Development of neuromuscular junctions in the mouse esophagus: focus on establishment and reduction of enteric co-innervation

The development of vagal and enteric innervation of esophageal motor endplates was examined in perinatal and adult BALB/c and NMRI mice using immunocytochemistry and confocal laser scanning microscopy. Nicotinic acetylcholine receptors were demonstrated with fluorochrome-tagged alpha-bungarotoxin, vagal motor terminals with antisera against vesicular acetylcholine transporter and calcitonin gene-related peptide, and enteric nerve terminals with antisera against neuronal nitric oxide synthase, vasoactive intestinal peptide and galanin. Results demonstrated that enteric and vagal innervations of striated esophageal muscle fibers develop in close spatiotemporal relationship, but with different courses. Connections between VAChT-positive vagal nerve terminals and growing acetylcholine receptor clusters were established from E17 to reach 100% motor endplate innervation at P14 and were maintained throughout adult life. CGRP immunoreactivity developed with a delay of several days after the appearance of VAChT in vagal terminals. From P14 to adulthood CGRP was colocalized with VAChT in almost all motor endplates. In contrast, enteric co-innervation rates increased from E17 to a maximum of 70-80% at P4, while their incidence at motor endplates progressively declined over the following 5 months to lower levels maintained throughout adulthood. Whereas adult enteric co-innervation rates in BALB/c and NMRI mice differed significantly (approximately 30% versus approximately 10%, respectively), their increase and reduction, respectively, during development showed an identical time course. These results suggest a well-ordered sequence of attraction of enteric nerve fibers to, and removal from motor endplates in the developing mouse esophagus. Thus, enteric co-innervation may subserve a functional role in the development and control of perinatal striated esophageal muscle rather than representing an unspecific "hangover" from the smooth muscle past of this organ.     

Neurotrophin and Neurotrophin Receptors in Vascular Smooth Muscle Cells: Regulation of Expression in Response to Injury

The neurotrophins, a family of related polypeptide growth factors including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin (NT)-3 and NT-4/5 promote the survival and differentiation of distinctive sets of embryonic neurons. Here we define a new functional role for neurotrophins, as autocrine or local paracrine mediators of vascular smooth muscle cell migration. We have identified neurotrophins, and their cognate receptors, the trk tyrosine kinases, in human and rat vascular smooth muscle cells in vivo. In vitro, cultured human smooth muscle cells express BDNF; NT-3; and trk A, B, and C Similarly, rat smooth muscle cells expressed all three trk receptors as well as all four neurotrophins. Moreover, NGF induces cultured human smooth muscle cell migration at subnanomolar concentrations. In the rat aortic balloon deendothelialization model of vascular injury, the expression of NGF, BDNF, and their receptors trk A and trk B increased dramatically in the area of injury within 3 days and persisted during the formation of the neointima. In human coronary atherosclerotic lesions, BDNF, NT-3, and NT-4/5, and the trk B and trk C receptors could be demonstrated in smooth muscle cells. These findings suggest that neurotrophins play an important role in regulating the response of vascular smooth muscle cells to injury.     

Muscarinic M(2) acetylcholine receptor distribution in the guinea-pig gastrointestinal tract

In the enteric nervous system, acetylcholine is the most common neurotransmitter to induce gastrointestinal smooth muscle contractions. Cholinergic signaling is mediated by muscarinic acetylcholine receptors on the surface of smooth muscle cells. Five different muscarinic receptor subtypes (M(1)-M(5)) have been identified and characterized, all of which belong to the superfamily of the G-protein-coupled receptor. The muscarinic M(2) acetylcholine receptor is the major muscarinic receptor subtype expressed by smooth muscle tissues in the gastrointestinal tract, where it is coexpressed with a smaller population of M(3) receptor. In this study, we examined the immunohistochemical distribution of the M(2) receptor using a specific antibody in the guinea-pig gastrointestinal tract. M(2) receptor-like immunoreactivity was mainly observed as associated with smooth muscle cells in the gastrointestinal tract. M(2) receptor-like immunoreactivity in smooth muscle cells was distributed throughout the cell membrane associated with caveolae. In the proximal colon, M(2) receptor-like immunoreactivity in the smooth muscle cells was weak. In the small intestine, interstitial cells of Cajal that possessed neurokinin 1 receptor-like immunoreactivity had intense M(2) receptor-like immunoreactivity. In the proximal colon, intramuscular and myenteric interstitial cells of Cajal exhibited M(2) receptor-like immunoreactivity. These findings indicate that, in the gastrointestinal musculature, M(2) receptors are distributed both in the smooth muscle cells and interstitial cells of Cajal, suggesting that the M(2) receptor elicits smooth muscle cell contraction and the interstitial cells of Cajal are the sites of innervation by enteric cholinergic neurons.     

