are enriched in the junctional folds of the postsynaptic membrane at the NMJ. GLT was relatively higher in the slow-twitch muscle soleus than in the fast-twitch muscle extensor digitorum longus, whereas GLAST was relatively higher in extensor digitorum longus than in soleus. The findings show--together with previous demonstration of vesicular glutamate, a vesicular glutamate transporter and glutamate receptors--that mammalian NMJs contain the machinery required for synaptic release and action of glutamate. This indicates a signaling role for glutamate at the normal NMJ and provides a basis for the ability of denervated muscle to be reinnervated by glutamatergic axons from the CNS.     

Neuromuscular transmission failure during postnatal development.

Neuromuscular transmission failure in rat diaphragm muscle was examined during the first two weeks of postnatal development, and was found to contribute significantly more to diaphragm fatigue induced by repetitive activation than in adult muscle. Intracellular analysis of evoked end-plate potentials indicated that neuromuscular transmission failure was due to both a block of action potential propagation and reduced synaptic efficacy.     

Functional innervation of cultured human skeletal muscle proceeds by two modes with regard to agrin effects

Nerve-derived agrin is a specific isoform of agrin that promotes clustering of nicotinic acetylcholine receptors (AChR) and other components of the neuromuscular junction (NMJ). We investigated the effects of agrin on functional maturation of NMJs at the early stages of synaptogenesis in human muscle. Specifically, we assessed the importance of agrin for the differentiation of developing NMJs to the stage where they are able to transmit signals that result in contractions of myotubes. We utilized an in vitro model in which human myotubes are innervated by neurons extending from spinal cord explants of fetal rat. This model is suitable for functional studies because all muscle contractions are the result of neuromuscular transmission and can be quantitated. An anti-agrin antibody, Agr 33, was applied to co-cultures during de novo NMJ formation. Quantitative analyses demonstrated that Agr 33 reduced the number of AChR clusters to 20% and their long axes to 50% of control, yet still permitted early, NMJ-mediated muscle contractions that are normally observed in 7-10-day-old co-cultures. However, at later times of development, the same treatment completely prevented the increase in the number of contracting units as compared with untreated co-cultures. It is concluded that there are two modes of functional maturation of NMJs with regard to agrin effects: one that is insensitive and the other that is sensitive to agrin blockade. Agrin-insensitive mode is limited to the small population of NMJs that become functional at the earlier stages of functional innervation. However, most of the NMJs become contraction-competent at the later stages of the innervation process. These NMJs become functional only if agrin action is uncompromised. This is the first characterization of the contribution of agrin to NMJ development on human muscle.     

Scanning electron microscope study of neuromuscular junctions in different muscle fiber types in the zebra finch and rat

Examination by scanning electron microscopy revealed differences between neuromuscular junctions in the muscle fibers of the zebra finch (bird) and rat. The neuromuscular junctions between the anterior and posterior latissimus dorsi muscles of the zebra finch were compared. The junctions of the former, exclusively slow tonic fibers, were small and numerous along the long axis of a single muscle fiber. The synaptic depressions per junction were few. The junctions of the latter, exclusively fast twitch fibers, were large and consisted of more synaptic depressions than the former. Junctional folds were occasionally found in some depressions. The neuromuscular junctions between the extensor digitorum longus and soleus muscles of the rat were also compared. The former consisted almost entirely of fast twitch muscle fibers, whereas the latter consisted of both slow twitch fibers (75%) and fast twitch fibers (25%). The junctions in the extensor digitorum longus muscle were almost all labyrinthine gutters containing exclusively slit-like junctional folds. In the soleus muscle, two types of junctions were observed. One type was similar to that of the extensor digitorum longus muscle; the other was characterized by labyrinthine gutters containing sparse, narrow slit-like and pit-like junctional folds. We suggest from these structural differences of the subneural apparatuses that the junction of the fast twitch muscle is characterized by the subneural apparatus containing numerous slit-like junctional folds, and that of the slow twitch muscle fiber characterized by the apparatus containing sparse, narrow slit-like and pit-like junctional folds.     

Persistence of hybrid fibers in rat soleus after spinal cord transection.

