Neuronal Control of Esophageal Peristalsis and Its Role in Esophageal Disease.

PURPOSE OF REVIEW: Esophageal peristalsis is a highly sophisticated function that involves the coordinated contraction and relaxation of striated and smooth muscles in a cephalocaudal fashion, under the control of central and peripheral neuronal mechanisms and a number of neurotransmitters. Esophageal peristalsis is determined by the balance of the intrinsic excitatory cholinergic, inhibitory nitrergic and post-inhibitory rebound excitatory output to the esophageal musculature. RECENT FINDINGS: Dissociation of the longitudinal and circular muscle contractions characterizes different major esophageal disorders and leads to esophageal symptoms. Provocative testing during esophageal high-resolution manometry is commonly employed to assess esophageal body peristaltic reserve and underpin clinical diagnosis. Herein, we summarize the main factors that determine esophageal peristalsis and examine their role in major and minor esophageal motility disorders and eosinophilic esophagitis.

Conduction block of mammalian myelinated nerve by local cooling to 15-30°C after a brief heating.

This study aimed at understanding thermal effects on nerve conduction and developing new methods to produce a reversible thermal block of axonal conduction in mammalian myelinated nerves. In 13 cats under α-chloralose anesthesia, conduction block of pudendal nerves (n = 20) by cooling (5-30°C) or heating (42-54°C) a small segment (9 mm) of the nerve was monitored by the urethral striated muscle contractions and increases in intraurethral pressure induced by intermittent (5 s on and 20 s off) electrical stimulation (50 Hz, 0.2 ms) of the nerve. Cold block was observed at 5-15°C while heat block occurred at 50-54°C. A complete cold block up to 10 min was fully reversible, but a complete heat block was only reversible when the heating duration was less than 1.3 ± 0.1 min. A brief (<1 min) reversible complete heat block at 50-54°C or 15 min of nonblock mild heating at 46-48°C significantly increased the cold block temperature to 15-30°C. The effect of heating on cold block fully reversed within ∼40 min. This study discovered a novel method to block mammalian myelinated nerves at 15-30°C, providing the possibility to develop an implantable device to block axonal conduction and treat many chronic disorders. The effect of heating on cold block is of considerable interest because it raises many basic scientific questions that may help reveal the mechanisms underlying cold or heat block of axonal conduction.

Involvement of catecholaminergic neurons in motor innervation of striated muscle in the mouse esophagus.

Enteric co-innervation is a peculiar innervation pattern of striated esophageal musculature. Both anatomical and functional data on enteric co-innervation related to various transmitters have been collected in different species, although its function remains enigmatic. However, it is unclear whether catecholaminergic components are involved in such a co-innervation. Thus, we examined to identify catecholaminergic neuronal elements and clarify their relationship to other innervation components in the esophagus, using immunohistochemistry with antibodies against tyrosine hydroxylase (TH), vesicular acetylcholine transporter (VAChT), choline acetyltransferase (ChAT) and protein gene product 9.5 (PGP 9.5), α-bungarotoxin (α-BT) and PCR with primers for amplification of cDNA encoding TH and dopamine-ß-hydroxylase (DBH). TH-positive nerve fibers were abundant throughout the myenteric plexus and localized on about 14% of α-BT-labelled motor endplates differing from VAChT-positive vagal nerve terminals. TH-positive perikarya represented a subpopulation of only about 2.8% of all PGP 9.5-positive myenteric neurons. Analysis of mRNA showed both TH and DBH transcripts in the mouse esophagus. As ChAT-positive neurons in the compact formation of the nucleus ambiguus were negative for TH, the TH-positive nerve varicosities on motor endplates are presumably of enteric origin, although a sympathetic origin cannot be excluded. In the medulla oblongata, the cholinergic ambiguus neurons were densely supplied with TH-positive varicosities. Thus, catecholamines may modulate vagal motor innervation of esophageal-striated muscles not only at the peripheral level via enteric co-innervation but also at the central level via projections to the nucleus ambiguus. As Parkinson's disease, with a loss of central dopaminergic neurons, also affects the enteric nervous system and dysphagia is prevalent in patients with this disease, investigation of intrinsic catecholamines in the esophagus may be worthwhile to understand such a symptom.

Urinary and ejaculatory dysfunction induced by denervation of specific striated muscles anatomically related to the urethra in male rats.

