In vitro Neuromuscular Junction Induced from Human Induced Pluripotent Stem Cells.

The neuromuscular junction (NMJ) is a specialized synapse that transmits action potentials from the motor neuron to skeletal muscle for mechanical movement. The architecture of the NMJ structure influences the functions of the neuron, the muscle and the mutual interaction. Previous studies have reported many strategies by co-culturing the motor neurons and myotubes to generate NMJ in vitro with complex induction process and long culture period but have struggled to recapitulate mature NMJ morphology and function. Our in vitro NMJ induction system is constructed by differentiating human iPSC in a single culture dish. By switching the myogenic and neurogenic induction medium for induction, the resulting NMJ contained pre- and post- synaptic components, including motor neurons, skeletal muscle and Schwann cells in the one month culture. The functional assay of NMJ also showed that the myotubes contraction can be triggered by Ca++ then inhibited by curare, an acetylcholine receptor (AChR) inhibitor, in which the stimulating signal is transmitted through NMJ. This simple and robust approach successfully derived the complex structure of NMJ with functional connectivity. This in vitro human NMJ, with its integrated structures and function, has promising potential for studying pathological mechanisms and compound screening.

Bojungikgi-tang Improves Muscle and Spinal Cord Function in an Amyotrophic Lateral Sclerosis Model.

Amyotrophic lateral sclerosis (ALS) is a motor neuron disease characterized by progressive motor function impairment, dysphagia, and respiratory failure. Owing to the complexity of its pathogenic mechanisms, an effective therapy for ALS is lacking. Herbal medicines with multiple targets have good efficacy and low adverse reactions for the treatment of neurodegenerative diseases. In this study, the effects of Bojungikgi-tang (BJIGT), an herbal medicine with eight component herbs, on muscle and spinal cord function were evaluated in an ALS animal model. Animals were randomly divided into three groups: a non-transgenic group (nTg, n = 24), a hSOD1G93A transgenic group (Tg, n = 24), and a hSOD1G93A transgenic group in which 8-week-old mice were orally administered BJIGT (1 mg/g) once daily for 6 weeks (Tg+BJIGT, n = 24). The effects of BJIGT were evaluated using a rotarod test, foot-printing, and survival analyses based on Kaplan-Meier survival curves. To determine the biological mechanism underlying the effects of BJIGT in hSOD1G93A mice, western blotting, transmission electron microscopy, and Bungarotoxin staining were used. BJIGT improved motor function and extended the survival duration of hSOD1G93A mice. In addition, BJIGT had protective effects, including anti-oxidative and anti-inflammatory effects, in both the spinal cord and muscle of hSOD1G93A mice. Our results demonstrated that BJIGT causes muscle atrophy and the denervation of neuromuscular junctions in the gastrocnemius of hSOD1G93A mice. The components of BJIGT may alleviate the symptoms of ALS via different mechanisms, and accordingly, BJIGT treatment may be an effective therapeutic approach.

The Krebs Cycle Enzyme Isocitrate Dehydrogenase 3A Couples Mitochondrial Metabolism to Synaptic Transmission.

Neurotransmission is a tightly regulated Ca2+-dependent process. Upon Ca2+ influx, Synaptotagmin1 (Syt1) promotes fusion of synaptic vesicles (SVs) with the plasma membrane. This requires regulation at multiple levels, but the role of metabolites in SV release is unclear. Here, we uncover a role for isocitrate dehydrogenase 3a (idh3a), a Krebs cycle enzyme, in neurotransmission. Loss of idh3a leads to a reduction of the metabolite, alpha-ketoglutarate (αKG), causing defects in synaptic transmission similar to the loss of syt1. Supplementing idh3a flies with αKG suppresses these defects through an ATP or neurotransmitter-independent mechanism. Indeed, αKG, but not glutamate, enhances Syt1-dependent fusion in a reconstitution assay. αKG promotes interaction between the C2-domains of Syt1 and phospholipids. The data reveal conserved metabolic regulation of synaptic transmission via αKG. Our studies provide a synaptic role for αKG, a metabolite that has been proposed as a treatment for aging and neurodegenerative disorders.

