Histomorphology of the proventriculus of three species of Australian passerines: Lichmera indistincta, Zosterops lateralis and Poephila guttata.

Histomorphology of the proventriculi of nectarivorous, granivorous and omnivorous passerines was studied. The proventriculus consisted of mucosal, submucosal, muscularis and serosal layers. Proventricular wall was thickest in omnivore, thinnest in granivore and intermediate in nectarivore. The openings of mucosal glands had a single spiral-like fold of mucosa in the omnivorous Silvereye, 2-3 spirals in the granivorous Zebra finch and 4-5 spirals in the nectarivorous Brown honeyeater. The mucosal glands were arranged in a uniform row in the wall of the organ and opened individually via a primary duct to the lumen of the proventriculus. The surface epithelial cells of the tunica mucosa contained secretory cells and the proventricular glands contained endocrine, neck and oxynticopeptic cells. The ultrastructural features of the oxynticopeptic cells changed from the oral to the aboral portion of the gland. In the oral region, the cytoplasm presented numerous, smaller (600-900 nm) homogenously dense zymogen secretory vesicles and larger (0.8-2.3 microm) pale floccular, tubular, mucin-like secretory granules, few small mitochondria and RER while in the aboral portion of the gland, the cytoplasm presented numerous, large mitochondria with closely packed cristae, secondary lysosome and infolding of the basal and apical cell membrane. The tunica sub mucosa was thin with occasional large blood vessels. The tunica muscularis consisted of inner longitudinal, middle circular and outer longitudinal layers. The external tunica serosa contained large bundles of myelinated and unmyelinated axons that were possibly branches of the intestinal nerve. The structural adaptations of the proventriculi of these three species to their various diets are discussed.

Pharmacological characterization of 5-hydroxytryptamine-induced contraction in the chicken gastrointestinal tract.

5-Hydroxytryptamine (5-HT) receptor subtypes involved in 5-HT-induced contraction of the chicken gastrointestinal tract were characterized pharmacologically using subtype-selective agonists and antagonists. The proventriculus (area of stomach adjacent to the oesophagus) and ileum are examined. 5-HT applied cumulatively caused sustained contraction of the proventriculus that was not decreased by tetrodotoxin, atropine or l-nitro-arginine methylester (l-NAME). alpha-Methyl-5-HT showed the same potency as that of 5-HT, indicating the involvement of the 5-HT(2) receptor. (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-amino-propane (DOI), 5-methoxytryptamine and 1-(3-chlorophenyl)piperazine hydrochloride (mCPP) were potent, and 2-methyl-5-HT, 5-carboxamidotryptamine, BW723C86 and 6-chloro-2-(1-piperazinyl)pyrazine hydrochloride (MK212) were moderate, but (+/-)-8-hydroxy-2-dipropylaminotetralin hydrobromide (8-OH-DPAT), [endo-N-8-methyl-8-azabicyclo-(3,2,1)oct-3-yl]-2,3-dihydro-(1-methyl)ethyl-2-oxo-1H-benzimidazol-1-carboxamide (BIMU-8) and cisapride were weak agonists. Correlation of pEC(50) values of these agonists with documented pEC(50) values for 5-HT(2C) receptor was higher than 5-HT(2A) and 5-HT(2B). Cinanserin, ketanserin, methiothepin, methysergide, mianserin, (8-[5-(2,4-dimethoxy-5-(4-trifluoromethylphenylsulphonamido)phenyl-5-oxopentyl)-1,3,8-triazaspiro[4,5]decane-2,4-dione hydrochloride (RS102221), N-(1-methyl-1H-indolyl-5-yl)-N'-(3-methyl-5-isothiazolyl)urea (SB204741), spiperone and N-desmethylclozapine concentration-dependently inhibited the contractile responses to 5-HT. Correlation of pK(b)/pA(2) of antagonists with documented pK(i) for 5-HT(2C) receptor was highest among 5-HT(2) receptor subtypes. In the methysergide- and ketanserin-treated proventriculus, 5-HT, 2-methyl-5-HT and cisapride did not enhance the electrical field stimulation (5 Hz)-induced cholinergic contractions. 5-HT applied non-cumulatively caused transient contraction of ileum, and the responses were partly decreased by atropine or tetrodotoxin. 5-Methoxytryptamine, alpha-methyl-5-HT, 5-carboxamidotryptamine, L692,247 and DOI were potent agonists. However, 2-methyl-5-HT, cisapride, BW723C86, 8-OH-DPAT and 5-nonyloxytryptamine, mCPP and MK212 were less effective. Ketanserin and methysergide decreased the 5-HT-induced ileal contraction, but neither GR113808 nor SB269970 inhibited the responses. In conclusion, 5-HT causes contraction of the proventriculus via 5-HT(2C)-like receptors present on smooth muscle. 5-HT also causes contraction of the ileum, but the underlying mechanisms are complex, involving neural and smooth muscle components, and both 5-HT(1)- and 5-HT(2)-like receptors. Neural 5-HT receptors similar to 5-HT(3)/5-HT(4) receptors were not found in the chicken proventriculus and ileum.