AIMS: The purpose of the present study was to determine the contribution of the external urethral sphincter (EUS), the ischiocavernous (IC), or the bulbospongiosus (BS) on the control of micturition, copulatory behavior and semen expulsion in male rats. We hypothesized that the EUS contributes to maintain urinary continence, while all three muscles participate in expulsive urethral functions. METHODS: In Experiment 1, it was analyzed the effects of bilateral denervation of IC, BS or EUS, or sham surgery, on voiding behavior and urinary parameters measured before surgery and 2 and 10 days post-surgery. In Experiment 2, copulatory behavior and the weight of the seminal plug expelled during ejaculation were recorded before and after sham surgery or bilateral denervation of the aforementioned muscles. Immediately after ejaculation, the animals were anesthetized to confirm the denervation and determine whether seminal material had accumulated in the lower urinary tract. RESULTS: In IC-denervated animals, voiding duration and the number of mounts was increased, and intromission or ejaculation patterns were absent. Denervation of BS induced signs of post-micturition dribble, decreased voiding frequency, increased urine volume and reduced the amount of semen ejaculated. Denervation of EUS induced signs of post-micturition dribble and urinary incontinence, as well as retrograde ejaculation. CONCLUSIONS: Striated muscles anatomically related to the urethra contribute differentially to the control of continence and expulsive urethral functions. Damages to the muscles or to their innervation, as may occur during pelvic surgery, would result in sexual and urinary dysfunctions.

Localization of sensory nerve terminals containing calcitonin gene-related peptide (CGRP) on striated muscle fibers in the rat esophagus: Evidence for triple innervation via motor endplates.

BACKGROUND: Many esophageal striated muscles of mammals are dually innervated by the vagal and enteric nerves. Recently, substance P (SP)-sensory nerve terminals with calcitonin gene-related peptide (CGRP) were found on a few striated muscle fibers in the rat esophagus, implying that these muscle fibers are triply innervated. In this study, we examined the localization and origin of CGRP-nerve endings in striated muscles to consider their possible roles in the esophagus regarding triple innervation. METHODS: Wholemounts of the rat esophagus were immunolabeled to detect CGRP-nerve endings in striated muscles. Also, retrograde tracing was performed by injecting Fast Blue (FB) into the esophagus, and cryostat sections of the medulla oblongata, nodose ganglion (NG), and the tenth thoracic (T10) dorsal root ganglion (DRG) were immunostained to identify the origin of the CGRP-nerve endings. RESULTS: CGRP-fine, varicose nerve endings were localized in motor endplates on a few esophageal striated muscle fibers (4 %), most of which received nitric oxide (NO) synthase nerve terminals, and most of the CGRP nerve endings were SP- and transient receptor potential vanilloid member 1 (TRPV1)-positive. Retrograde tracing showed many FB-labeled CGRP-neurons positive for SP and TRPV1 in the NG and T10 DGR. CONCLUSIONS: This study suggests that the CGRP-varicose nerve endings containing SP and TRPV1 in motor endplates are sensory, and a few esophageal striated muscle fibers are triply innervated. The nerve endings may detect acetylcholine-derived acetic acid from the vagal motor nerve endings and NO from esophageal intrinsic nerve terminals in the motor endplates to regulate esophageal motility.

Striated muscle involvement in experimental oral infection by herpes simplex virus type 1.

Herpes simplex virus type 1 is one of the most frequent causes of oral infection in humans, especially during early childhood. Several experimental models have been developed to study the pathogenesis of this virus but all of them employed adult animals. In this work, we developed an experimental model that uses mice younger than 4 days old, to more closely resemble human infection. Mice were infected subcutaneously with the prototype strain McIntyre of Herpes simplex-1, and the progression of infection was studied by immunoperoxidase. All animals died within 24-72 h post-infection, while viral antigens were found in the oral epithelium, nerves and brain. The most striking result was the finding of viral antigens in the nucleus and cytoplasm of cells belonging to striated muscles. Organotypic cultures of striated muscles were performed, and viral replication was observed in them by immunocytochemistry, electron microscopy and viral isolation. We conclude that the infection of striated muscles is present from the onset of oral infection and, eventually, could explain some clinical observations in humans.

Readiness discharge for spontaneous initiation of walking in crayfish.