Protective effect of Sijunzi decoction on neuromuscular junction ultrastructure in autoimmune myasthenia gravis rats.

OBJECTIVE: To investigate the protective role of Sijunzi decoction in neuromuscular junction (NMJ) and muscle cell mitochondria ultrastructure; as well as its effects on the amount of adenosine triphosphate (ATP) and the activities of mitochondrial respiratory chain complexes I, II, III, and IV in autoimmune myasthenia gravis rats. METHODS: An experimental autoimmune myasthenia gravis (EAMG) rat model was established by inoculating rats with acetylcholine receptors extracted from Torpedo. Rats were divided into three groups: model, prednisone, and Sijunzi decoction, and were fed physiological saline, prednisone, or Sijunzi decoction, respectively. NMJ and muscle cell mitochondria ultrastructure were observed by transmission electron microscope. The amount of ATP was assessed by high performance liquid chromatography. The activities of mitochondrial respiratory chain complexes I, II, III, and IV was determined using the Clark oxygen electrode method. RESULTS: In the model group, there were sparse muscle fibers, with decreased mitochondria, and sparse, diffluent, or absent NMJ folds. After intervention with Sijunzi decoction, the above pathology changes were improved: muscle fiber structure was clear and complete; the mitochondria count was higher; and the NMJ structure was close to normal. Gastrocnemius muscle mitochondria in the model group produced significantly less ATP than those in the prednisone group (P < 0.01). Conversely, the ATP of Sijunzi decoction group was significantly higher than prednisone group (P < 0.01). The activities of gastrocnemius muscle mitochondrial respiratory chain complexes I, II, III, and IV in both the prednisone and Sijunzi decoction groups was dramatically higher compared with the model group (P < 0.05). The activities of complexes I and III in the Sijunzi decoction group were significantly higher than those in the prednisone group (P < 0.05), but there was no obvious difference in complex II or IV activities between the two groups (P > 0.05). CONCLUSION: Sijunzi decoction improved pathological changes in muscle mitochondria and NMJ, enhanced the amount of ATP in gastrocnemius muscle mitochondria, and improved the activities of respiratory chain complexes I, II, III, and IV (especially I and III) of the EAMG rats.

Neuromuscular synapses on the dactyl opener muscle of the lobster Homarus americanus.

The crustacean dactyl opener neuromuscular system has been studied extensively as a model system that exhibits several forms of synaptic plasticity. We report the ultrastructural features of the synapses on dactyl opener of the lobster (Homarus americanus) as determined by examination of serial thin sections. Several innervation sites supplied by an inhibitory motoneuron have been observed without nearby excitatory innervation, indicating that excitatory and inhibitory inputs to the muscle are not always closely matched. The ultrastructural features of the lobster synapses are generally similar to those described previously for the homologous crayfish muscle, with one major distinction: few dense bars are seen at the presynaptic membranes of these lobster synapses. The majority of the lobster neuromuscular synapses lack dense bars altogether, and the mean number of dense bars per synapse is relatively low. In view of the finding that the physiology of the lobster dactyl opener synapses is similar to that reported for crayfish, these ultrastructural observations suggest that the structural complexity of the synapses may not be a critical factor determining synaptic plasticity.

Recruitment of synapses in the neurosecretory process during long-term facilitation at the lobster neuromuscular junction.

We investigated long-term facilitation at the lobster neuromuscular synapse employing a combination of FM1-43 staining of synaptic vesicles, electron microscopy analysis, and electrical recordings of synaptic activity. Synaptic terminals were loaded with the fluorescent dye FM1-43 producing clusters of activity-dependent fluorescent spots. Electron microscopy analysis of synaptic ultrastructure suggested that fluorescent spots represent compartments of synaptic terminals filled with vesicles. Excitatory postsynaptic currents were recorded from the stained synaptic terminals using focal macropatch electrodes. Terminals were stained during the nerve stimulation at a low stimulation frequency (2, 5 or 10 Hz) before and after long-term facilitation was elicited by high-frequency stimulation (20 or 30 Hz for 5 min). We found that staining after long-term facilitation results in the appearance of new fluorescent spots, as well as in the increase in fluorescence of the spots that appeared before long-term facilitation. This increase in fluorescence accounted for the increase in quantal release. Activation of individual fluorescent spots was found to be non-uniform. In spite of overall increase in fluorescence, some synaptic compartments decreased their staining after long-term facilitation. Thus, our study demonstrates that long-term facilitation produces non-uniform activation of FM1-43 uptake in synaptic compartments that correlates with the increase in quantal neurosecretion.

