Neuroendocrine epithelial cells in the broncho-parabronchial transition of embryonic, immature and mature quail (Coturnix coturnix).

The main objective of this integrated light microscopic, transmission and scanning electron microscopic study was to describe in greater detail the structural pattern and developmental stages of pulmonary neuroendocrine epithelial cells (PNECs) in the broncho-parabronchial transition (BPT) of both developing and mature quail. In mature quail the BPT appeared as a diaphragm opening into the parabronchial vestibulum. Perpendicular sections revealed two bilayered crest-like entrance folds invested by a uniform population of granular cells with lamellar bodies and a brush border of blunt microvilli. Solitary PNECs were found interlaced between the granular cells on both sides of the BPT. In addition, PNECs with small dense-cored vesicles (DCVs) were found subepithelially in the lamina propria mucosae of the parabronchial compartment of the BPT, which was surrounded by a capsule of granular cells. Furthermore, clusters of PNECs devoid of any epithelial capsule, but with large DCVs were located in the tunica propria mucosae. The first signs of the developing BPT could be identified as early as embryonic day (ED) 13. By ED 17, the area of the future BPT was seen to be invested by a uniform population of granular cells but the entrance fold proper did not appear until after hatching. Solitary or clustered PNECs were demonstrated in parabronchial buds growing into the mesenchyme on ED 12 and 13: either type I PNECs with small DCVs measuring about 80-120 nm or type II PNECs with large dense granules measuring approximately 150-280 nm. Both types of PNECs represented a temporary cell population in the prehatching period. In the parabronchus no PNECs or neuroendocrine epithelial bodies could be demonstrated in mature and immature quail beyond the BPT.

Lamellar inclusions and trilaminar substance in the parabronchial epithelium of the quail (Coturnix coturnix).

The fine structure of the epithelial cells of the parabronchus and their secretory products have been the subject of many studies and have given rise to considerable controversy about their configuration and ultrastructure. The aim of the present study was to investigate the mode of formation and discharge of lamellar bodies of granular cells and the trilaminar substance produced and discharged by the embryologically related squamous atrial and respiratory epithelial cells. The material for light and transmission electron microscopic analysis was collected from 10 mature quail and 3 individuals aged 2 days. The parabronchial atria harbour two ultrastructurally distinct types of epithelial cells. The granular cells (analogous to type II cells of the mammalian pulmonary alveolus) produce and discharge balls of lamellar bodies. The squamous atrial cells produce and discharge sheets of trilaminar substance sandwiched between long tentacle-like processes, viz. the microvilli. The infundibula and air capillaries of the gas exchange tissue are invested with squamous respiratory cells which extend very thin, long processes that cover the air capillaries and constitute, together with the blood capillaries, the blood-air barrier. The squamous respiratory cells produce and discharge trilaminar substance as an extracellularly located acellular lining layer which is found in close contact with their cell membrane. Both squamous atrial and respiratory cells hence synthetize and discharge trilaminar substance, the basic unit of which has the ultrastructural appearance of a 7.5 to 8.0 nm membrane unit. The formation of trilaminar substance originates in the agranular endoplasmic reticulum, while the origin of the lamellar bodies of granular cells is related to the granular endoplasmic reticulum, the Golgi complex and the multivesicular bodies. Their structural unit is composed of a 4.5 to 5.0 nm thread-like structure which is concentrically arranged around a spherical core of granular substance.

A scanning and transmission electron microscopy study of the parabronchial unit in quail (Coturnix coturnix) and town pigeons (Columba livia).

A combined scanning electron (SEM) and transmission electron microscopy (TEM) investigation was undertaken to gain insight into the complex structural pattern of the atrial compartment and the gas exchange tissue of parabronchial units in quail and town pigeons. The aim was also to depict the changes taking place in the parabronchial unit in the late prehatching and early posthatching periods in quail. The standard SEM and TEM investigation was carried out in 13 mature quail and 8 town pigeons. The developmental study involved embryonic quail (Days 15, 16, 17), newly hatched quail, quail 24 h after hatching, and quail aged 2, 10, 19, and 25 days (3 individuals per group). The luminal relief of the parabronchus is formed by anastomosing interatrial septa delineating the atrial pits, which are thinner and shallower in pigeons. The atrial bottom opens in mature individuals into 3-6 infundibula. The extracellular material represented by trilaminar substance, which does not appear until hatching, veils the surface relief of the parabronchial epithelium, which is consequently hardly accessible to three-dimensional visualization. Only in town pigeons with fewer discontinuous layers of extracellular material was it possible to visualize the surface of the atrial epithelium, that is, of the granular and squamous atrial cells. The SEM analysis has convincingly shown the intricate spatial organization of atria, infundibula, and air and blood capillaries of the gas exchange tissue. The retinacula, that is, parallelly arranged processes of squamous respiratory cells bridging the air-capillary lumina, were evidenced by SEM and TEM. The complex structure of the avian parabronchus has been successfully demonstrated in the present SEM and TEM study.