Animals initiate behavior not only reflexively but also spontaneously in the absence of external stimuli. In vertebrates, electrophysiological data on the neuronal activity associated with the self-initiated voluntary behavior have accumulated extensively. In invertebrates, however, little is known about the neuronal basis of the spontaneous initiation of behavior. We investigated the spike activity of brain neurons at the time of spontaneous initiation of walking in the crayfish Procambarus clarkii and found neuronal signals indicative of readiness or preparatory activities in the vertebrate brain that precede the onset of voluntary actions. Those readiness discharge neurons became active >1 s before the initiation of walking regardless of stepping direction. They remained inactive at the onset of mechanical stimulus-evoked walking in which other descending units were recruited. These results suggest that the parallel descending mechanisms from the brain separately subserve the spontaneous and stimulus-evoked walking. Electrical stimulation of these different classes of neurons caused different types of walking. In addition, we found other descending units that represented different aspects of walking, including those units that showed a sustained activity increase throughout the walking bout depending on its stepping direction, as well as one veto unit for canceling out the output effect of the readiness discharge and three termination units for stopping the walking behavior. These findings suggest that the descending activities are modularized in parallel for spontaneous initiation, continuation, and termination of walking, constituting a sequentially hierarchical control.

Nonmuscle Myosin II helps regulate synaptic vesicle mobility at the Drosophila neuromuscular junction.

BACKGROUND: Although the mechanistic details of the vesicle transport process from the cell body to the nerve terminal are well described, the mechanisms underlying vesicle traffic within nerve terminal boutons is relatively unknown. The actin cytoskeleton has been implicated but exactly how actin or actin-binding proteins participate in vesicle movement is not clear. RESULTS: In the present study we have identified Nonmuscle Myosin II as a candidate molecule important for synaptic vesicle traffic within Drosophila larval neuromuscular boutons. Nonmuscle Myosin II was found to be localized at the Drosophila larval neuromuscular junction; genetics and pharmacology combined with the time-lapse imaging technique FRAP were used to reveal a contribution of Nonmuscle Myosin II to synaptic vesicle movement. FRAP analysis showed that vesicle dynamics were highly dependent on the expression level of Nonmuscle Myosin II. CONCLUSION: Our results provide evidence that Nonmuscle Myosin II is present presynaptically, is important for synaptic vesicle mobility and suggests a role for Nonmuscle Myosin II in shuttling vesicles at the Drosophila neuromuscular junction. This work begins to reveal the process by which synaptic vesicles traverse within the bouton.

Hox genes regulate muscle founder cell pattern autonomously and regulate morphogenesis through motor neurons.

The differentiation of myoblasts to form functional muscle fibers is a consequence of interactions between the mesoderm and ectoderm. The authors examine the role of segment identity in directing these interactions by studying the role of Hox genes in patterning adult muscles in Drosophila. Using the 'four-winged fly' to remove Ultrabithorax function in the developing adult, the authors alter the identity of the ectoderm of the third thoracic segment towards the second and show that this is sufficient to inductively alter most properties of the mesoderm-myoblast number, molecular diversity, and migration pattern-to that of the second thoracic segment. Not all aspects of myogenesis are determined by the segment identity of the ectoderm. The autonomous identity of the mesoderm is important for choosing muscle founder cells in the correct segmental pattern. The authors show this by removal of the function of Antennapedia, the Hox gene expressed in the mesoderm of the third thoracic segment. This results in the transformation of founder cells to a second-thoracic pattern. The authors also report a role for the nervous system in later aspects of muscle morphogenesis by specifically altering Ultrabithorax gene expression in motor neurons. Thus, ectoderm and mesoderm segment identities collaborate to direct muscle differentiation by affecting distinct aspects of the process.

Repeatability of innervation zone identification in the external anal sphincter muscle.