Stochastic modeling of facilitated neurosecretion.

Two models of neurosecretion were evaluated in terms of their ability to predict the dependency of quantal content (m) on the frequency of repetitive stimulation of a lobster motoneuron. First, the hypothesis that neurosecretion is limited by a fixed number of release sites was tested by the fit of the distribution of m by uniform and nonuniform binomial statistics. The obtained release probabilities suggest that frequency facilitation can be due to activation of a group of sites with high release probabilities. However, the fit obtained using this model is not statistically significant due to a large number of fitting parameters. Second, the hypothesis that neurosecretion is limited by the rates of exchange between the releasable pool and the total store of quanta and that each stimulus enhances quantal mobilization was tested. Monte Carlo simulation was carried out in accordance with this model and reproduced the observed distribution of m with very few fitting parameters and therefore with a high level of significance (>0.1). This result demonstrates that mobilization of extra vesicles with each stimulus is a mechanism that allows a very accurate and parsimonious quantitative description of frequency facilitation.

Facilitation of neurotransmitter release at the spiny lobster neuromuscular junction.

Stimulation-induced facilitation of transmitter release was examined at spiny lobster (Palinurus japonicus) neuromuscular junctions by measuring excitatory junctional potentials (EJPs). We found three components of facilitation with the decay time constants about 16, 200 and 1000 ms, respectively. The decay time constants of the nerve terminal free Ca(2+) concentration after stimulation agreed well with the two slower time constants of facilitation. The relationship among the three components of facilitation and a yet slower component, S, was investigated on the basis of several models. The models that have a multiplicative relationship between any two components of facilitation, and the additive model between all components could not account for the results of experiments. Only the 'unified power model', which assumes that facilitation and S are described by the 3-4th power of the sum of underlying components, could account for both the growth process of EJPs during stimulation and the effects of Ca(2+)-chelators. Loading Ca(2+)-chelators, BAPTA and EGTA, into the presynaptic terminals resulted in reduction, but not elimination, of any component of the facilitation. These results suggest that in the spiny lobster neuromuscular junction, the 'unified power model' can describe the relationship between three components of facilitation and S, and that residual free Ca(2+) enhances all three components of facilitation, although it may not be essential for them.

Epidermal tendon cells require Broad Complex function for correct attachment of the indirect flight muscles in Drosophila melanogaster.

Drosophila Broad Complex, a primary response gene in the ecdysone cascade, encodes a family of zinc-finger transcription factors essential for metamorphosis. Broad Complex mutations of the rbp complementation group disrupt attachment of the dorsoventral indirect flight muscles during pupal development. We previously demonstrated that isoform BRC-Z1 mediates the muscle attachment function of rbp(+) and is expressed in both developing muscle fibers and their epidermal attachment sites. We now report two complementary studies to determine the cellular site and mode of action of rbp(+) during maturation of the myotendinous junctions of dorsoventral indirect flight muscles. First, genetic mosaics, produced using the paternal loss method, revealed that the muscle attachment phenotype is determined primarily by the genotype of the dorsal epidermis, with the muscle fiber and the ventral epidermis exerting little or no influence. When the dorsal epidermis was mutant, the vast majority of muscles detached or chose ectopic attachment sites, regardless of the muscle genotype. Conversely, wild-type dorsal epidermis could support attachment of mutant muscles. Second, ultrastructural analysis corroborated and extended these results, revealing defective and delayed differentiation of rbp mutant epidermal tendon cells in the dorsal attachment sites. Tendon cell processes, the stress-bearing links between the epidermis and muscle, were reduced in number and showed delayed appearance of microtubule bundles. In contrast, mutant muscle and ventral epidermis resembled the wild type. In conclusion, BRC-Z1 acts in the dorsal epidermis to ensure differentiation of the myotendinous junction. By analogy with the cell-cell interaction essential for embryonic muscle attachment, we propose that BRC-Z1 regulates one or more components of the epidermal response to a signal from the developing muscle.

