Effect of schistosomiasis on CX3CR1-expressing mononuclear phagocytes in the ileum and mesenteric lymph nodes of the mouse.

BACKGROUND: Intestinal dendritic cells (DCs) maintain immune homeostasis, only initiating an active immune response against invading pathogens. However, little information is available on the reaction of mononuclear phagocytes (MNP) to intestinal trematode infection, a reaction equally important in helminth-based therapies. The CD11c(+)  CX3CR1(+)  F4/80(-) DCs in the ileal lamina propria (LP) of the mouse were proven to migrate to the mesenteric lymph nodes (MLNs). We analyzed all MNP subsets present in the mouse LP and MLNs, under steady-state conditions and during acute Schistosoma mansoni-induced inflammation. Furthermore, we studied the uptake of schistosomal antigens by MNP in vivo in the LP and MLNs. METHODS: Using a combination of immunohistochemistry and multiparametric flow cytometry, we investigated distributional changes of the MNP during acute intestinal schistosomiasis. Next, S. mansoni-derived products, i.e., S. mansoni soluble worm proteins (SmSWP) and S. mansoni soluble egg antigens (SmSEA) were intraperitoneally injected into CX3CR1(+/) (GFP) C57BL/6 mice and antigen uptake was analyzed using confocal microscopy. KEY RESULTS: The CD11c(+)  CX3CR1(+)  F4/80(-) DCs significantly increased during intestinal schistosomiasis in the LP and MLNs. Only CX3CR1-expressing DC and MФ subsets, but not other LP DCs, are involved in both SmSWP and SmSEA antigen uptake and processing. CONCLUSIONS & INFERENCES: The significant upregulation of CD11c(+)  CX3CR1(+)  F4/80(-) DCs during intestinal schistosomiasis and the restriction of phagocytosis of parasitic antigens to CX3CR1-expresssing MNP indicate a crucial role for this immune cell niche in response to trematodiasis. These findings add insight into the functional specialization of LP immune cells and add to the understanding of cellular mechanisms behind helminth-based therapies.

Chronic alcohol consumption induces an overproduction of NO by nNOS- and iNOS-expressing myenteric neurons in the murine small intestine.

BACKGROUND: There are indications that alterations in the nitric oxide (NO) system of relaxation mediate gastrointestinal motor disturbances induced by chronic alcohol consumption (CAC). As CAC is known to inhibit the motility of the mouse small intestine, we investigated in this model if CAC affects basal NO synthesis by myenteric neurons and which NOS isoforms are involved. METHODS: The instantaneous NO synthesis of individual neurons was optically measured in whole-mount preparations loaded with the NO synthesis indicator DAF-FM, and the expression of nNOS, iNOS and eNOS was determined by immunohistochemistry. KEY RESULTS: The DAF-FM recordings showed that CAC induced an increase in neuronal NO synthesis (absolute fluorescence: control 34±12; CAC 140±56; mean±SD; P<0.0004). Neurons of control mice expressed the nNOS (29±3% of total) and iNOS (28±1%) isoforms. eNOS expression was observed in <0.5% of the neurons. Chronic alcohol consumption caused an increase in the proportion of iNOS-expressing neurons (to 33±5%; P<0.01) and a decrease in nNOS-expressing neurons (to 22±3%; P<0.0001), without altering the proportion of NO-producing neurons (control 55±13%; CAC 56± 11%; P=0.82). CONCLUSIONS & INFERENCES: Chronic alcohol consumption induces a marked increase in NO synthesis by jejunal myenteric neurons, accompanied by an up-regulation of iNOS-expressing neurons and a downregulation of nNOS neurons. We conclude that the overproduction of NO may be a direct cause of gastrointestinal motility disturbances.

The influence of ileitis on the neurochemistry of the caudal mesenteric ganglion in the pig.