AIMS: Knowledge of the distribution of the innervation zones (IZs) of the external anal sphincter (EAS) may be useful for preventing anal sphincter incompetence during vaginal delivery. A method proposed for the automatic estimation of the distribution of IZs of EAS from high-density surface electromyography (EMG) was evaluated for repeatability in continent volunteers. METHODS: In 13 healthy female subjects (age: 35 +/- 11 years) surface EMG signals were acquired using an anal probe with three circumferential electrode arrays (of 16 contacts each) at different depths within the anal canal (15 mm distance between the centers of adjacent arrays), during four independent experimental sessions. Three maximal voluntary contractions (MVCs) of 10 sec were performed for each session for a total of 12 contractions per subject. Repeatability of the estimation of the distribution of IZ was tested by evaluating the coefficient of multiple correlations (CMC) between the IZ distributions estimated from the signals recorded from each subject. RESULTS: A high repeatability (CMC > 0.8) was found comparing IZ distributions estimated from signals recorded by each array within the same session. A slightly lower value was obtained considering signals recorded during different sessions (CMC > 0.7), but a higher value (CMC > 0.8) was obtained after aligning the estimated IZ distributions. The realignment compensates for the operator's error in repositioning the probe in the same position during different sessions. CONCLUSION: This result justifies clinical studies using high-density surface EMG in routine examinations, providing information about IZs of EAS and assessing the possibilities of preventing neuronal trauma during vaginal delivery.

Voltage-gated sodium channels mediating conduction in vagal motor fibers innervating the esophageal striated muscle.

The vagal motor fibers innervating the esophageal striated muscle are essential for esophageal motility including swallowing and vomiting. However, it is unknown which subtypes of voltage-gated sodium channels (NaV1s) regulate action potential conduction in these efferent nerve fibers. The information on the NaV1s subtypes is necessary for understanding their potential side effects on upper gut, as novel inhibitors of NaV1s are developed for treatment of pain. We used isolated superfused (35 °C) vagally-innervated mouse esophagus striated muscle preparation (mucosa removed) to measure isometric contractions of circular striated muscle evoked by electrical stimulation of the vagus nerve. NaV1 inhibitors were applied to the de-sheathed segment of the vagus nerve. Tetrodotoxin (TTX) applied to the vagus nerve completely abolished electrically evoked contractions. The selective NaV1.7 inhibitor PF-05089771 alone partially inhibited contractions and caused a >3-fold rightward shift in the TTX concentration-inhibition curve. The NaV1.1, NaV1.2 and NaV1.3 group inhibitor ICA-121431 failed to inhibit contractions, or to alter TTX concentration-inhibition curves in the absence or in the presence of PF-05089771. RT-PCR indicated lack of NaV1.4 expression in nucleus ambiguus and dorsal motor nucleus of the vagus nerve, which contain motor and preganglionic neurons projecting to the esophagus. We conclude that the action potential conduction in the vagal motor fibers to the esophageal striated muscle in the mouse is mediated by TTX-sensitive voltage gated sodium channels including NaV1.7 and most probably NaV1.6. The role of NaV1.6 is supported by ruling out other TTX-sensitive NaV1s (NaV1.1-1.4) in the NaV1.7-independent conduction.

The interaction of positive and negative sensory feedback loops in dynamic regulation of a motor pattern.

In many rhythmic behaviors, phasic sensory feedback modifies the motor pattern. This modification is assumed to depend on feedback sign (positive vs. negative). While on a phenomenological level feedback sign is well defined, many sensory pathways also process antagonistic, and possibly contradictory, sensory information. We here model the locust flight pattern generator and proprioceptive feedback provided by the tegula wing receptor to test the functional significance of sensory pathways processing antagonistic information. We demonstrate that the tegula provides delayed positive feedback via interneuron 301, while all other pathways provide negative feedback. Contradictory to previous assumptions, the increase of wing beat frequency when the tegula is activated during flight is due to the positive feedback. By use of an abstract model we reveal that the regulation of motor pattern frequency by sensory feedback critically depends on the interaction of positive and negative feedback, and thus on the weighting of antagonistic pathways.

Time course of adult castration-induced changes in soma size of motoneurons in the rat spinal nucleus of the bulbocavernosus.

The spinal nucleus of the bulbocavernosus (SNB) innervates striated muscles, the bulbocavernosus and levator ani (BC/LA), which control penile reflexes. Castration results in shrinkage in the size of SNB somata and dendrites, as well as BC/LA muscle mass. However, there is no information about how quickly these regressive changes occur compared to the rapid effects of castration upon penile reflexes, which are greatly diminished a few days after surgery. Therefore we examined the time course of change in the size of SNB somata after castration of adult male rats. Males were sacrificed 2, 14, or 28 days after either castration or sham surgery and somata were measured in the SNB and in a control population of motoneurons, the retrodorsolateral nucleus (RDLN). BC/LA weight was reduced in castrates compared to intact males 14 and 28 days post surgery, but SNB somata were significantly smaller in castrates only at 28 days after surgery. As has been previously observed, castration did not affect soma size in the RDLN. These data indicate that SNB somata respond more slowly after castration than BC/LA mass or penile reflexes, suggesting that the size of SNB somata cannot account for the loss of penile reflexes. Androgenic effects on SNB somata may contribute to aspects of reproductive behavior that are not apparent in penile reflexes tested ex copula.

