Development of neuromuscular junctions in the mouse esophagus: morphology suggests a role for enteric coinnervation during maturation of vagal myoneural contacts

The time course of establishment of motor endplates and the subsequent developmental changes in their enteric and vagal innervation were examined in esophageal striated muscle of perinatal and adult C57/Bl6 mice by using immunocytochemistry and confocal laser scanning microscopy. Nicotinic acetylcholine receptors were visualized with alpha-bungarotoxin; vagal motor nerve terminals with antisera against vesicular acetylcholine transporter; and enteric nerve fibers with antisera against neuronal nitric oxide synthase, vasoactive intestinal peptide, and galanin. Because the various stages of esophageal striated myogenesis advance caudocranially, i.e., more mature stages are found cranial to immature stages, longitudinal cryosections through the esophagus were investigated. Synaptogenesis was divided into several distinct stages. 1) Mononucleated cells express acetylcholine receptors over their entire surface. 2) They start to cluster receptors without nerve fiber contacts. 3) The first nerve contact on a growing receptor cluster is made by a vagal nerve terminal, followed by an enteric terminal. 4) Vagal terminals grow until they match the size of endplate areas, and one to three enteric terminals intertwine with them on every receptor cluster. 5) After vagal terminals have covered the whole endplate area, enteric terminals are withdrawn from the majority of motor endplates. In a minority of endplates, enteric coinnervation persists through adulthood. The enteric innervation of all developing motor endplates, shortly after vagal terminals have contacted them, and the removal of enteric nerve fibers from the majority of mature motor endplates suggest a major role of enteric nerve fibers during maturation of esophageal neuromuscular junctions.     

Motor innervation of striated oesophageal muscle. Part 2. Characteristics of the oesophagomotor fibres in the rat studied by implanting the vagus nerve into a skeletal muscle

In 12 rats the right vagus nerve distal to its recurrent laryngeal branch was implanted into the inferior segment of the denervated sternohyoid muscle. One month after implantation the first signs of neuromuscular transmission at the vagal motor endings could be recorded. Two months after implantation the reinnervated muscles showed vigorous contractions on electrical stimulation of the vagus nerve. During the performance of propulsive waves of the oesophagus the implanted vagus nerve caused clonic to tetanic contractions of the sternohyoid muscle, thus proving the oesophagomotor genesis of the reinnervating nerve fibres. In addition, the vagus-innervated motor end-plates were shown to exhibit the same ultrastructural peculiarities as the original neuromuscular junctions of the oesophagus. In sections stained for cholinesterase it could be demonstrated that the oesophagomotor fibres had preferentially reinnervated the denervated motor end-plates. In many instances the subneural apparatus was not completely covered by the vagal axon terminals. Newly formed, ectopic vagal motor endings were few in number and confined to muscle fibres immediately adjacent to the site of nerve-implantation. Six months after implantation some of the vagal motor endings showed signs of degeneration. As in the oesophagus, the reinnervating oesophagomotor fibres proved to be unmyelinated, sometimes forming a plexus-like intramuscular network before terminating at motor end-plates. Myelinated vagal nerve fibres were also observed running between the skeletal muscle fibres, but they did not establish any demonstrable form of neuromuscular contacts. It was concluded that, in the rat, the myelinated fibres of the oesophageal nerves are afferent, whereas the oesophagomotor fibres, although supplying striated muscle, are unmyelinated.     

Endomorphin-1 and -2, endogenous ligands for the μ-opioid receptor, inhibit striated and smooth muscle contraction in the rat oesophagus