BACKGROUND: Some literature data suggest that there is a regulatory neuronal circuit between the small and the large bowel. To verify this hypothesis the present study investigated: (i) the distribution, chemical coding and routing of caudal mesenteric ganglion (CaMG) neurons participating in an intestinointestinal reflex pathway involving ileal descending neurons and viscerofugal colonic neurons and (ii) possible changes in the neuroarchitecture of this pathway evoked by chemically induced ileitis in juvenile pigs (n=16). METHODS: Combined retrograde tract tracing and transections of the intermesenteric or caudal colonic nerves were applied. In addition, double immunostainings was used to investigate the chemical coding of retrogradely labeled CaMG neurons and intraganglionic nerve terminals apposed to them, under normal and inflammatory conditions. KEY RESULTS: The majority of the ileum-projecting neurons were found in the caudal part of CaMG. Disruption of particular nerve pathways resulted in diminished number of retrogradely labeled neurons, ipsilateral to the side of manipulation. In normal pigs, ileum-projecting CaMG neurons stained for tyrosine hydroxylase, dopamine-ß-hydroxylase, neuropeptide Y (NPY), somatostatin and galanin (GAL). The number and chemical coding of the neurons in the inflamed animals were similar to those observed in the normal pigs. However, in the inflamed pigs, the number of NPY-, GAL- or substance P-positive nerve terminals supplying retrogradely labeled neurons was increased. CONCLUSIONS & INFERENCES: The present results suggest that inflammatory processes of the porcine ileum are able to induce changes in the intraganglionic architecture of a sympathetic ganglion located at discrete distance from the affected bowel segment.

Development of galanin-containing nerve fibres in rat tibia.

Galanin exerts tonic inhibition of nociceptive input to the central nervous system. Recently, this peptide was demonstrated in several neuronal and non-neuronal structures in bones and joints. In this study, the time of appearance and topographic localization of galanin-containing nerve fibres in bone were studied in rats from gestational day 16 (GD16) to postnatal day 21 (PD21). The tibia was chosen as a model of developing long bone and indirect immunofluorescence combined with confocal laser scanning microscopy was used to identify galanin-immunoreactive (GAL-IR) nerve fibres. The earliest, sparse GAL-IR fibres were observed on GD21 in the perichondrium of both epiphyses and in the periosteum of the diaphysis. From PD1 onwards, GAL-IR fibres were also seen in the bone marrow cavity and in the region of the inter-condylar eminence of the knee joint. Intramedullary GAL-IR fibres in proximal and distal metaphyses appeared around PD1. Some of them accompanied blood vessels, although free fibres were also seen. GAL-IR fibres located in the cartilage canals of both epiphyses were observed from PD7, in the secondary ossification centres from PD10 and in the bone marrow of both epiphyses from PD14. The time course and localization of galanin-containing nerve fibres resemble the development of substance P- and CGRP-expressing nerve fibres, thus suggesting their sensory origin.

Selective visualisation of neuroepithelial bodies in vibratome slices of living lung by 4-Di-2-ASP in various animal species.

Pulmonary neuroepithelial bodies (NEBs) are extensively innervated organoid groups of neuroendocrine cells that lie in the epithelium of intrapulmonary airways. Our present understanding of the morphology of NEBs is comprehensive, but direct physiological studies have so far been challenging because the extremely diffuse distribution of NEBs makes them inaccessible in vivo and because a reliable in vitro model is lacking. Our aim has been to optimise an in vitro method based on vibratome slices of living lungs, a model that includes NEBs, the surrounding tissues and at least part of their complex innervation. This in vitro model offers satisfactory access to pulmonary NEBs, provided that they can be differentiated from other tissue elements. The model was first optimised for living rat lung slices. Neutral red staining, reported to stain rabbit NEBs, proved unsuccessful in rat slices. On the other hand, the styryl pyridinium dye, 4-(4-diethylaminostyryl)-N-methylpyridinium iodide (4-Di-2-ASP), showed brightly fluorescent cell groups, reminiscent of NEBs, in the airway epithelium of living lung slices from rat. In addition, nerve fibres innervating the NEBs were labelled. The reliable and specific labelling of pulmonary NEBs by 4-Di-2-ASP was corroborated by immunostaining for protein gene-product 9.5. Live cell imaging and propidium iodide staining further established the acceptable viability of 4-Di-2-ASP-labelled NEB cells in lung slices, even over long periods. Importantly, the in vitro model and 4-Di-2-ASP staining procedure for pulmonary NEBs appeared to be equally reproducible in mouse, hamster and rabbit lungs. Diverse immunocytochemical procedures could be applied to the lung slices providing an opportunity to combine physiological and functional morphological studies. Such an integrated approach offers additional possibilities for elucidating the function(s) of pulmonary NEBs in health and disease.