Effects of sphincterotomy and pudendal nerve transection on the anal sphincter in a rat model.

PURPOSE: Our objective was to define anal resting pressure and electromyography of the normal rat anal sphincter and investigate the short-term effects of both mechanical trauma to the anal sphincter muscles and pudendal nerve transection. METHODS: Forty-five virgin female Sprague-Dawley rats were randomly allotted to three groups: controls (n = 21), sphincterotomy (n = 12), and pudendal nerve transection (n = 12). Anal pressure was monitored using a saline-filled balloon connected to a pressure transducer. Anal pressure and electromyography of the anal sphincter with use of a needle electrode were recorded both before and after injury or succinylcholine administration. RESULTS: Anal pressure data were consistent with rhythmic pressure contractions. Succinylcholine significantly reduced both pressure and electromyography signals. Electromyography amplitude and frequency decreased after nerve transection but not after sphincterotomy. The histology showed that the rat anal anatomy has muscular components that compare with human anatomy. The sphincterotomy group showed injury to the anal sphincters and the sphincter anatomy of the nerve transection group appeared similar to the control group. The anal pressure wave appears to be created by synergistic activity of both striated and smooth muscle of the anal sphincter. CONCLUSION: The female rat is a suitable and reliable model for studying effect of direct and indirect injury to the anal sphincters.

From action potential to contraction: neural control and excitation-contraction coupling in larval muscles of Drosophila.

The neuromuscular system of Drosophila melanogaster has been studied for many years for its relative simplicity and because of the genetic and molecular versatilities. Three main types of striated muscles are present in this dipteran: fibrillar muscles, tubular muscles and supercontractile muscles. The visceral muscles in adult flies and the body wall segmental muscles in embryos and larvae belong to the group of supercontractile muscles. Larval body wall muscles have been the object of detailed studies as a model for neuromuscular junction function but have received much less attention with respect to their mechanical properties and to the control of contraction. In this review we wish to assess available information on the physiology of the Drosophila larval muscular system. Our aim is to establish whether this system has the requisites to be considered a good model in which to perform a functional characterization of Drosophila genes, with a known muscular expression, as well as Drosophila homologs of human genes, the dysfunction of which, is known to be associated with human hereditary muscle pathologies.

Organization of Buccal Cone Musculature in the Pteropod Mollusc Clione limacina.

The pteropod mollusc Clione limacina is a feeding specialist, preying on shelled pteropods of the genus Limacina. Specialized prey-capture structures, called buccal cones, are hydraulically everted from within the mouth to capture the prey. Once captured, the prey is manipulated so the shell opening is over the mouth of Clione. Analyses of high-speed cine sequences of prey capture suggest that the mouth is actively opened rather than passively forced open by buccal cone eversion. The inflated buccal cones are initially straight and form a wide angle (maximum, 113°) prior to prey contact. Individual buccal cones bend orally following prey contact, suggesting a sensory trigger. To determine the muscular basis of buccal cone movements, the musculature of the buccal cones is described. Three distinct muscle fiber types include circular smooth muscle, longitudinal smooth muscle, and longitudinal striated muscle. The organization, distribution, and innervation of the muscle types suggest that circular muscle is used during buccal cone eversion, longitudinal smooth muscle is used for buccal cone withdrawal, and longitudinal striated muscle is used for oral bending of the buccal cones after prey contact and for manipulation of the prey.

Regional variations in the number distribution of intrinsic myenteric neurons and coinnervated motor endplates on the striated muscles in the rat esophagus.