Colocalization of numerous immunoreactivities in endocrine cells of the chicken proventriculus at hatching.

The colocalization of regulatory peptide immunoreactivities in endocrine cells of the chicken proventriculus at hatching has been investigated using the avidin-biotin technique in serial sections and double immunofluorescence in the same section for light microscopy, and double immunogold staining for electron microscopy. In addition to the eight immunoreactivities previously described in this organ, cells immunoreactive for peptide histidine isoleucine (PHI), peptide gene product 9.5 (PGP), and the amidating enzyme, peptidylglycine alpha-amidating monooxygenase (PAM) were observed. All the cells immunoreactive to glucagon were also immunostained by the PHI antiserum. In addition, all the glucagon-like peptide 1, avian pancreatic polypeptide, and some of the neurotensin-like cells costored also glucagon- and PHI-immunoreactive substances. PGP- and PAM-immunoreactivities were also found in the glucagon-positive cells. A small proportion of the somatostatin-containing cells were positive for PHI but not for other regulatory peptides. These results could suggest either the existence of a very complex regulatory system or that the endocrine system of the newborn chickens is not yet fully developed.

The nervous system of the chicken proventriculus: an immunocytochemical and ultrastructural study.

The proventriculus constitutes the glandular region of the chicken stomach. This organ is innervated by two parasympathetic networks, the myenteric and submucous plexus, and here we present a systematic study of this system by immunohistochemistry and electron microscopy. All the neurons and fibres were positive for the neural markers, protein gene product 9.5 and the amidating enzymes. Immunoreactivities for the constitutive neuronal isoform of the enzyme nitric oxide synthase and the vasoactive intestinal peptide were present in neuronal bodies suggesting an intrinsic origin for the similarly immunoreactive fibres found in the proventriculus. On the other hand, immunoreactivity to gastric inhibitory peptide was only found in varicose fibres making contact with the blood vessels and the glandular epithelium, but never in the neuronal somas, suggesting that this substance may be provided by an extrinsic nervous system whose neuronal bodies are located elsewhere. Electron microscopy revealed frequent neuromuscular and neuroepithelial connections in the muscle layers, the wall of the blood vessels and the epithelium. In addition, synapsis-like structures were identified in the proximity of cells belonging to the diffuse endocrine system, providing a new example of neuroendocrine contacts. No positivity was found for antibodies against other neural substances including somatostatin, peptide histidine-isoleucine, peptide tyrosine-tyrosine, neuropeptide tyrosine, bombesin, met-enkephalin, serotonin, substance P, galanin, calcitonin gene-related peptide and S-100 protein.

Ontogeny of galanin-immunoreactive elements in the intrinsic nervous system of the chicken gut.

Galanin is a brain-gut peptide that is present in the central and peripheral nervous systems. In the gut, it is contained exclusively in intrinsic and extrinsic nerve supplies, and it is involved overall in the regulation of gut motility. To obtain information about the ontogeny of galanin, we undertook an immunohistochemical study of chicken embryos. The time of first appearance and the distribution patterns of galanin were investigated with fluorescence and streptavidin-biotin-peroxidase (ABC) immunohistochemical protocols by using a galanin polyclonal antiserum. The various regions of the gut and the pancreas were obtained from chicken embryos aged from 3 days of incubation to hatching. All specimens were fixed in buffered picric acid-paraformaldehyde, frozen, and cut with a cryostat. Galanin-immunoreactive neuroblasts were first detected at 4 days in the mesenchyme of the proventriculus/gizzard primordium and within the Remak ganglion. They then extended cranially and caudally, reaching all of the other gut regions at 6.5 days. Galanin-immunoreactive nerve elements mainly occupied the sites of myenteric and submucous plexuses. From day 15, galanin-immunoreactive nerve fibers tended to invade the circular muscular layer and part of the lamina propria of the mucosa. In the pancreas, weak galanin-immunoreactive nerve elements were detected at 5.5 days. They tended to be distributed among the glandular lobules according to the organ differentiation. The widespread distribution during the earlier embryonic stages represents evidence indicating that the neuropeptide galanin may have a role as a differentiating or growth factor. From late embryonic life, its predominant presence in sympathetic nerves and in muscular layers fits with the functions demonstrated previously in adults of other vertebrates for galanin as a modulator of intestinal motility.