Recently, morphological evidence for an interaction of autonomic nerve fibres and extrinsic motor innervation of the rat oesophagus has emerged. The aim of the present study was to investigate the possible influence of endogenous and exogenous opioids on rat oesophageal smooth and striated muscle function in vitro. The entire oesophagus (excluding the lower oesophageal sphincter) with both Nervi (Nn) vagi, including the Nn recurrentes, was dissected and placed in an organ bath (100 mL, 37 degrees) with oxygenated Krebs-Ringer buffer. Contractile activity was measured in a longitudinal direction with a force transducer. Both Nn vagi were placed on a bipolar platinum electrode 2 cm distant from the oesophagus. Vagal stimulation (VS), applied for 1 s (40 V, 0.5 ms, 20 Hz) resulted in a biphasic contractile response that was completely blocked by 10(-6) M tetrodotoxin. The first part consisted of a tetanic striated muscle contraction, as it was abolished by tubocurarine (10(-5) M, n=5) but unaffected by atropine (10(-6) M, n=3) or hexamethonium (10(-4) M, n=4). In contrast, the second part was completely inhibited by hexamethonium (10(-4) M) and atropine (10(-6)M), whereas tubocurarine (10(-5) M) showed no influence, indicating a stimulation of preganglionic nerve fibres supplying oesophageal smooth muscle (muscularis mucosae) via relays in myenteric ganglia. In order to characterize opioid influence on the oesophageal striated and smooth muscle contractility, the following experiments were carried out. 10(-6) M endomorphin-1 and -2, endogenous mu-opioid-receptor agonists, reduced the contractile response of the striated (EM-2, -25.1+/-5.3%; n=16), and the smooth muscle (EM-2, -81.9+/-3.3%; n=11). Both effects were reversible by the opioid receptor antagonist naloxone (10(-6) M) and therefore, mediated via opioid receptors. Neither SNC-80, an agonist on the delta-opioid-receptor, U-69593, an agonist on the kappa-opioid-receptor, nor nociceptin, an agonist at the ORL1 (opioid receptor-like) receptor, had a significant effect on the striated muscle contraction. In contrast to SNC-80, U-69593 and nociceptin inhibited smooth muscle contraction but this relaxation could not be antagonized by naloxone. None of the opioid receptor antagonists used had an effect on basal tonus or muscle contraction following VS. Our data provide evidence for an autonomic modulation of vagal motor innervation of the striated and smooth oesophageal muscle. Endomorphin-1 and -2, both selective mu-opioid receptor agonists, cause an inhibition of striated and smooth muscle response which is reversible by naloxone, an opioid receptor antagonist. The location of the mu-opioid receptor still has to be established.     

The origin and development of the vagal and spinal innervation of the external muscle of the mouse esophagus

Retrograde and anterograde tracing and immunohistochemical techniques were used to examine the origin of the extrinsic innervation, and the development of the vagal innervation to the mouse esophagus. Cholinergic nerve terminals were localised using an antiserum to the vesicular acetylcholine transporter and cholinergic cell bodies were localised using an antiserum to choline acetyltransferase. Cholinergic nerve terminals, which also contained calcitonin gene-related peptide, were present at the motor end plates in the external (striated) muscle of the esophagus. Following injection of Fast Blue into subdiaphragmatic or cervical levels of the esophagus, the only retrogradely-labelled cholinergic nerve cell bodies that also contained calcitonin gene-related peptide were found in the nucleus ambiguus. Neurons in the dorsal motor nucleus of the vagus, the nodose ganglia and dorsal root ganglia gave rise to a number of different types of nerve terminals within the myenteric plexus. Retrogradely-labelled neurons in the dorsal motor nucleus of vagus contained cholinergic markers only, nitric oxide synthase only or cholinergic markers plus nitric oxide synthase, retrogradely-labelled neurons in the dorsal root ganglia contained calcitonin gene-related peptide only, and a small number of retrogradely-labelled neurons in the nodose ganglia contained tyrosine hydroxylase. The development of the vagal innervation to the esophagus was examined following application of DiI to the vagus nerve of fixed mouse embryos. Anterogradely-labelled nerve fibres, which arose from both nodose ganglia and the medulla, were already present in the esophagus of embryonic day 12 (E12) mice. Some of the DiI-labelled vagal nerve fibres were present in among the smooth muscle cells of the external muscle layer prior to their transdifferentiation to striated muscle. We conclude that the neurons in the nucleus ambiguus that project to the esophagus differ from other extrinsic neurons in their chemistry as well as their targets within the esophagus. The development of the extrinsic innervation precedes the transdifferentiation of the external muscle to striated muscle, raising the possibility that, during development, smooth muscle of the esophagus is innervated transiently by vagal neurons.     