Expression of neurotrophins and their receptors tropomyosin-related kinases (Trk) under tension-stress during distraction osteogenesis

The localization and expression of neurotrophins and their receptors during distraction osteogenesis was investigated in 72 male rat femurs (11 weeks old) to further clarify the concurrence of cellular and molecular events of new bone formation. After osteotomy, a 7-day lag phase was followed by distraction at the rate of 0.25 mm/12 h for 21 days (distraction phase), and a 7-day consolidation phase. The localization of neurotrophins (NGF, BDNF and NT-3) and their receptors tropomyosinrelated kinases (TRKA, TRKB and TRKC) by immunostaining showed positive staining in bone forming cells in each stage, although the presence and staining intensity varied by cell type and phase. The expressions of NGF, BDNF and NT-3 by real-time polymerase chain reaction (real-time PCR) showed that the peak of the mRNA expression of NGF occurred 10 days after distraction. NT-3 increased during bone extension, but decreased when distraction stopped. In contrast, BDNF continued to increase gradually throughout the distraction and consolidation phases. These findings suggest that neurotrophins and their receptors may play different roles in endochondral and intramembranous ossification in distraction osteogenesis. The tension stress caused by distraction may stimulate the expression of neurotrophins and their receptors, and promote osteogenesis.     

Neurotrophins and neurotrophin receptors in some neural crest-derived tumours (ganglioneuroma, phaeochromocytoma and paraganglioma)

AIM: This study analyses the occurrence and distribution of neurotrophins and their receptors in some types of tumours of neural-crest derived cells. METHODS AND RESULTS: Light microscopy immunohistochemistry associated with quantitative image analysis was used to study the expression of neurotrophins (nerve growth factor, brain-derived neurotrophic factor and neurotrophin (NT)-3) and their cognate receptors (p75(LNGFR), TrkA, TrkB and TrkC) in histologically defined ganglioneuroma, phaeochromocytoma and paraganglioma. The material was fixed in 10% formaldehyde, paraffin-embedded and processed for indirect peroxidase immunohistochemistry using a battery of poly- and monoclonal antibodies to detect neurotrophins and their receptors, as well as some neuronal, endocrine and glial cell markers. A subpopulation of cells in phaeochromocytomas and ganglioneuromas expressed NT-3, but not other neurotrophins, while in paragangliomas no neurotrophins were detected. Regarding neurotrophin receptors, all tumours lacked p75(LNGFR), except for the ganglionic part of a case of mixed phaeochromocytoma, whereas they displayed TrkA (two of two ganglioneuromas, six of nine phaeochomocytomas and three of four paragangliomas). Furthermore, TrkC was regularly detected in a neuronal subpopulation in ganglioneuroma. Interestingly, the percentage of neurones expressing TrkA and TrkC was increased with respect to normal tissues in ganglioneuromas, as well as the percentage of the area occupied by TrkA-immunoreactive cells in the phaeochromocytomas. CONCLUSION: The pattern of expression of neurotrophins and neurotrophin receptors in the analysed tumours basically matches that of sympathetic neurones, adrenal chromaffin cells and paraganglionic cells, and suggests responsiveness of these cells to neurotrophins. Nevertheless, the function of TrkA and TrkC in regulating the biology of these tumours, if any, remains to be elucidated.     