Anatomical and neurochemical features of the extrinsic and intrinsic innervation of the striated muscle in the porcine esophagus: evidence for regional and species differences.

Studies of the intrinsic and extrinsic innervation patterns of esophageal motor endplates (MEPs) are mainly confined to small rodents. Therefore, an immunocytochemical, denervation and tracing study was conducted on the pig, an experimental model in which the distribution of the striated esophageal muscle portion more closely resembles the human situation. The purpose of this study was to analyze the origin and neurochemical content of the nerve fibers participating in the myoneural synapse. Fifteen 6-week-old domestic pigs were studied by immunohistochemistry combined with alpha-bungarotoxin labeling to define the co-innervation patterns of nitrergic and peptidergic nerve terminals in MEPs. Some animals were subjected to unilateral infra- or supranodose vagotomy to determine the origin of the nerve terminals in MEPs. Special attention was paid to the interregional differences in terms of co-innervation rates, and these findings were compared with literature data on small mammals. Double stainings revealed that most of the nNOS-immunoreactive (ir) terminals in MEPs co-stained for VIP, GAL and NPY, but not for PACAP and L-ENK. PACAP- and L-ENK-ir terminals were coarser than nNOS-ir terminals, and largely co-localized VAChT. High percentages of MEPs at the cervical level were contacted by PACAP- (approximately 94%) and L-ENK-ir (approximately 78%) terminals, but the proportion of both decreased in the rostrocaudal direction. Vagotomy significantly reduced their presence in MEPs at the thoracic and abdominal levels, while nNOS-ir terminals observed in approximately 30% of the MEPs were unaffected by vagotomy. Immunostainings on brainstem cryosections after retrograde tracing from the cervical esophagus showed that a large number of FB-positive cells in the nucleus ambiguus were PACAP-ir (approximately 72%). C-kit-positive interstitial cells of Cajal were seen adjacent to the striated muscle fibers, apparently without direct relationship to MEPs. Similar to mouse esophagus, intrinsic nitrergic fibers were found to run close to, or even spiral around, these interstitial cells, an association that might point to a role as specialized spindle proprioceptors. In conclusion, the cholinergic terminals-part of which coexpress PACAP and/or L-ENK-that innervate MEPs in the porcine esophagus have a vagal origin, whereas the nNOS/VIP/GAL/NPY-ir fibers co-innervating these MEPs are intrinsic in nature. The regional differences observed along the esophageal length pertain to the neurochemical content of the vagal motor innervation of the MEPs.

Neither axotomy nor target-tissue inflammation changes the NOS- or VIP-synthesis rate in distal bowel-projecting neurons of the porcine inferior mesenteric ganglion (IMG).

The present study was aimed at disclosing axotomy- and inflammation-induced changes in the chemical coding of retrogradely labelled distal bowel-projecting neurons in the porcine IMG. Particular attention was paid to the changes in the expression pattern of vasoactive intestinal polypeptide and nitric oxide synthase (as a marker of nitric oxide) in affected cells, as these substances are thought to play a crucial role in the regeneration of injured sympathetic neurons. However, while both pathological processes failed to induce an increase in the number of sympathetic bowel-projecting neurons exhibiting vasoactive intestinal polypeptide or nitric oxide synthase, axotomy, but not target-tissue inflammation, led to the upregulation in the expression pattern of galanin, pituitary adenylate cyclase-activating peptide and/or Leu5-enkephalin in the affected perikarya. On the other hand, axotomy resulted in a diminished density of vasoactive intestinal polypeptide-immunoreactive intraganglionic nerve fibres, whilst target-tissue inflammation evoked a distinct increase in the number of visible vasoactive intestinal polypeptide-immunoreactive terminals, especially in those regions where bowel-projecting neurons were located. Thus, the data obtained in the present study run counter to the results of the injury-related responses observed in neurons of the sympathetic chain ganglia, suggesting the existence of either species- or target tissue-dependent differences in the injury-induced responses of the affected sympathetic neurons.