Immunohistochemical localization of voltage-activated calcium channels in the rat oesophagus

Voltage-activated calcium channels play an important role in the physiology of the enteric nervous system. To determine which types of voltage-activated calcium channels are present in the rat oesophagus, an immunohistochemical study was performed using specific antibodies for the alpha1 subunits of Cav2.1 (P/Q-type), Cav2.2 (N-type), Cav1.2 and Cav1.3 (L-type) calcium channels. All myenteric cell bodies showed Cav2.2 immunoreactivity, whereas labelling for this N-type channel was absent in nerve fibres. Cav1.2 immunoreactivity was found on nerve fibres in the myenteric plexus and on fibres innervating the striated muscle of the rat oesophagus, whereas no labelling was detected on neuronal somata. Immunoreactivity against Cav1.3 was not detected in the myenteric plexus or at the level of the striated muscle. Labelling for Cav2.1 was absent at the level of the myenteric plexus, but present in the striated muscle layer at the level of the motor endplates. Comparison with recent literature data from rat small intestine reveals region-specific distribution patterns of the various subtypes of voltage-activated calcium channels within the enteric nervous system. In addition, the present immunohistochemical data corroborate our physiological data (see accompanying paper), which indicate that the Cav2.2 (N-type) channel is the predominant channel involved in the generation of the calcium-dependent action potential evoked by intrasomatic depolarizing current pulses in all rat oesophageal myenteric neurones.     

Serotonin-immunoreactive neurons and mast cells in the mouse esophagus suggest involvement of serotonin in both motility control and neuroimmune interactions

Background Serotonin is a major transmitter in the gastrointestinal tract, but little is known about the serotonergic system in the esophagus. Methods The aim of this study was to use multilabel immunofluorescence to characterize serotonin‐positive nerve cell bodies and fibers and their relationship with other neuronal and non‐neuronal elements in the mouse esophagus. Antibodies against serotonin, vesicular acetylcholine transporter (VAChT), choline acetyltransferase (ChAT), protein gene product 9.5 (PGP 9.5), and α‐bungarotoxin (α‐BT), were used. Key Results Serotonin‐containing perikarya represented ～10% of all PGP 9.5‐positive myenteric neurons. Serotonin‐positive varicose nerve fibers were found in the lamina muscularis mucosae and present on ～13% of α‐BT–labeled motor endplates in addition to VAChT‐immunoreactive motor terminals. As ChAT‐positive neurons of the compact formation of the nucleus ambiguus were negative for serotonin, serotonin‐positive varicosities on motor endplates are presumed to be of enteric origin. On the other hand, cholinergic ambiguus neurons were densely supplied with serotonin‐positive varicosities. The tela submucosa and tunica adventitia contained large numbers of serotonin‐positive mast cells, a few of which were in close association with serotonin‐positive nerve fibers. Conclusions & Inferences The mouse esophagus is endowed with a rich serotonin‐positive intrinsic innervation, including enteric co‐innervation of striated muscles. Serotonin may modulate vagal motor innervation of esophageal‐striated muscles not only at the central level via projections of the raphe nuclei to the nucleus ambiguus but also at the peripheral level via enteric co‐innervation. In addition, mast cells represent a non‐neuronal source of serotonin, being involved in neuroimmune processes.     

Motor innervation of the striated oesophagus muscle: Part 1. Intramural distribution of the right and left vagus nerve in the rat oesophagus as revealed by the glycogen depletion technique