Ultrastructure of synapses in the first-evolved nervous systems.

The phylum Cnidaria represents the first group of animals to evolve a recognizable nervous system. A comparison of the ultrastructural features of synaptic loci in animals representing all four classes of the cnidaria has provided an overview of the first-evolved synapses that can be compared morphologically to synapses in higher forms. Synapses in these watery jellylike animals with unmyelinated axons are sparse and difficult to fix well. However, we now have sufficient evidence to define an early synapse as one with paired electron dense plasma membranes separated by a 13-25 nm gap containing intracleft filaments and with vesicles on one or both sides of the synaptic cleft. The vesicles are of three types: dense-cored, clear, and opaque. Neuromuscular synapses resemble neuronal synapses and lack the postsynaptic specializations of higher animals. However, some coelenterates, such as the jellyfish Chrysaora, have a postsynaptic cisterna in the muscle. Neuromuscular and neuronematocyte synapses can have either clear or dense-cored vesicles. Opaque vesicles at two-way interneuronal synapses and at neuromuscular synapses in the oral sphincter muscle of sea anemones can be labelled with antisera to the neuropeptides Antho-RFamide (Antho-Arg-Phe-NH2) and Antho-RWamides (Antho-Arg-Trp-NH2) I and II, respectively. That suggests that neuropeptides evolved as neurotransmitters early in the animal kingdom. The basic differences between first evolved synapses and synapses of higher animals are the lack of postjunctional folds at neuromuscular synapses and the presence of fewer and somewhat larger synaptic vesicles, generally containing granular cores, in the more primitive animals.

Monitoring of black widow spider venom (BWSV) induced exo- and endocytosis in living frog motor nerve terminals with FM1-43.

The neurotoxin Black Widow Spider Venom (BWSV) triggers massive release of neurotransmitter at synapses. Here we demonstrate that the action of BWSV on the frog neuromuscular junction can be visualized in vivo by the use of the fluorescent styryl dye FM1-43. This vital dye stains recycled synaptic vesicles upon nerve stimulation. Motor nerve terminals were stained with FM1-43 via electrical stimulation, washed and then exposed to BWSV or alpha-Latrotoxin. All terminals destained completely, independent of external calcium. Exposure of frog nerve terminals to BWSV in the presence of FM1-43 and calcium led to staining of terminals. The staining pattern appeared to be exactly the same as in control preparations, stimulated electrically via the nerve. When the same experiment was performed in the absence of calcium, only a minute quantity of dye was taken up into the nerve terminals, and the synapses looked swollen and puffed. Addition of external calcium to these preparations elicited an immediate shrinking of the nerve terminals, indicating endocytosis. These observations support electron-microscopic data that suggest an important role for extracellular calcium in endocytosis of BWSV poisoned nerve terminals.

Cisplatin-induced peptic ulcers, vagotomy, adrenal and calcium modulation.

Cytochemical and autoradiographic studies in Wistar rats [Crl:(WI)BR] show that cisplatin treatment (9 mg/kg) inhibits the release of acetylcholine from the axonal endings of the stomach smooth muscle resulting in bloating of the stomach and ulceration. Cisplatin also induces corticosteroid release from the adrenal gland stimulating peptic ulceration. Vagotomy helps ameliorate the effect but not eliminate it. Calcium supplementation restores normal neuromuscular function to gastric smooth muscle, thereby eliminating the gastro-intestinal toxicity due to cisplatin.

Two structural adaptations for regulating transmitter release at lobster neuromuscular synapses.