Ca2+ involvement in the action potential generation of myenteric neurones in the rat oesophagus.

Intracellular recordings were used to study the physiological behaviour of rat oesophageal myenteric neurones, which are embedded in striated muscle. Injection of depolarizing pulses evoked action potentials with a clear 'shoulder' in all neurones. This shoulder disappeared under low Ca2+/high Mg2+ conditions. Tetrodotoxin (TTX; 1 micromol L-1) did not impede spike firing, whereas under combined TTX and low Ca2+/high Mg2+ conditions the action potentials were completely abolished, indicating that TTX- resistant action potentials are mediated by a Ca2+ current. Further experiments with omega-conotoxin GVIA (100 nmol L-1) revealed that these Ca2+ currents enter the cell via N-type voltage-activated Ca2+ channels (see also accompanying paper). Tetraethylammonium (10 mmol L-1) caused broadening of the action potentials, which probably resulted from prolonged Ca2+ influx due to blockade of the delayed rectifier K+ channel. Although Ca2+ appears to be involved in the spike generation of all rat oesophageal myenteric neurones, only a minority (14%) shows a slow afterhyperpolarization. Thus, no strict correlation exists between the presence of a shoulder and a slow afterhyperpolarization. Furthermore, morphological identification of 25 of the impaled neurones revealed that there was no strict correlation between morphology and electrophysiological behaviour. Consequently, rat oesophageal myenteric neurones appear to differ in several aspects from myenteric neurones in smooth muscle regions of the gastrointestinal tract.

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.

Functional implications of extensive new data on the innervation of pulmonary neuroepithelial bodies.

Pulmonary neuroepithelial bodies (NEBs) are innervated organoid groups of neuroendocrine cells, present in the epithelial lining of intrapulmonary airways of man and all air-breathing vertebrates studied so far. NEBs receive a vagal nodose sensory innervation that is considered by other authors as their main, if not their only innervation, although apparently not needed for the normal development and maintenance of NEBs. In the present study of NEBs in the developing rat lung (gestational day 16 - adult), immunoreactivity (IR) for calcitonin gene-related peptide (CGRP; a marker for NEBs), protein gene product 9.5 (PGP9.5; a marker for NEBs and neuronal elements), calbindin D28k (CB; a calcium binding protein), and for the growth-associated protein 43 (GAP-43; a marker for growing or remodelling nerve fibers) was combined with vagal denervation experiments. GAP-43 and CB IR revealed that the vagal sensory innervation of airways precedes the prenatal development of NEBs by several generations of branching. Unlike several other nerve fiber populations innervating NEBs, the vagal sensory component apparently does not express GAP-43 IR in mature lungs. GAP-43 labelling, however, did reveal newly ingrowing intraepithelial vagal sensory fibers, specifically reinnervating NEBs in adult rats 2 weeks following a cervical vagal crush. In conclusion, details of the innervation of pulmonary NEBs were disclosed that had been invisible so far, shedding new light on its complexity and probable involvement in the normal development and functional maintenance of NEBs throughout life.

Outer submucous plexus: an intrinsic nerve network involved in both secretory and motility processes in the intestine of large mammals and humans.

The architecture of the enteric nerve networks in the gastrointestinal tract appears to be more complex in large mammals, including humans, than in small laboratory animals. At least two distinct ganglionic nerve plexuses could be identified in the submucous layer in the digestive tract of large mammals. While functionally and morphologically similar neuron populations are found in the intestinal wall of both small and large mammals, significant differences in their topographical organization and neurochemical features may be present. This short review clearly illustrates that the close and exclusive association, which has been assumed so far between the efferent pathways of the submucous plexus and regulation of intestinal secretion/absorption on the one hand and between the myenteric plexus and regulation of intestinal motility on the other hand, cannot be interpreted that strictly. An attempt has been made to give a briefoverview of the current status of the identification of distinct functional enteric neuronal classes in the gastrointestinal tract of large mammals using the pig and human intestine as references, and to compare these data with the more extensive information gathered from the guinea-pig intestine.