Distribution of BDNF, NT-3, trkB and trkC in the developing retino-tectal system of the pigeon (Columba livia)

The distribution of the neurotrophins BDNF and NT-3 as well as their corresponding high-affinity receptors trkB and trkC was characterized by immunohistochemistry in the developing retino-tectal system of the pigeon. These neurotrophins are known to be important for survival and development of neuronal tissues, but also for activity-dependent neuronal plasticity. In pigeons visual asymmetry is established at the morphological and behavioral level due to a natural asymmetrical light input before hatch, which is followed by a posthatch period of consolidation with unbiased light stimulation. Since the retino-tectal system is the crucial entity of these events, we studied the retinal and the tectal distribution of these neurotrophins and their receptors during retino-tectal formation, to analyze the developmental sequences to which these neurotrophins are tuned. Here we demonstrate that in altricial pigeons no retinal immunolabeling of BDNF, NT-3 or their receptors could be detected before hatch, although a prominent tectal labeling pattern throughout most layers was evident. After hatch, both neurotrophins and their receptors showed a dramatic increase of retinal and tectal distribution. While the tectal and retinal protein synthesis of NT-3 vanished after 2 weeks, that of BDNF could still be revealed in adults. Therefore, the establishment of the retino-tectal system does not seem to depend on these neurotrophins before hatch, although they are probably utilized to shape the intratectal wiring pattern. In contrast, BDNF and NT-3 could play a prominent role in posthatch retino-tectal plasticity, as the consolidation of tectal asymmetries requires posthatch modifications of tectal circuits and proceeds within the first two posthatching weeks. These data are comparable with the distribution of neurotrophins in the retino-tectal system of chicks, although the onset of neurotrophin synthesis seems to be earlier in precocial chicks.     

Distribution of BDNF, NT-3, trkB and trkC in the developing retino-tectal system of the pigeon (Columba livia)

The distribution of the neurotrophins BDNF and NT-3 as well as their corresponding high-affinity receptors trkB and trkC was characterized by immunohistochemistry in the developing retino-tectal system of the pigeon. These neurotrophins are known to be important for survival and development of neuronal tissues, but also for activity-dependent neuronal plasticity. In pigeons visual asymmetry is established at the morphological and behavioral level due to a natural asymmetrical light input before hatch, which is followed by a posthatch period of consolidation with unbiased light stimulation. Since the retino-tectal system is the crucial entity of these events, we studied the retinal and the tectal distribution of these neurotrophins and their receptors during retino-tectal formation, to analyze the developmental sequences to which these neurotrophins are tuned. Here we demonstrate that in altricial pigeons no retinal immunolabeling of BDNF, NT-3 or their receptors could be detected before hatch, although a prominent tectal labeling pattern throughout most layers was evident. After hatch, both neurotrophins and their receptors showed a dramatic increase of retinal and tectal distribution. While the tectal and retinal protein synthesis of NT-3 vanished after 2 weeks, that of BDNF could still be revealed in adults. Therefore, the establishment of the retino-tectal system does not seem to depend on these neurotrophins before hatch, although they are probably utilized to shape the intratectal wiring pattern. In contrast, BDNF and NT-3 could play a prominent role in posthatch retino-tectal plasticity, as the consolidation of tectal asymmetries requires posthatch modifications of tectal circuits and proceeds within the first two posthatching weeks. These data are comparable with the distribution of neurotrophins in the retino-tectal system of chicks, although the onset of neurotrophin synthesis seems to be earlier in precocial chicks. Accepted: 1 March 2001     

The myogenic factor Myf5 supports efficient skeletal muscle regeneration by enabling transient myoblast amplification

The myogenic factor Myf5 defines the onset of myogenesis in mammals during development. Mice lacking both Myf5 and MyoD fail to form myoblasts and are characterized by a complete absence of skeletal muscle at birth. To investigate the function of Myf5 in adult skeletal muscle, we generated Myf5 and mdx compound mutants, which are characterized by constant regeneration. Double mutant mice show an increase of dystrophic changes in the musculature, although these mice were viable and the degree of myopathy was modest. Myf5 mutant muscles show a small decrease in the number of muscle satellite cells, which was within the range of physiological variations. We also observed a significant delay in the regeneration of Myf5 deficient skeletal muscles after injury. Interestingly, Myf5 deficient skeletal muscles were able to even out this flaw during the course of regeneration, generating intact muscles 4 weeks after injury. Although we did not detect a striking reduction of MyoD positive activated myoblasts or of Myf5-LacZ positive cells in regenerating muscles, a clear decrease in the proliferation rate of satellite cell-derived myoblasts was apparent in satellite cell-derived cultures. The reduction of the proliferation rate of Myf5 mutant myoblasts was also reflected by a delayed transition from proliferation to differentiation, resulting in a reduced number of myotube nuclei after 6 and 7 days of culture. We reason that Myf5 supports efficient skeletal muscle regeneration by enabling transient myoblast amplification. Disclosure of potential conflicts of interest is found at the end of this article.     