The distal accessory flexor muscle (DAFM) in the lobster (Homarus americanus) walking leg consists of 5 muscle fiber bundles. All five bundles, one proximal, one distal, and 3 medial, are innervated by one excitatory and one inhibitory motor neuron. Both neurons release more transmitter on the distal bundle than on the proximal bundle. The aim of our studies was to investigate the structural basis of this differentiation. Thin sections cut at 50 microns intervals showed a similar number of excitatory synapses on the two bundles. Freeze-fracture views of excitatory synapses showed that synapse size, active zone number per synapse, and intramembrane particle density in the postsynaptic membrane are similar proximally and distally. Active zones at synapses on the distal bundle are larger and contain about 50% more large intramembrane particles, which are thought to include the voltage-gated Ca2+ channels that couple the action potential to transmitter release, than their counterparts on the most proximal bundle. This difference in channel number appears to produce a disproportionate increase in the probability of transmitter release sufficient to account for most of the proximal-distal disparity in the amplitude of the excitatory postsynaptic potential. In contrast, staining the inhibitor for antibodies to the inhibitory neurotransmitter, GABA, showed that it forms more varicosities on the distal bundle than on the proximal bundle. Because most of the synapses are located in the varicosities, differences in synapse number likely regulate the proximal-distal disparity in the amount of inhibitory transmitter released. Therefore, the regional differentiation in the amount of transmitter released in the DAFM appears to be based on two distinct mechanisms. In the inhibitor, transmitter release appears to be regulated differentially by differences in synapse number. In the excitor, transmitter release appears to be regulated differentially from a similar number of synapses by differences in active zone structure.

Facilitation and depression at different branches of the same motor axon: evidence for presynaptic differences in release.

This study provides evidence that a neuron can exhibit differences in activity-dependent transmitter release at two synaptic sites due to variations in the properties of its presynaptic terminals. Two muscles in the stomatogastric system of the lobster Homarus americanus are innervated by a single motor neuron but respond differently to that motor neuron's input, resulting in two different movements evoked by one motor neuron. During continued motor neuron stimulation, the gm8 muscle contracts slowly and maintains contraction, while the gm9 muscle contracts rapidly and then relaxes. These different muscle responses can be accounted for, in large part, by the properties of the respective neuromuscular synapses: the excitatory junctional potentials recorded in gm8 are initially small but summate and facilitate with repeated stimulation, while those in gm9 are initially large but depress with repeated stimulation. Presynaptic differences in neurotransmitter release contribute strongly to the divergent responses; reduction of the excitatory junction potential amplitude by partial postsynaptic receptor blockade or by desensitization does not change the amount of depression at gm9. However, reduction of neurotransmitter release with low-Ca2+, high-Mg2+ saline removes gm9 synaptic depression and reveals that both neuromuscular junctions exhibit frequency-dependent homosynaptic facilitation. Postsynaptic differences in muscle input resistance and muscle composition may enhance the effects of the divergent release properties, but are not responsible for the activity-dependent changes. Ultrastructural features of the nerve terminals on the two muscles are consistent with the differential output of the terminals; the synapses on gm9 are larger and have more presynaptic dense bars than their counterparts on gm8. These data suggest that the basis for the differences in transmitter release between the two muscles may be a higher density of release sites in the gm9 synapses that leads to a higher output of neurotransmitter, rapid depletion of transmitter stores, and synaptic depression.

Early innervation of abdominal swimmeret muscles in developing lobsters.

The swimmerets in the abdomen of the lobster Homarus americanus are paired external appendages whose back and forth propulsive movements are brought about largely by a group of power and return stroke muscles located in the lateral abdominal cavity. We find functional innervation of these muscles by several excitatory axons and a single inhibitor in embryonic and stage 1 larval lobsters before the external appendages are even formed. This early innervation is via a few nerve bundles in which branches of the motor axons are intertwined in a complex manner. As the swimmerets develop to maturity in later larval and juvenile stages, the innervation consisting usually of several excitor and a single inhibitor synaptic terminals becomes localized to individual muscles. Patterned synaptic activity in these muscles was not seen in the embryonic and larval stages but has been shown in early juvenile stages, when it coincides with the onset of rhythmic movement of the swimmerets. Consequently, such early innervation of the swimmeret muscles may be influential in establishing the central circuitry for the generation of patterned activity, a possibility that was discounted in a previous study (Proc. Natl. Acad. Sci. USA, 70:954-958).

Age-related remodeling of lobster neuromuscular terminals.