Development of the innervation of long bones: expression of the growth-associated protein 43.

It has been known from clinical and experimental observations that the peripheral nervous system is involved in the development of long bones. Expression of growth-associated protein 43 (GAP-43/B-50) was found in axonal growth cones during embryonic and postnatal ontogeny as well as in regenerating axons after nerve injury. The aim of the present study was to examine the occurrence of growing nerve fibers in rat tibia from gestational day 16 (GD 16) to postnatal day 28 (PD28). An indirect immunoenzymatic reaction using antibodies raised against GAP-43 was applied to detect outgrowing nerve fibers penetrating into the developing bone. On GD 16 and GD 17 no GAP-43-immunoreactive (IR) fibers were observed in the close vicinity of bone rudiments. On GD19 GAP-43-IR fibers were scarcely present within the periosteum of the central portion of the diaphysis. In the perichondrium surrounding the proximal epiphysis, nerve fibers were first detected around birth. From PD1 onward, numerous fibers were seen in the fibrous buds of the perichondrium at the epi-metaphyseal junction (Ranvier's grooves), some of them being adjacent to the blood vessels. Nerve fibers penetrating into the bone and located in the bone marrow, predominantly associated with blood vessels, were first observed on GD21 and their number increased with further development. They were initially located in the central portion of the diaphysis and later extended towards the metaphyses. On PD4 an increased number of GAP-43-IR fibers appeared in the perichondrium of proximal and distal epiphyses. In the fibrous strands penetrating into the epiphyses and in the secondary ossification centers, nerve fibers were first observed on PD10. From PD14 onward the pattern of tibial innervation remained unchanged but the intensity of GAP-43 immunostaining visibly decreased. The present study demonstrates that developing long bones of rat hindlimbs are supplied by growing nerve fibers immunoreactive for GAP-43 from GD 19 onward. Time and location of their appearance were at least partially correlated with known events taking place during long bone development, e.g. formation of primary and secondary ossification centers. Decreased expression of GAP-43 immunoreactivity in later developmental stages is believed to reflect nerve fiber maturation.

Intraepithelial vagal sensory nerve terminals in rat pulmonary neuroepithelial bodies express P2X(3) receptors.

The neurotransmitters/modulators involved in the interaction between pulmonary neuroepithelial bodies (NEBs) and the vagal sensory component of their innervation have not yet been elucidated. Because P2X(3) purinoreceptors are known to be strongly expressed in peripheral sensory neurons, the aim of the present study was to examine the localization of nerve endings expressing P2X(3) purinoreceptors in the rat lung in general and those contacting pulmonary NEBs in particular. Most striking were intraepithelial arborizations of P2X(3) purinoceptor-immunoreactive (IR) nerve terminals, which in all cases appeared to ramify between calcitonin gene-related peptide (CGRP)- or calbindin D28k (CB)-labeled NEB cells. However, not all NEBs received nerve endings expressing P2X(3) receptors. Using CGRP and CB staining as markers for two different sensory components of the innervation of NEBs, it was revealed that P2X(3) receptor and CB immunoreactivity were colocalized, whereas CGRP-IR fibers clearly formed a different population. The disappearance of characteristic P2X(3) receptor-positive nerve fibers in contact with NEBs after infranodosal vagal crush and colocalization of tracer and P2X(3) receptor immunoreactivity in vagal nodose neuronal cell bodies in retrograde tracing experiments further supports our hypothesis that the P2X(3) receptor-IR nerve fibers contacting NEBs have their origin in the vagal sensory nodose ganglia. Combination of quinacrine accumulation in NEBs, suggestive of the presence of high concentrations of adenosine triphosphate (ATP) in their secretory vesicles, and P2X(3) receptor staining showed that the branching intraepithelial P2X(3) receptor-IR nerve terminals in rat lungs were exclusively associated with quinacrine-stained NEBs. We conclude that ATP might act as a neurotransmitter/neuromodulator in the vagal sensory innervation of NEBs via a P2X(3) receptor-mediated pathway. Further studies are necessary to determine whether the P2X(3) receptor-expressing neurons, specifically innervating NEBs in the rat lung, belong to a population of P2X(3) receptor-IR nociceptive vagal nodose neurons.