Effects of neurotrophins on human bronchial smooth muscle cell migration and matrix metalloproteinase-9 secretion

The neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) have been found to be upregulated in inflammatory pulmonary diseases, including asthma. The functional role for the neurotrophins in the airways is still not known, but it has been proposed that neurotrophins induce airway hyperreactivity and tissue remodeling. Bronchial smooth muscle cells have been suggested to be involved in the remodeling process through their capacity to proliferate, migrate, and secrete inflammatory mediators and matrix metalloproteinases (MMPs). Therefore, we studied the effect of NGF, BDNF, and NT-3 on human bronchial smooth muscle cell (HBSMC) migration and MMP-2 and MMP-9 secretion. Immunocytochemistry studies showed that HBSMCs expressed the neurotrophin receptors TrkA, TrkB, and TrkC. BDNF, NT-3, and NGF increased MMP-9, but not MMP-2, secretion as shown by zymography. BDNF and NT-3, but not NGF, stimulated HBSMC migration as evaluated by Boyden chamber. Taken together, our data indicate that the neurotrophins may stimulate events important for airway remodeling.     

The expression of nestin delineates skeletal muscle differentiation in the developing rat esophagus

The muscularis externa of the developing rodent esophagus is initially composed of smooth muscle, and later replaced by skeletal muscle in a craniocaudal progression. There is growing evidence of distinct developmental origins for esophageal smooth and skeletal muscles. However, the identification of skeletal muscle progenitor cells is controversial, and the detailed cell lineage of their descendants remains elusive. In the current study, we carried out multiple labeling immunofluorescence microscopy of nestin and muscle type-specific markers to characterize the dynamic process of rat esophageal myogenesis. The results showed that nestin was transiently expressed in immature esophageal smooth muscle cells in early developing stages. After nestin was downregulated in smooth muscle cells, a distinct population of nestin-positive cells emerged as skeletal muscle precursors. They were mitotically active, and subsequently co-expressed MyoD, followed by the embryonic and later the fast type of skeletal muscle myosin heavy chain. Thus, the cell lineage of esophageal skeletal muscle differentiation was established by an immunotyping approach, which revealed that skeletal myocytes arise from a distinct lineage rather than through transdifferentiation of smooth muscle cells during rat esophageal myogenesis.     

Presence of neurotrophic factors in skeletal muscle correlates with survival of spinal cord motor neurons.

To determine which combination of skeletal muscle-derived neurotrophic factors may be important for the survival of specific subpopulations of developing spinal cord motor neurons, we used Myf5 and MyoD (myogenic regulatory factors) knockouts, containing differentially committed myogenic precursor cells (MPCc) and immunohistochemistry against several muscle-secreted neurotrophic factors. At the peak of motor neuron cell death, skeletal muscle development is delayed in the back and body wall muscles of Myf5-/- embryos and in the limb muscles of MyoD-/- embryos. We hypothesized that, if the skeletal muscle was indeed an important source of survival factors for motor neurons, the back, the abdominal wall, and the forelimb MPCs of Myf5-/- or MyoD-/- embryos should produce at least some neurotrophic factors necessary for the survival of motor neurons. In this report, we demonstrate that (1) different MPCs lacking Myf5, MyoD, or Myf5/MyoD have different capabilities in providing factors potentially required for the survival of motor neurons and intramuscular nerve branching, (2) MPCs in double-mutant embryos do not contain neurotrophic factors in the absence of myogenic specification, and (3) different subpopulations of MPCs contain different combinations of neurotrophic factors potentially required for the survival of the specific subpopulations of innervating motor neurons.     