Multiterminal innervation of a lobster limb muscle by an identified excitor motoneuron was examined during primary development and adult growth. To keep pace with the growth in the target muscle, axonal branches proliferate by sprouting from synaptic terminals; an increasingly complex branching pattern results. Neuromuscular synapses multiply in number, enlarge in size, and become perforated. Concomitantly, synapses tend to appear on the more distal axonal branches and to disappear on more proximal branches, providing for continual remodeling of multiterminal innervation. This plasticity in an identified motoneuron occurs over a long life span of several decades.

Inhibitory innervation of a lobster muscle.

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

Long-term survival of decentralized axons and incorporation of satellite cells in motor neurons of rock lobsters.

Previous electrophysiological experiments have shown that in the abdominal extensor muscles of rock lobsters, axons which were cut in surviving animals do not degenerate peripherally for several months, but conduct action potentials and release transmitter quanta on stimulation closely distal to the scar. Electron micrographs from the axon distal to the scar (in a reliably conducting region) show invasion of the axoplasmic space by nucleated cells, probably glia. After several months, the cell membranes of the invaders have vanished and apparently functional multiple nuclei remain. We suggest that decentralized axons may survive for months with the help of 'donated' nuclei.

Morphogenesis of nuclear inclusions in the soleplate region of rat skeletal muscle fibers following chronic daily administration of neostigmine.

In the course of ultrastructural investigations of motor endplate pathology mediated by calcium ions, intranuclear sarcoplasmic inclusions, either membrane-free (true type) or membrane-delimited (false type), were observed during chronic daily high-dose exposure to the anticholinesterase neostigmine. At the stage in which subjunctional components, including soleplate nuclei, were severely damaged (day 7), the true nuclear inclusions were frequently associated with the disrupted nuclear envelope (fragmentation, vesiculation etc.) and nuclear pores. At a subsequent stage, in which muscle repair was accelerated and most soleplate nuclei were less severely affected (day 21), formation of the false inclusions in these nuclei was enhanced. Analysis of serial sections of the less severely affected nuclei, where only a true inclusion type was present, revealed no sign of invaginated nuclear envelopes or other membranes enclosing the inclusions. Our findings indicate that morphogenesis of true inclusions depends upon the severity of nuclear degeneration, i.e., in severely affected nuclei there is disruption in the nuclear envelope and/or nuclear pores, while in less severely affected nuclei, either a pinched-off invagination or diffusion of excessive sarcoplasmic proteins into the nucleus via nuclear pores occurs.

Highly active neuromuscular system in developing lobsters with programmed obsolescence.

The primary locomotory apparatus in the three larval stages of the lobster, Homarus americanus, are paddlelike structures on the thoracic appendages called exopodites, which beat almost continuously. Consequently their power and return-stroke muscles are examples of highly active but short-lived neuromuscular systems. The muscles, which are well vascularized, are of the fast type with 2-3-micron sarcomere lengths and 6 thin filaments surrounding a thick one. The most striking feature, however, is the large volume of mitochondria making up 40-50% of the fiber. They appear as simple cylinders packed several layers deep along the periphery of the fiber and as large, multibranched forms distributed throughout the fiber and subdividing it into smaller units. The motor innervation to the return-stroke muscle is via 3 excitatory axons, which generate large junctional potentials and twitch contractions. The muscle is densely populated with large neuromuscular synapses, most of which have a well-defined active site or dense bar denoting the site of transmitter release. Altogether this motor system is specialized for prolonged activity. Atrophy of the neuromuscular system occurs by the late larval third stage. The muscle fibers lose their identity, fuse, and become vacuolated. The myofibrils condense and erode and the mitochondria are lost. Atrophy of motor innervation is gradual with individual axons dropping out. The largest axon providing most of the innervation is the first to degenerate. Early degenerative changes affect the axon and neuromuscular terminals but not the synaptic contacts, dense bars, and vesicles, which appear intact. Continued atrophy in the postlarval fourth stage reduces the exopodites to vestiges. Thus the return-stroke muscle of the larval exopodites in which muscle fiber and motoneurons are identifiable permits study of the interaction between a neuron and its target muscle undergoing programmed obsolescence.