Mucosal projections of enteric neurons in the porcine small intestine.

In the present study, a combination of immunohistochemistry and retrograde 1,1;-didodecyl-3,3,3;,3;-tetramethylindocarbocyanine perchlorate (DiI) tracing was used to unravel the morphology, distribution, and neurochemical coding of submucous and myenteric neurons with axonal projections to the mucosa of the porcine small intestine. The majority of traced neurons was located in the inner submucous plexus (ISP; 78%), whereas the remaining part was distributed between the outer submucous plexus (OSP; 10%) and myenteric plexus (MP; 12%). Among these traced neurons, some distinct neuronal populations could be distinguished according to their morphologic and neurochemical properties. In the ISP, several types of traced neurons were detected: 1) morphologic type II neurons expressing choline acetyltransferase (ChAT) immunoreactivity, calcitonin gene-related peptide (CGRP) immunoreactivity, and substance P (SP) immunoreactivity; 2) ChAT/SP-immunoreactive (-IR) small neurons; 3) vasoactive intestinal polypeptide (VIP) -IR small neurons; and 4) multidendritic ChAT/somatostatin (SOM) -IR neurons. The traced neuronal populations of the OSP and MP were similar to each other. In both plexuses, the following DiI-labelled neurons were found: 1) ChAT/CGRP/(SP)-IR type II neurons; 2) multidendritic ChAT/SP-IR neurons; and 3) multidendritic ChAT/SOM-IR neurons. Comparison of the present findings with previously obtained data concerning the mucosal innervation pattern of the intestine of small mammals, revealed significant species differences with respect to the morphologic and neurochemical features of the involved enteric neuronal classes. Although not identical, a closer resemblance between pig and human enteric nervous system seems to be at hand, as far as the anatomic organization and the presence of neurochemically identified neuronal subtypes within the enteric nervous system are concerned.

Species-dependent features of Dogiel type II neurones in the mammalian enteric nervous system.

Although autonomic gastrointestinal reflex movements, which occur in all mammalian species, have been described almost a century ago, little was known on the mechanisms underlying this behaviour. Recently, however, intrinsic primary afferent neurones, functioning as the first relay in the reflex arches embedded in the intestinal wall, have been identified in the guinea pig ileum. In guinea pig, such neurones display a Dogiel type II morphology and behave electrophysiologically as slow AHP neurones. In other gastrointestinal regions, in both guinea pig and rat, Dogiel type II cells are also encountered, but the strong correlation with slow AHP neuronal features seems less strict. In large mammals, a correlation of the cellular morphology with intracellular el ectrophysiological recordings has only been obtained in the pig small intestine. Surprisingly, in these experiments aberrant electrophysiological behaviour of Dogiel type II neurones is even more striking since the majority of these cells display electrophysiological features considered typical of S neurones. Furthermore, in those rare cases in which a slow afterhyperpolarization (AHP) could be recorded in porcine Dogiel type II cells, its amplitudes were negligible. This has led us to the conclusion that the differences in electrophysiological behaviour of neurones with comparable morphology in different species are most probably due to the modulating influence of the neurotransmitter substances present. This seems to be the most likely hypothesis in view of the considerable differences in neurotransmitter content of neurones with comparable functions throughout the species.

Postnatal development of nitrergic neurons in the myenteric plexus of rat stomach.

The effect of age on the proportion of nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd)-positive neurons was investigated in the myenteric plexus of five different gastric areas of 1-day-, 1-week-, 2-week-, 1-month- and 2-month-old rats. Protein gene product 9.5 immunocytochemistry was used as a marker for the total enteric neuron population in order to establish the percentage of gastric nitrergic neurons in relation to age. The percentage of NADPHd-positive neurons in the proximal parts of the rat stomach (34-38%) is significantly higher than in the antral part (29%). This difference persists in all the age groups investigated. No significant relative increase with age of NADPHd-positive neurons could be observed in any of the areas studied. These findings imply that the increased nitrergic response in the rat proximal stomach as seen in pharmacological studies cannot be explained by an increased relative number of nitrergic neurons.