5-Hydroxytryptamine participation in the vagal inhibitory innervation of the stomach

1. Intraluminal pressure was recorded from the isolated guinea-pig and mouse stomach with the vagus and sympathetic nerves attached.2. The response to vagal stimulation, which consists of an excitatory and an inhibitory component, resembled the response to 5-hydroxytryptamine (5-HT), which has no direct action on the muscle but acts on intrinsic excitatory and inhibitory ganglia.3. In the presence of hyoscine, the effect of vagal stimulation, of nicotinic compounds and of 5-HT were all purely relaxant. Competitive block of ganglionic receptors for acetylcholine reduced the vagal relaxation without antagonizing 5-HT. Specific desensitization of ganglionic receptors for 5-HT reduced the vagal relaxation without antagonizing nicotinic compounds.4. During the early phase of the blocking action of nicotine, responses to vagal stimulation and to 5-HT were both abolished. As the non-specific antagonism changed to the later phase of specific antagonism to acetylcholine, the inhibitory (but not the excitatory) component of the vagal response recovered partially, in parallel with the recovery of the relaxant effect of 5-HT.5. The vagal inhibitory effect was completely abolished only when competitive block of acetylcholine receptors was combined with desensitization of 5-HT receptors.6. Stimulation of the mouse stomach (after asphyxiation of the mucosa and exclusion of the luminal content) in the presence of hyoscine caused the release of 5-HT; this release was blocked by tetrodotoxin.7. The results, together with previous observations that 5-HT is contained within preganglionic nerve fibres in the myenteric plexus, are consistent with the hypothesis that 5-HT, with acetylcholine, may be a neurotransmitter in the vagal inhibitory innervation of the stomach.     

Enteric co-innervation of striated muscle fibers in the esophagus: just a "hangover"?

Striated muscle of the esophagus was until recently considered to consist of "classical" skeletal muscle fibers innervated by cholinergic vagal motoneurons. The recently described co-innervation originating from enteric neurons expressing nNOS, VIP, NPY, and galanin added a new dimension of complexity. The aim of this study was to summarize current knowledge about, and to get further hints as to the possible function of enteric co-innervation of striated esophageal muscle fibers. Aldehyde fixed rat esophagi were processed for immunocytochemistry for CGRP or VAChT (to demonstrate vagal motor terminals), nNOS/NADPH-d, VIP, NPY, and galanin (to demonstrate enteric terminals), met-enkephalin, mu opiate receptor, muscarinic receptors m1-3, soluble guanylyl cyclase, and cGMP dependent kinase type I and II. Motor endplates were visualized using fluorochrome tagged alpha-bungarotoxin to label nicotinic receptors, or with AChE histochemistry. Besides light and confocal laser scanning microscopy, immuno electron microscopy was also employed. Up to 80% of motor endplates were co-innervated. In addition to nNOS, VIP, NPY, and galanin, many enteric terminals in esophageal motor endplates expressed met-enkephalin. Some appeared to stain for the muscarinic m(2) receptor. There was prominent immunostaining for the micro opioid receptor in the sarcolemma at both junctional and extrajunctional sites. Immunostaining for soluble guanylyl cyclase was prominent immediately beneath the clusters of nicotinic receptors. Enteric varicosities and vagal terminals intermingled in motor endplates often without intervening teloglial processes. During ontogeny, initially high co-innervation rates were reduced to adult levels in a cranio-caudally progressing manner. We conclude that, in addition to a possible nitrergic, VIP-, NPY-, and galaninergic modulation of neuromuscular transmission by enteric neurons, opioidergic mechanisms could play a role. On the other hand, cholinergic influence on enteric neurons may be exerted also by the nucleus ambiguus via motor endplates, in addition to the input from the dorsal motor nucleus. The observations that enteric nerve fibers contact striated muscle fibers at specialized sites, i.e., motor endplates, and that these contacts appear in an ordered cranio-caudal sequence after cholinergic motor endplates have been established point to a specific function in neuronal control of esophageal muscle rather than to be an unspecific "hangover" from the smooth muscle past of this organ.     