The effects of a chronic (up to 360 days) reduction in neuromuscular activity (defined as electrical activation and loading) on myosin heavy chain (MHC) isoform expression in the rat soleus muscle were studied. A complete mid-thoracic (T7-T8) spinal cord transection (ST) was used to induce a reduction in soleus muscle neuromuscular activity. Electrophoretic analyses revealed that MHC-I was progressively decreased after ST, accounting for approx. 90% of the total soleus MHC in controls and only approx. 12% 1 year after ST. The reductions in the proportion of MHC-I were countered by increases in MHC-IIa and MHC-IIx with the increase in MHC-IIx preceding the increase in MHC-IIa. Curiously, MHC-IIb was expressed only at very low levels. Thus, a complete transformation from predominantly MHC-I to MHC-IIb did not occur. Many fibers (up to approx. 80%) contained multiple MHCs (hybrid fibers) after ST. The proportion of hybrid fibers was maintained at a high level (approx. 50%) 1 year after ST. These data suggest that: 1) a prolonged reduction in neuromuscular activity was not sufficient to induce high level MHC-IIb expression by the soleus muscle; and 2) hybrid fibers were not simply transitional fibers. Thus, it appears that under appropriate conditions hybrid fibers may represent a "stable" fiber phenotype.     

Muscle power failure in mobility-limited older adults: preserved single fiber function despite lower whole muscle size, quality and rate of neuromuscular activation

This study investigated the physiological and gender determinants of the age-related loss of muscle power in 31 healthy middle-aged adults (aged 40-55 years), 28 healthy older adults (70-85 years) and 34 mobility-limited older adults (70-85 years). We hypothesized that leg extensor muscle power would be significantly lower in mobility-limited elders relative to both healthy groups and sought to characterize the physiological mechanisms associated with the reduction of muscle power with aging. Computed tomography was utilized to assess mid-thigh body composition and calculate specific muscle power and strength. Surface electromyography was used to assess rate of neuromuscular activation and muscle biopsies were taken to evaluate single muscle fiber contractile properties. Peak muscle power, strength, muscle cross-sectional area, specific muscle power and rate of neuromuscular activation were significantly lower among mobility-limited elders compared to both healthy groups (P ≤ 0.05). Mobility-limited older participants had greater deposits of intermuscular adipose tissue (P < 0.001). Single fiber contractile properties of type I and type IIA muscle fibers were preserved in mobility-limited elders relative to both healthy groups. Male gender was associated with greater decrements in peak and specific muscle power among mobility-limited participants. Impairments in the rate of neuromuscular activation and concomitant reductions in muscle quality are important physiological mechanisms contributing to muscle power deficits and mobility limitations. The dissociation between age-related changes at the whole muscle and single fiber level suggest that, even among older adults with overt mobility problems, contractile properties of surviving muscle fibers are preserved in an attempt to maintain overall muscle function.     

Age affects reciprocal cellular interactions in neuromuscular synapses following peripheral nerve injury

Studies of the influence of age on regeneration and reinnervation in the peripheral nervous system (PNS) and neuromuscular junction (NMJ) are reviewed, with a particular focus on aged and denervated skeletal muscles. The morphological and functional features of incomplete regeneration and reinnervation are compared between adult and aged animals. In addition, some possible mechanisms of the age-related defects will be discussed. Increased fragmentation or damage in individual components of the NMJ (terminal Schwann cells (TSCs), axon terminals and acetylcholine receptor sites occurs during muscle reinnervation following PNS injury in the aged animals. The capacity to produce ultraterminal sprouting or multiple innervation secondary to PNS injury is maintained, but not the capacity to eliminate such anomalous axonal profiles. The frequency and accuracy of reoccupation of the synaptic sites by TSCs and axon terminals are impaired. Thus, despite the capability of extending neural processes, the rate at which regenerating nerve fibers grow, mature and precisely appose the postsynaptic muscle fiber is impaired, resulting in the failure of re-establishment of the normal single motor innervation in the NMJ. A complex set of cellular interactions in the NMJ are known to participate in the neurotrophism and neurotrophism to support growth of the regenerating and sprouting axons and their pathfinding to direct the target muscle fiber. Besides the capability of α-motoneurons, signaling originating from the TSCs and muscle may be impaired during aging.     