A neurochemical characterisation of the golden hamster myenteric plexus.

The neurochemical composition of nerve fibres and cell bodies in the myenteric plexus of the proventriculus, stomach and small and large intestines of the golden hamster was investigated by using immunohistochemical and histochemical techniques. In addition, the procedures for localising nitric-oxide-utilising neurones by histochemical (NADPH-diaphorase) and immunohistochemical (nitric oxide synthase) methods were compared. The co-localisation of vasoactive intestinal polypeptide and nitric oxide synthase in the myenteric plexus of all regions of the gut was also assessed. The results demonstrated the presence of nerve fibres and nerve cell bodies immunoreactive to protein gene product, vasoactive intestinal polypeptide, substance P, calcitonin gene-related peptide, tyrosine hydroxylase, 5-hydroxytryptamine and nitric oxide synthase in all regions of the gastrointestinal tract examined. The pattern of distribution of immunoreactive nerve fibres and nerve cell bodies containing the above markers was found to vary in different regions of the gut. Myenteric neurones and nerve fibres containing immunoreactivity to nitric oxide synthase and NADPH-diaphorase reactivity, however, were shown to have an identical distribution throughout the gut. In contrast to some studies on the guinea-pig and rat, the co-existence of vasoactive intestinal polypeptide and nitric oxide synthase was seen in only a small population of myenteric neurones.

Distribution and colocalization of NADPH-diaphorase activity, nitric oxide synthase immunoreactivity, and VIP immunoreactivity in the newly hatched chicken gut.

BACKGROUND: The distribution and colocalization of nitric oxide synthase and NADPH-diaphorase have been investigated quite extensively in the mammalian gut; however, no such study has been undertaken in the avian gut. In the present report, we have therefore studied the distribution and coexpression of nitric oxide synthase (NOS), NADPH-diaphorase, and vasoactive intestinal polypeptide (VIP) in enteric neurons of the newly hatched chicken gut. METHODS: Immunohistochemical methods were used to detect NOS immunoreactivity (NOS-IR) and VIP immunoreactivity (VIP-IR). NADPH-diaphorase activity was detected using a histochemical technique. RESULTS: Neurons expressing NADPH-diaphorase activity, NOS-IR, and VIP-IR were detected in both the myenteric and submucous plexus of all regions of the gastrointestinal tract examined. All NADPH-diaphorase positive neurons were also NOS-IR and all NOS-IR neurons were NADPH-diaphorase positive, in both plexuses, indicating that NADPH-diaphorase can be used as a marker for NOS containing neurons in the chicken gut. The majority of VIP-IR neurons also expressed NADPH-diaphorase activity. Only few neurons that expressed NADPH-diaphorase activity did not express VIP-IR. The proportion of VIP immunopositive neurons that were NADPH-diaphorase negative increased anally and these neurons were more prominent in the submucous than the myenteric plexus ganglia. NADPH-diaphorase positive, NOS-IR, and VIP-IR nerve fibres were detected in the circular muscle, but very few, if any, were present in the longitudinal muscle. VIP-IR, but not NOS-IR or NADPH-diaphorase activity, was detected in mucosal fibres, in contrast to the situation in the mammalian gut. CONCLUSIONS: These results indicate that in birds, as in mammals, nitric oxide may play a role in the neural control of the gut musculature, but that it is unlikely to be involved in the nervous control of mucosal activity.

Vagal preganglionic neurons innervating the different gastric regions in the Japanese quail (Coturnix japonica).