The neural regulation of the mammalian esophageal motility and its implication for esophageal diseases

In contrast to the tunica muscularis of the stomach, small intestine and large intestine, the external muscle layer of the mammalian esophagus contains not only smooth muscle but also striated muscle fibers. Although the swallowing pattern generator initiates the peristaltic movement via vagal preganglionic neurons that project to the myenteric ganglia in the smooth muscle esophagus, the progressing front of contraction is organized by a local reflex circuit composed by intrinsic neurons similarly to other gastrointestinal tracts. On the other hand, the peristalsis of the striated muscle esophagus is both initiated and organized by the swallowing pattern generator via vagal motor neurons that directly innervate the muscle fibers. The presence of a distinct ganglionated myenteric plexus in the striated muscle portion of the esophagus had been enigmatic and neglected in terms of peristaltic control for a long time. Recently, the regulatory roles of intrinsic neurons in the esophageal striated muscle have been clarified. It was reported that esophageal striated muscle receives dual innervation from both vagal motor fibers originating in the brainstem and varicose intrinsic nerve fibers originating in the myenteric plexus, which is called ‘enteric co-innervation’ of esophageal motor endplates. Moreover, a putative local neural reflex pathway that can control the motility of the striated muscle was identified in the rodent esophagus. This reflex circuit consists of primary afferent neurons and myenteric neurons, which can modulate the release of neurotransmitters from vagal motor neurons in the striated muscle esophagus. The pathogenesis of some esophageal disorders such as achalasia and gastroesophageal reflux disease might be involved in dysfunction of the neural networks including alterations of the myenteric neurons. These evidences indicate the physiological and pathological significance of intrinsic nervous system in the regulation of the esophageal motility. In addition, it is assumed that the components of intrinsic neurons might be therapeutic targets for several esophageal diseases.     

Myogenin (Myf4) upregulation in trans-differentiating fibroblasts from a congenital myopathy with arrest of myogenesis and defects of myotube formation

Congenital myopathies often have an unclear aetiology. Here, we studied a novel case of a severe congenital myopathy with a failure of myotube formation. Polymerase chain reaction-based analysis was performed to characterize the expression patterns of the Desmin, p21, p57, and muscle regulatory factors (MRFs) MyoD, Myf4, Myf5 and Myf6 in differentiating skeletal muscle cells (SkMCs), normal human fibroblasts and patient-derived fibroblasts during trans-differentiation. The temporal and spatial pattern of MRFs was further characterized by immunocyto- and immunohistochemical stainings. In differentiating SkMCs, each MRF showed a characteristic expression pattern. Normal trans-differentiating fibroblasts formed myotubes and expressed all of the MRFs, which were detected. Interestingly, the patient's fibroblasts also showed some fusion events during trans-differentiation with a comparable expression profile for the MRFs, particularly, with increased expression of Myf4 and p21. Immunohistochemical analysis of normal and patient-derived skeletal musculature revealed that Myf4, which is downregulated during normal fetal development, was still present in patient-derived skeletal head muscle, which was also positive for Desmin and sarcomeric actin. The abnormal upregulation of Myf4 and p21 in the patient who suffered from a severe congenital myopathy suggests that the regulation of Myf4 and p21 gene expression during myogenesis might be of interest for further studies.     

Neurotrophins and their receptors in early development of the mammalian nervous system

Neurotrophins belonging to the class of growth factors and including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4/5 (NT-4/5) are widely recognized as essential factors in the developing central nervous system (CNS). Neurotrophins are synthesized as precursor forms (proneurotrophins). Mature forms of neurotrophins exert their effect by binding to specific tyrosine kinases receptors (TrkA, TrkB and TrkC) as well as via the p75 receptor, a member of the tumor necrosis factor receptor superfamily while proneurotrophins interact with the receptor p75 or co-receptor complex of p75 and sortilin, that is a Vps10p domain-containing transmembrane protein. Expression of neurotrophins corresponds with the onset of neurogenesis in developing mammalian species. BDNF is low in early embryonic stages of development, while NT-3 highly expresses in the developing CNS. Expression of neurotrophins receptors mainly overlaps at early development. Data concerning early distribution of neurotrophins and their receptors in the nervous system and results in mice with targeted disruptions of neurotrophin or receptor genes show that neurotrophins and their receptors play distinct roles in control and regulation of the most crucial developmental processes such as proliferation, migration, differentiation, survival, apoptosis and synaptic plasticity.