The muscularis propria of the rat oesophagus is entirely made up of striated muscle fibres. All fibres are of the same histochemical type, which is characterized by high activity of actomyosin ATPase, medium activity of oxidative enzymes and relatively strong reaction for phosphorylase. Prolonged stimulation (10 Hz, 30 min) of the vagus nerves causes depletion of the glycogen content of the oesophageal muscle fibres. This stimulation effect can be visualized by means of the PAS technique as well as by the histochemical reaction for phosphorylase.In 8 animals the right and in 8 animals the left vagus nerve were stimulated repetitively and the stimulated muscle fibres were identified in transverse sections of the oesophagi, stained for phosphorylase. The muscle fibres supplied by one vagus nerve are distributed all over the circumference of the oesophagus. In the upper third of the oesophagus stimulation of either vagus nerve depletes slightly less than 50% of the muscle fibres, whereas in the lower two-thirds the right vagus nerve seems to predominate to a certain degree.In 3 animals both vagus nerves were stimulated simultaneously. Bilateral stimulation produced a very extensive depletion. Only a few muscle fibres remained unaffected. Functional implications of the results, the question of polyneuronal innervation and the role of the myenteric plexus are discussed.     

Characterization of vagal input to the rat esophageal muscle

There is recent morphological evidence for an interaction of autonomic nerve fibers and extrinsic motor nerves of the rat esophagus. The aim of the present study was to investigate a possible functional role of this autonomic innervation of vagal motor fibers on rat esophageal smooth and striated muscle function in vitro. The entire esophagus with both Nn vagi, including the Nn recurrentes, was dissected and placed in an organ bath with oxygenated Krebs-Ringer buffer. Contractile activity was measured in longitudinal direction with a force transducer. Both Nn vagi were placed on a bipolar platinum electrode 2 cm apart from the esophagus. Vagal stimulation, applied for 1 s (40 V, 0.5 ms, 20 Hz) resulted in a biphasic contractile response, which was completely blocked by tetrodotoxin (10(-6) M). The first part consisted of a tetanic striated muscle contraction, which was abolished by tubocurarin (10(-5) M) but unaffected by atropine (10(-6) M) or hexamethonium (10(-4) M). In contrast, the second part was completely abolished by hexamethonium (10(-4) M) and atropine (10(-6) M), whereas tubocurarine (10(-5) M) showed no influence, suggesting a stimulation of preganglionic nerve fibers supplying esophageal smooth muscle (muscularis mucosae). In order to characterize possible autonomic transmitters of the ENS of the esophagus, the following experiments were carried out. The magnitude of the striated muscle response was unaffected by VIP (10(-7) M), 5-HT (10(-6) M) and galanin (10(-8) - 10(-7) M), whereas they caused an inhibition of the smooth muscle response (VIP: -53.8 +/- 4.2%; galanin 10(-8) M: - 18.5 +/- 2.2%; 10(-7) M: -40.4 +/- 2.9%; 5-HT: -78.2 +/- 2.1%). The inhibitory effects of VIP and galanin on smooth muscle were reversible by the antagonists VIP 10-28 and galanin 1-15. In the presence of the nitric oxide synthase (NOS) inhibitor L-NNA (10(-4) M), the smooth and striated muscle contraction were not significantly influenced. Exogenous application of the NO-donor DEA-NO (10(-4) M) reduced the smooth muscle contraction by -81.6 +/- 7.4%, but had no significant effect on the striated muscle contraction. Though immunohistochemical findings are highly suggestive of an nitrergic autonomic modulation of striated muscle contraction by enteric neurons, we could not demonstrate a NO-mediated action on striated muscle activity. Therefore, the physiological relevance of the immunohistochemical findings remain unclear.     