Pulmonary intraepithelial vagal nodose afferent nerve terminals are confined to neuroepithelial bodies: an anterograde tracing and confocal microscopy study in adult rats.

Our present understanding of the morphology of neuroepithelial bodies (NEBs) in mammalian lungs is comprehensive. Several hypotheses have been put forward regarding their function but none has been proven conclusively. Microscopic data on the innervation that appears to affect the reaction of NEBs to stimuli have given rise to conflicting interpretations. The aim of this study has been to check the validity of the hypothesis that pulmonary NEBs receive an extensive vagal sensory innervation. The fluorescent neuronal tracer DiI was injected into the vagal sensory nodose ganglion and NEBs were visualized in toto by using immunocytochemistry and confocal microscopy on 100-micrometer-thick frozen sections of the lungs of adult rats. The most striking finding was the extensive intraepithelial terminal arborizations of DiI-labelled vagal afferents in intrapulmonary airways, apparently always co-appearing with calcitonin gene-related peptide (CGRP)-immunoreactive NEBs. Not all NEBs received a traced nerve fibre. Intrapulmonary CGRP-containing nerve fibres, including those innervating NEBs, always appeared to belong to a nerve fibre population different from the DiI-traced fibres and hence did not arise from the nodose ganglion. Therefore, at least some of the pulmonary NEBs in adult rats are supplied with sensory nerve fibres that originate from the vagal nodose ganglion and form beaded ramifications between the NEB cells, thus providing support for the hypothesis of a receptor function for NEBs.

Structural organization and neuropeptide distribution in the mammalian enteric nervous system, with special attention to those components involved in mucosal reflexes.

Gastrointestinal events such as peristalsis and secretion/absorption processes are influenced by the enteric nervous system, which is capable of acting largely independently from other parts of the nervous system. Several approaches have been used to further our understanding of the underlying mechanisms of specific enteric microcircuits. Apart from pharmacological and physiological studies, the deciphering of the chemical coding of distinct morphological and functional enteric neuron classes, together with a detailed analysis of their projections by the application of immunocytochemistry, of tracing, and of denervation techniques, have substantially contributed to our knowledge. In view of existing interspecies and regional differences, it is of major importance to expand our knowledge of the enteric nervous system in mammals other than the guinea-pig, the most commonly used experimental animal in this research area. This will increase our chances of finding a valid model, from which well-founded extrapolations can be made regarding the precise function of distinct enteric neuron types regulating motility and ion transport in the human gastrointestinal tract.

Distribution pattern, neurochemical features and projections of nitrergic neurons in the pig small intestine.

The presence and topographical distribution of nitrergic neurons in the enteric nervous system (ENS) of the pig small intestine have been investigated by means of nitric oxide synthase (NOS) immunocytochemistry and nicotinamide dinucleotide phosphate diaphorase (NADPHd) histochemistry. Both techniques yielded similar results, thus confirming that within the pig ENS the neuronal isoform of NOS corresponds to NADPHd. Intrinsic nitrergic neurons were not confined to the myenteric plexus; considerable numbers were also present in the outer submucous plexus. In the inner submucous plexus, NOS immunoreactivity or NADPHd staining was restricted to a few nerve fibres and nerve cell bodies. The nitrergic neurons displayed a wide variety in size and shape, but could all be characterized as being multidendritic uniaxonal. Nerve lesion experiments showed that the majority of the myenteric nitrergic neurons project in an anal direction. Evidence is at hand to show that a substantial proportion of these neurons contribute to the dense nitrergic innervation of the tertiary plexus and the circular smooth muscle layer. Some of the nitrergic neurons of the outer submucous plexus were equally found to send their axons towards the circular muscle layer. In some of the nitrergic enteric neurons, VIP, neuropeptide Y, galanin or protein 10 occurred colocalized, but not calbindin or serotonin. The present findings provide morphological evidence for the presence of NOS in a proportion of the enteric neurons in the small intestine of a large omnivorous mammal, i.e. the pig. The topographical features of the staining patterns of NOS and NADPHd are in accord with the results of neuropharmacological studies and argue for the existence of distinct nitrergic subpopulations acting either as interneurons or as motor neurons.