The roles of intrinsic neurons and the significance of the coinnervated striated muscles in the esophagus are unclear. We examined the number distribution of intrinsic neurons and coinnervated motor endplates on the striated muscles in the rat esophagus using immunohistochemistry to investigate whether these neurons and coinnervated striated muscles may be relevant to the local control of esophageal motility. The number of PGP9.5-positive neurons was higher in the cervical esophagus (segment 1) and gradually decreased toward the aboral, with a moderate increase in the abdominal (segment 5). This pattern was similar to that of NOS-positive neurons, while the number of ChAT-positive neurons decreased toward the aboral, but it was not significantly different among segments 3 to 5. The number of ChAT-positive motor endplates increased toward the aboral, with the highest number in segment 5. The proportion of coinnervated motor endplates was approximately 80% in segments 1 to 4, but approximately 66% in segment 5. NPY-IR was localized in some nerve terminals among the smooth muscles of the muscularis mucosa and some NOS- or ChAT-positive esophageal intrinsic neurons. ENK-8-IR was found in some NOS- or ChAT-positive intrinsic neurons, and nerve terminals surrounding intrinsic neurons in the esophagus, but not in motor neurons at the NA or DMV. This study suggests that regional variations in the number of intrinsic neurons and coinnervated striated muscles in the rat esophagus may be involved in local regulations of esophageal motility, and that the rat esophageal intrinsic neurons may contain, at least, motor neurons and interneurons.

Serotonergic transmission and gap junctional coupling in proventricular muscle cells in the American cockroach, Periplaneta americana.

The visceral muscle tissues of insects consist of striated muscle cells. The mechanisms responsible for delivering signals to the contractile muscles in the insect digestive tract remain unclear. We found that serotonergic nerves innervate the hemocoel surfaces of foregut and midgut muscles in the American cockroach. Electron microscopy of the neuromuscular junctions in the proventriculus (gizzard) revealed typical synaptic structures, the accumulation of large core/cored vesicles (neuropeptides) and small clear vesicle (neurotransmitter) at presynapses, and synaptic clefts. However, only a limited number of muscle cells, which were located in the outer part of the muscle layer, came into contact with synapses, which contained classical neurotransmitters, such as glutamate. A gap junction channel-permeable fluorescent dye, Lucifer yellow, was microinjected into single muscle cells, and it subsequently spread to several neighboring muscle cells. The dye movement occurred in the radial (hemocoel-lumen) direction rather than tangential directions. A gap junction blocker, octanol, reversibly inhibited the dye coupling. Messenger RNA for innexin 2, a gap junction-related protein, was detected in the proventriculus. These results suggest that motile signals in the insect digestive tract only reach the outermost part of the visceral muscles and are propagated to the inner muscle cells via gap junctions. Therefore, invertebrate gap junction-related proteins have potential as new targets for pest control.

Rhythmic motor patterns accompanying ejaculation in spinal cord-transected male rats.

A spinal pattern generator controls the ejaculatory response. Activation of this spinal generator elicits rhythmic motor patterns of the striated musculature that surrounds the genital tract that contributes to the expulsion of seminal secretions. In the present study, we elicited ejaculation in spinal cord-transected male rats by mechanically stimulating the urethra and registered rhythmic motor patterns in the cremasteric, iliopsoas and pubococcygeus muscles. The rhythmic motor activity recorded in these muscles was compared with that elicited in the bulbospongiosus muscles; the results revealed similarities in the motor parameters among all the muscles. Data of this study, showing the occurrence of rhythmic motor behaviour in the cremasteric, iliopsoas and pubococcygeus muscles during ejaculation, suggest that these muscles might be under the control of the spinal generator for ejaculation.

The ultrastructure of the striated muscles of Derocheilocaris typica (Mystacocarida, Crustacea).

The striated muscles of Derocheilocaris typica consist of mononucleated cells, each containing one filament bundle. Large muscles consist of two or more cells adjacent to each other. The mitochondria line up along the filament bundle on one side. The nucleus is situated in the mitochondrial row and has a small cytoplasmic area around it filled with glycogen. The sarcomeres are between 3 and 6 µm long. The Z-line and H band are present. Six thin filaments surround one thick filament. All muscles belong to the phasic type. The tubular system emanates from the ends of the muscle cell and penetrates the whole cell. The tubules are formed as cisterns, which also open at the cell membrane at the level of the I bands. They have sarcoplasmic cisterns on both sides forming a continuous triad system. Partially transformed epidermal cells mediate muscle insertions on the cuticle. Tendons are formed with the transformed epidermal cells being supplemented by fibroblasts forming collagen fibers. Dorsal and ventral abdominal muscles are innervated from the dorso-lateral nerve arising from the nerve chain. Each muscle cell receives one axon, which forms one synapse on the mitochondrial-free side of the muscles. Axons form terminal spines, which make axo-axonal synapses.