Galanin modulates vagally induced contractions in the mouse oesophagus

Abstract Nitrergic myenteric neurons co‐innervating motor endplates were previously shown to inhibit vagally induced contractions of striated muscle in the rodent oesophagus. Immunohistochemical demonstration of putative co‐transmitters, e.g. galanin, in enteric neurons prompted us to study a possible role of galanin in modulating vagally mediated contractions in an in vitro vagus nerve‐oesophagus preparation of the mouse. Galanin (1–16) (1–100 nmol L^?1), in the presence of the peptidase inhibitor, phenanthroline monohydrate, inhibited vagally induced contractions in a concentration‐dependent manner (control: 100%; galanin 1 nmol L^?1: 95.6 ± 1.6%; galanin 10 nmol L^?1: 57.3 ± 6.5%; galanin 100 nmol L^?1: 31.2 ± 8.1%, n = 5). The non‐selective galanin receptor antagonist, galantide (100 nmol L^?1), blocked the inhibitory effect of galanin (10 nmol L^?1) while the selective non‐galanin receptor 1 and galanin receptor 3 antagonists, M871 (1 μmol L^?1) and SNAP37889 (100 nmol L^?1), respectively, and the nitric oxide synthase inhibitor, NG‐nitro‐l ‐arginine methyl ester (l ‐NAME) (200 μmol L^?1), failed to affect this galanin‐induced response. Simultaneous application of galantide (100 nmol L^?1) and l ‐NAME (200 μmol L^?1) significantly reduced the inhibitory effect of capsaicin (30 μmol L^?1) on vagally induced contractions when compared with its effect in the presence of l ‐NAME alone or in combination with the selective galanin receptor 2 or 3 antagonists. An inhibitory effect of piperine on vagally induced contractions was reduced neither by galantide nor by l ‐NAME. Immunohistochemistry revealed galanin immunoreactive myenteric neurons and nerve fibres intermingling with cholinergic vagal terminals at motor endplates. These data suggest that galanin from co‐innervating enteric neurons co‐operates with nitric oxide in modulating vagally induced contractions in the mouse oesophagus.     

Motor innervation and acetylcholine receptor distribution of human extraocular muscle fibres

Motor innervation and acetylcholine receptor distribution of human extraocular muscle (EOM) fibres were investigated by using combined silver and acetylcholinesterase (AChE) staining method and [125I]alpha-bungarotoxin autoradiography. Three types of motor endplates were distinguished; (A) a large compact ending on a large-diameter fibre, (B) several endings regularly spaced on an intermediate-diameter fibre, each of which consisted of small stained particles and (C) numerous small endings scattered as a chain of beads on a small-diameter fibre. Type A fibre was singly innervated, while type B and C had multiterminal (possibly polyneuronal) innervation. These results indicate that the organization of human EOM endplates is quite different from that of limb muscle endplates and may provide some implications to understand the pathophysiology of the neuromuscular diseases, for example, myasthenia gravis.     

Abnormalities in the vagus nerve in canine acrylamide neuropathy

Dogs exposed to acrylamide develop a sensorimotor peripheral neuropathy and megaoesophagus. The presence of neuropathy was confirmed electrophysiologically and histologically. Hindlimb motor conduction velocity was reduced and there was a loss of large diameter myelinated fibres in the dorsal common digital nerve and the tibial nerve. The conduction velocity of vagal motor fibres innervating the thoracic oesophagus was not decreased; there was a reduction in the conduction velocity of the mixed nerve action potential of the vagus. Degenerating nerve fibres were observed in the vagus in the midthoracic region. The damage to vagal nerve fibres may be an important factor in the causation of megaoesophagus.     

Intramural nerves and interstitial cells revealed by the Champy-Maillet stain in the opossum esophagus

The 3 layers of smooth muscle of the opossum esophagus exhibit distinctly different and characteristic functions in response to nerve stimulation. These behaviors might be related to differences in patterns of innervation. Serial sections, stained with osmic acid and zinc iodide, were examined from all parts of the esophagus to describe in full the innervation of opossum esophageal smooth muscle. Linear beaded structures identified as terminal nerves were abundant in all 3 layers of smooth muscle. They were aligned with muscle bundles in bundles of 1-5 in mucosal muscle and up to about 10 in the longitudinal layer of muscularis propria. In the circular layer of muscularis propria they were less dense and more arborized and they crossed the muscle obliquely to also innervate bipolar interstitial cells; these cells resembled type III interstitial cells of Cajal, were specific for the circular muscle layer and were uniformly distributed throughout the thickness of that layer. Their distribution density in the circular muscle was slightly greater, 8777/cm2 in the most caudad 1.5 cm of the esophagus (which encompasses the sphincter) than in the 6 cm above that level. Terminal nerves also innervated submucosal glands, vessels and the epithelium. Ganglion cells and nerve bundles in the submucous and myenteric plexuses were variably stained. Ganglia of the latter plexus contained intraganglionic laminar endings as described previously in other species. In the striated muscle there were nerves terminating in motor end-plates. Other leafy stellate cells resembling interstitial cells of Cajal of other types were stained, as were oval interstitial cells.     