The cytoarchitecture and distribution patterns of the vagal preganglionic neurons within the dorsal motor nucleus of the vagus nerve (DMNX) innervating the proventriculus and the gizzard of the Japanese quail were examined by Nissl staining and the horseradish peroxidase (HRP) method. A 30% solution of HRP was injected into nine different gastric regions: the ventral and dorsal parts of the proventriculus, the caudodorsal and cranioventral thick muscles, the craniodorsal and caudoventral thin muscles, and the pylorus, and the ventral and dorsal tendons of the gizzard. Nissl preparations showed that the DMNX is composed of two cell groups, the dorsal magnocellular and mediocellular subnucleus (Xd) and the ventral parvicellular subnucleus (Xv). After injection of HRP into the ventral and dorsal parts of the proventriculus, HRP-labeled cells were predominantly observed in the left and right DMNX, respectively. The rostrocaudal distribution patterns of HRP-labeled cells in the Xd and Xv were symmetric on the left and right sides. The distribution patterns of labeled cells following the injection of HRP into each region of the gizzard showed that there was very little difference in the number of neurons between the left and right DMNX, and no topographic localization was found in the Xd and Xv. The vagal preganglionic neurons projecting to the gizzard lay more caudal than the ones for the proventriculus. This study suggested topographic localization in the distribution patterns of the vagal preganglionic neurons innervating the proventriculus and the gizzard.

The inhibitory action of lead on mechanical responses of the proventricular smooth muscle in the chick.

The purpose of the present experiments was to examine the mechanism of the proventricular dilatation caused by lead in the isolated vagus nerve-proventricular smooth muscle preparation of the chick. Lead caused dose- and time-dependent inhibition of contractions induced by vagal stimulation, transmural stimulation and externally applied acetylcholine (ACh). Vagally evoked contraction was much more sensitive to the inhibitory action of lead than the contractile response to ACh. The lower the frequency of transmural stimulation, or the lower the concentration of ACh was applied, the greater the inhibitory action of lead on the evoked smooth muscle contraction. The results suggest that proventricular impaction occurring in lead poisoning results from the pre- and post-synaptic inhibition of the vagus nerve-smooth muscle transmission.

Development and birthdates of vasoactive intestinal peptide immunoreactive neurons in the chick proventriculus.

To gain insight into the mechanisms regulating expression of transmitter phenotypes in the enteric nervous system, we have studied the development and birthdate of vasoactive intestinal peptide immunoreactive (VIP-IR) myenteric neurons in the chicken proventriculus (secretory portion of the avian stomach) by a combination of immunocytochemistry and radioautography. The appearance and numbers of VIP-IR neurons in whole mounts of the myenteric plexus from chick embryos and chickens were examined. We found that VIP-IR neurons first appeared at embryonic day (E) 5.5-6.5 in the distal part of the proventriculus. At E7.5, VIP-IR neurons were found singly, in pairs, or in small groups, which together with unlabeled cells formed primitive myenteric ganglia. VIP-IR fibers were found within the developing fiber tracts which connected the ganglia. The number of VIP-IR neurons was found to be maximum in the E15.5 embryo and to decline to 68% of maximum in the 4 week old chicken. Birthdate studies were performed by application of either single pulses or cumulative doses of [3H]-thymidine to embryos between E3 and E14. Whole mounts of the myenteric plexus from the proventriculus of these embryos were immunostained for VIP at E10 or E17. The whole mounts were subsequently sectioned and processed for radioautography. We found that VIP-IR myenteric neurons were born between E3 and E10 with a peak at E7. Most cells underwent terminal division between E5 and E9. These data will be useful in determining the time and conditions when cells make decisions about transmitter phenotypes.

Topographic representation of visceral target organs within the dorsal motor nucleus of the vagus nerve of the pigeon Columba livia.

Our previous work (Katz and Karten, '83a J. Comp. Neurol. 217:31-46 demonstrated that the dorsal motor nucleus of the vagus nerve (DMN complex) in the pigeon is composed of cytoarchitecturally distinct subnuclei that are distinguished by the size, shape, position, and cytochemical characteristics of their constituent neurons. In view of the diversity of target organs innervated by the vagus nerve, we sought to determine whether the subnuclear heterogeneity of the DMN complex is related to the pattern of target innervation. To test this possibility, retrograde tracing techniques were used to define the subnuclear localization of vagal motoneurons that innervate individual vagal target organs. The distribution of horseradish peroxidase (HRP)-labeled motoneurons within the DMN complex was studied following application of HRP to the cut central end of individual vagal nerve branches and after injection of the tracer into vagal target tissues. In addition, we examined the distribution of acetylcholinesterase depletion within the DMN complex following transection of individual vagal branches. Our data demonstrate that individual vagal target organs have discrete and topographic representations within cytoarchitecturally distinct subnuclei of the DMN complex. Therefore, in the pigeon, the subnuclear distribution of vagal motoneurons plays a critical role in the organization of descending vagal motor pathways. Segregation of visceral representations within the DMN complex may provide a mechanism for organizing functionally diverse afferent inputs to target-specific populations of vagal motoneurons.