Actions of MDMA at glutamatergic neuromuscular junctions

3,4-Methylenedioxymethamphetamine (MDMA, "Ecstasy") compels mammalian serotonergic neurons to release serotonin (5-HT). In this study, MDMA altered synaptic transmission presynaptically by enhancing quantal release in two model glutamatergic synapses-the neuromuscular junction (NMJ) of the crayfish opener muscle, which is enhanced by exogenous 5-HT application, and the NMJ of a larval body wall muscle in Drosophila melanogaster, which is insensitive to exogenous 5-HT application. At the crayfish NMJ, MDMA mimicked the actions of 5-HT but only at a substantially higher concentration. At the Drosophila NMJ, MDMA altered synaptic transmission but not through a 5-HT receptor. Using simple invertebrate preparations, we have demonstrated an additional non-serotonergic mechanism of MDMA activity that has not yet been addressed in vertebrate systems and that may play an important role in understanding the mechanism of action for a commonly abused drug.     

Persistence of hybrid fibers in rat soleus after spinal cord transection

The effects of a chronic (up to 360 days) reduction in neuromuscular activity (defined as electrical activation and loading) on myosin heavy chain (MHC) isoform expression in the rat soleus muscle were studied. A complete mid‐thoracic (T7‐T8) spinal cord transection (ST) was used to induce a reduction in soleus muscle neuromuscular activity. Electrophoretic analyses revealed that MHC‐I was progressively decreased after ST, accounting for approx. 90% of the total soleus MHC in controls and only approx. 12% 1 year after ST. The reductions in the proportion of MHC‐I were countered by increases in MHC‐IIa and MHC‐IIx with the increase in MHC‐IIx preceding the increase in MHC‐IIa. Curiously, MHC‐IIb was expressed only at very low levels. Thus, a complete transformation from predominantly MHC‐I to MHC‐IIb did not occur. Many fibers (up to approx. 80%) contained multiple MHCs (hybrid fibers) after ST. The proportion of hybrid fibers was maintained at a high level (approx. 50%) 1 year after ST. These data suggest that: 1) a prolonged reduction in neuromuscular activity was not sufficient to induce high level MHC‐IIb expression by the soleus muscle; and 2) hybrid fibers were not simply transitional fibers. Thus, it appears that under appropriate conditions hybrid fibers may represent a “stable” fiber phenotype. Anat Rec 255:188–201, 1999. © 1999 Wiley‐Liss, Inc.     

Degeneration and regeneration of neuromuscular junction architecture in rat skeletal muscle fibers damaged by bupivacaine hydrochloride

We evaluated the degeneration and regeneration of neuromuscular junctions (NMJs) on the extensor digitorum longus muscle of Fischer 344 rats between 4 h and 3 weeks after bupivacaine hydrochloride (BPVC) injection, which induces muscle fiber necrosis, using histochemical staining by acetylcholine esterase (AchE)-silver and electron microscopy. Degeneration of muscle fibers and NMJs was observed 4 h after BPVC injection. One week after BPVC injection, some terminal axons were almost completely retracted, and the level of basal lamina-associated AchE in some NMJ regions had gradually disappeared. At that time, the depression contained a few, mostly pit-like or elongated oval invaginations: the incipient junctional folds and some NMJs did not have any secondary junctional fold. By 2 weeks after the BPVC injection, secondary junctional folds began to develop: however, the number of secondary junctional folds was clearly less than that in normal NMJs. At 3 weeks when regeneration of muscle fibers was well advanced, the staining for AchE at the end-plates became stronger and better-defined. The volume density of mitochondria in the terminal area of the terminal significantly decreased upon BPVC-induced destruction of the NMJ, and the density reached the lowest value 24 h after BPVC injection. Significant changes in the ultrastructural features of the architecture of NMJs occurred in skeletal muscle fibers damaged by BPVC during both the degeneration and regeneration processes. The changes in the ultrastructural and morphological features of the NMJ architecture during the regeneration of degenerated muscle fibers resembled those that occur during the differentiation of normal muscle fibers.