Vasoactive intestinal peptide and neuropeptide Y coexist in non-noradrenergic sympathetic neurons to guinea pig trachea.

Vasoactive intestinal peptide (VIP) has been suggested to be a mediator of vagal inhibition of airway tone and it has been assumed that VIP-containing nerve fibres in the airway arise from intrinsic ganglia. We have used a combination of double- and triple-labelling immunohistochemistry, retrograde axonal tracing, organotypic culture and nerve lesion studies, to identify the origin and distribution of neurons containing immunoreactivity (IR) to VIP in guinea pig airway smooth muscle. We also investigated whether immunoreactivity to other neuropeptides coexisted with VIP-IR within these neurons. We found that all VIP-IR nerve fibres in guinea pig tracheal smooth muscle also contained IR to neuropeptide Y (NPY) but not to tyrosine hydroxylase (TH), a marker for noradrenergic neurons. Both VIP-IR and NPY-IR were absent from nerve cell bodies in the tracheal plexus. After maintenance of isolated trachea in organotypic culture for 4 days, to allow degeneration of extrinsic nerve fibres, nerve fibres containing VIP-IR or NPY-IR were almost completely absent from tracheal smooth muscle. Of ganglia known to supply the trachea, coexistence of VIP-IR and NPY-IR was found only in cell bodies of the stellate ganglion. Retrograde tracing studies using the fluorescent tracer, DiI, confirmed that the stellate ganglion was the site of origin of neurons containing VIP-IR and NPY-IR supplying the airways. These neurons projected to the airways from the stellate ganglion both directly through the mediastinum, and via the cervical sympathetic trunk and vagus nerves. These results suggest that nerve fibres containing both VIP-IR and NPY-IR in the tracheal smooth muscle of the guinea pig are derived from non-noradrenergic cell bodies in the stellate ganglion. The absence of VIP-IR from vagal post-ganglionic neurons suggests that VIP cannot be a mediator of vagal inhibitory transmission in tracheal smooth muscle of this species.     

Innervation of avian latissimus dorsi muscles and axonal outgrowth pattern in the posterior latissimus dorsi motor nerve during embryonic development

The distribution of the innervation to the anterior latissimus dorsi (ALD) and posterior latissimus dorsi (PLD) muscles of the chicken are described on the day of hatching and 6 weeks later using electron microscopy. In the ALD muscle, there are 5,000 muscle fibres and 374,400 endplates supplied by about 169 skeletomotor axons; in the PLD muscle, there are 12,000 focally innervated muscle fibers supplied by about 20 skeletomotor axons. On the cell surface of the muscle fibers the mean total subsynaptic area contacted by each motor axon is comparable in the ALD and PLD muscles. The growth pattern of the axons in the PLD motor nerve was described from the ninth day in ovo up to 6 weeks after hatching. The axons arrive in the PLD muscle in two successive waves: first, the large somatic axons which are already present before the ninth day in ovo and second, the small autonomic axons which continue to accumulate until hatching. The total number of somatic axons decreases from the ninth day until the hatching day when it reaches its definitive value. This decrease takes place during a period when the numbers of myofibers and of endplates dramatically increase, and it coincides with the axonal segregation by the Schwann cells. The myelination of the axons starts on the 15th day in ovo and is essentially complete upon hatching. Despite the decreasing number of somatic axons in the PLD nerve, the decrease in number of nerve endings per PLD endplate and the increasing number of PLD endplates per PLD muscle, it was found that between the 16th day in ovo and 6 weeks after hatching the mean number of axonal branches per PLD motor axon does not decrease.     

Terminal sprouting of mouse motor nerves when the post-synaptic membrane degenerates

Injection of ecothiopate, 4-aminopyridine and caffeine into the mouse calf produced necrosis in the endplate region of approximately 40% of soleus muscle fibres. Within two days terminal sprouting, as seen by zinc iodide/osmium tetroxide staining, had occurred at nearly a quarter of such endplates, but not at neighbouring intact ones. Almost half of these sprouts were greater than 50 μm in length. Terminal sprouting at degenerating endplates was also seen in identically treated silver-stained gluteus maximus muscles. Muscle degeneration caused by mechanical damage produced similar effects. In transverse sections of the gluteus maximus preparation, the terminals could be found within the necrotic muscle fibres, having penetrated the synaptic basal lamina. It is concluded that motor nerve terminals have an intrinsic tendency to grow, and are normally prevented from doing so by their formation of synapses with muscle fibres. Destruction of this relationship alone can cause terminal sprouting.     

Development of the vagal innervation of the gut: steering the wandering nerve

Background The vagus nerve is the major neural connection between the gastrointestinal tract and the central nervous system. During fetal development, axons from the cell bodies of the nodose ganglia and the dorsal motor nucleus grow into the gut to find their enteric targets, providing the vagal sensory and motor innervations respectively. Vagal sensory and motor axons innervate selective targets, suggesting a role for guidance cues in the establishment of the normal pattern of enteric vagal innervation. Purpose This review explores known molecular mechanisms that guide vagal innervation in the gastrointestinal tract. Guidance and growth factors, such as netrin‐1 and its receptor, deleted in colorectal cancer, extracellular matrix molecules, such as laminin‐111, and members of the neurotrophin family of molecules, such as brain‐derived neurotrophic factor have been identified as mediating the guidance of vagal axons to the fetal mouse gut. In addition to increasing our understanding of the development of enteric innervation, studies of vagal development may also reveal clinically relevant insights into the underlying mechanisms of vago‐vagal communication with the gastrointestinal tract.     

Reinnervation of a striated muscle by vagal sensory axons

Fibres of the sterno-cleido-mastoid (s.c.m.) muscle normally innervated by effects of the accessory nerve have been reinnervated by afferent fibres of the vagus nerve after supranodose vagal-accessory nerve anastomoses or direct implantation of the vagus nerve into the s.c.m. in 58% of the rabbits, 60% of the cats and 75% of sheep in which experiments were performed. Afferents of the vagus growing from cell bodies of the nodose ganglion after severance of central connections can replace the efferent of motor supply to the muscle. Evidence that there was reinnervation of the s.c.m. muscle by vagal afferent fibres was provided from the observations that: (i) electrical stimulations of the anastomosed cervical vagus nerve elicited potentials in the s.c.m. muscle which were abolished by local anaesthesia or final section of the nerve proximal to the site of stimulation; (ii) discharges recorded as bursts of electromyographic potentials occurred during spontaneous movements of larynx, respiratory tract, oesophagus and stomach and on their mechanical or evoked stimulation; and (iii) horseradish peroxidase injected into the reinnervated s.c.m. muscle was detected in somata of ipsilateral nodose ganglia cells. The afferent fibres contributing to the reinnervation were confirmed to be cholinergic as transmission was blocked by gallamine and histochemical evidence obtained of cholinergic motor end-plates. Factors which may have limited the small extent of reinnervation--only one vagal sensory axon out of 600 is able to form functional connections--are discussed.     

Anatomical demonstration of vagal input to nicotinamide acetamide dinucleotide phosphate diaphorase-positive (nitrergic) neurons in rat fundic stomach

Recent pharmacological evidence suggests that the nonadrenergic, noncholinergic (NANC) vagal inhibitory input responsible for receptive relaxation of the fundic stomach is mediated by nitric oxide-synthesizing enteric neurons. To demonstrate anatomically such direct vagal inputs to neurochemically identified enteric neurons, we utilized the nicotinamide acetamide dinucleotide phosphate (NADPH)-diaphorase histochemical reaction in conjunction with selective anterograde labeling of vagal efferents or afferents. Approximately 30% of all myenteric neurons of the fundic myenteric plexus stained positive for NADPH diaphorase, and the principal recipient of axonal projections from NADPH diaphorase-positive neurons was the circular muscle layer. In a group of animals showing the most complete labeling of vagal efferent preganglionics with the carbocyanine dye DiA, quantitative analysis of the half of the ventral fundic wall closer to the greater curvature revealed that 46.8% ± 4.4% of all myenteric neurons received some degree of vagal contacts and that 30.5% ± 6.6% of such vagally contacted neurons were also NADPH diaphorase positive. In another group of rats with the most successful selective labeling of vagal afferents through DiI injections into the left nodose ganglion, analysis of select ganglia throughout the ventral fundic wall revealed that, of a total of 454 neurons with vagal afferent contacts, 34.8% ± 2.8% were NADPH diaphorase positive. These findings support the view that, in the fundic stomach, some vagal preganglionic efferents terminate on nitric oxide-synthesizing neurons that, in turn, project to and relax the external smooth muscle layers. Furthermore, vagal afferent endings also contact NADPH diaphorase-positive neurons, suggesting the possibility of local axon reflexes originating from smooth muscular in-series tension receptors and terminating on nitrergic neurons of the myenteric plexus. © 1995 Wiley-Liss, Inc.     

Distribution of NADPH diaphorase in intramural plexuses of cat and opossum esophagus

Nonadrenergic noncholinergic (NANC) nerves, releasing nitric oxide (NO), regulate peristalsis in esophageal smooth muscle. The NADPH diaphorase reaction identifies nitric oxide synthase (NOS). NO-synthesizing (NANC) nerves should be more abundant in esophageal smooth muscle than in striated muscle. We used NADPH diaphorase histochemistry in whole-mounts of the esophagus of cat (predominantly striated muscle) and opossum (predominantly smooth muscle) to compare striated and smooth muscle regions. In the opossum myenteric plexus, 90% of nerve cells were stained, with two populations, dark-staining and light-staining cells. Dark cells constituted 35–51% of stained cells with no difference between striated muscle and smooth muscle regions. In the cat myenteric plexus, 50% of cells were unstained in the striated muscle segment, but only 10% in the smooth muscle segment. Stained cells constituted two populations, dark cells and light cells, dark cells making up 48–60% of stained cells in both striated and smooth muscle segments, with no difference between segments. Both dark cells and light cells lay in clusters in opossum, but not in cat. Dark cells showed no selectivity for large or small ganglia of myenteric plexus in either region in opossum. Many more NADPH diaphorase positive nerve fibers lay in the circular smooth muscle layer than in the other layers. They were not found in striated muscle. Interstitial cells of Cajal in the circular layer of smooth muscle were also positive. In the submucosal plexus of opossum, all cells were stained, 10% being dark cells in both striated and smooth muscle segments. The cat has almost no nerve cells in the submucosal plexus. The similarity of the myenteric plexus in striated muscle and smooth muscle regions of the esophagus suggests that NADPH diaphorase positive nerves have other functions besides regulation of smooth muscle. Also, a positive reaction may not dictate that NO is the sole transmitter released.     

Calcitonin gene-related peptide-like immunoreactivity, in botulinum toxin-paralysed rat muscles

Changes in calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) at the motor endplates of botulinum toxin-paralysed rat muscles were investigated using immunohistochemistry. One day following toxin injection, a dramatic increase in CGRP-LI was detected at the motor endplates and within preterminal axons of the soleus and gastrocnemius muscles. The upregulation of CGRP-LI persisted throughout the period during which muscle fibres were paralysed and new neuromuscular junctions were being formed by the growing sprouts. Decline of CGRP-LI at the motor endplates coincided with clinical recovery. Both up- and down-regulation of CGRP-LI took place earlier in the soleus than in the gastrocnemius muscle. Up-regulation of CGRP-LI was also detected in a subpopulation of motor axons in the sciatic nerves and in the spinal motor neurons innervating the paralysed muscles. These results indicate that levels of CGRP are regulated, at least partly, by changes in the target innervation. They also suggest an important role for CGRP in the regenerative processes following muscle paralysis.