An SEM and TEM study of the transition of the bronchus to the parabronchus in quail (Coturnix coturnix).

The main objective was to analyse the transition of the bronchus to the parabronchus in birds and to describe its specific structure in an integrated light microscopic, transmission electron microscopic (TEM) and scanning electron microscopic (SEM) study. Lung tissue from immature and mature quail was subjected to standard processing for paraffin light microscopy, TEM and SEM after intratracheal inflation with fixative. In transverse paraffin and Durcupan semithin sections, the partition incompletely closing the broncho-parabronchial transition has the appearance of a crest-like fold delineating the entrance to the underlying parabronchial vestibulum. The core of the entrance fold is composed of loose connective tissue with free cells, and has a well-developed blood supply and innervation. Voluminous groups of smooth muscle cells are interconnected with those of neighbouring entrance folds and the interatrial septa. On the parabronchial surface and partly on the bronchial surface the entrance fold is invested with simple cuboid epithelium consisting exclusively of granular cells with lamellar inclusions. On the bronchial surface, they pass into ciliated columnar pseudostratified epithelium. At the root of the parabronchially orientated surface, they continue into the mixed population of granular and squamous atrial cells of the parabronchus. Among the granular cells of the entrance fold, scattered epithelial neuroendocrine cells are consistently present. The three-dimensional visualization demonstrated the oval form of the entrance window with a circular field of non-ciliated cells delineating the entrance to the parabronchial labyrinthine system. The general structural pattern of the entrance fold, together with the complex system of interatrial trabecles of the parabronchi underline the multifactorial function of a complex system submitted to the skeletal, regulatory and host defense of the gas exchange tissue.

The development and differentiation of the parabronchial unit in quail (Coturnix coturnix).

The present study has been inspired by the conflicting data in the relevant literature concerning the embryogenesis of cell types of the parabronchial epithelium and the formation, discharge and distribution of trilaminar substance and lamellar bodies. Lung tissue from embryonic, newly hatched, immature and mature quail was subjected to standard processing for light and transmission electron microscopy. The parabronchial rudiments form shallow primitive atria on embryonic day 13. The precursors of granular cells differentiate with lamellar bodies in their cytoplasm. The residual population of non-granular epithelial cells is the common source for the differentiation of primitive squamous atrial and respiratory cells, the potential producers of trilaminar substance. The primitive squamous atrial cells sprout as branching infundibular canaliculi into the mesenchyme on embryonic day 14. The infundibular epithelium differentiates into the squamous respiratory cells that constitute with blood capillaries the blood-air barrier. Not until the time of hatching could the trilaminar substance be visualized being produced by squamous atrial and respiratory cells. In the late prehatching and early posthatching period the granular cells intensely escalate the production and discharge of lamellar bodies. The lamellar bodies form, together with sheets of trilaminar substance, mixed multilayered masses in atria. They disappear fast in the successive posthatching period. The formation of trilaminar substance in squamous atrial and respiratory cells is governed by the agranular endoplasmic reticulum, the cisternae of which take part in the formation of trilaminar units. The gas exchange tissue is predominantly represented by infundibula in immature quail. The posthatching growth of the gas exchange tissue of immature to mature quail occurs via intense multiplication of air and blood capillaries.

An electron microscopic study of the parabronchial epithelium in the mature lung of four bird species.

BACKGROUND: No integrated comprehensive description of the ultrastructure of the parabronchial epithelium is available. The origin, discharge, and occurrence of the trilaminar substance have not yet been sufficiently studied. Therefore, the main objectives were to classify the cell types of the parabronchial epithelium and to describe their role in manufacturing the trilaminar substance. METHODS: Lung tissue of mature quail, domestic fowl, town pigeon, homing pigeon, and barn owl was subjected to standard processing for transmission electron microscopy, both after intratracheal inflation and intravascular perfusion. RESULTS: The atrial epithelium is constituted by granular and squamous atrial cells. Granular cells (1) are confined to the atrial wall; they produce and discharge osmiophilic lamellar bodies. Squamous atrial cells (2) manufacture and discharge a trilaminar substance in sheets sandwiched between the long microvilli emerging from the apical cytoplasm. Their attenuating cell outgrowths overlap granular cells. At the bases of atria, they pass as intermediate squamous atrial cells to the infundibula, contacting the extensions of squamous respiratory cells. The squamous atrial cells undergo distinct structural variations depending on age and environment. Squamous respiratory cells (3) (cellulae squamosae) continuously line the air capillaries and neighboring infundibula. They constitute the epithelial compartment of the blood-gas barrier. The cell bodies extend long, very thin cell outgrowths. The apical surface is smooth and the basal part is invested with a very thin basement membrane. The trilaminar substance originates from granular and agranular endoplasmic reticulum in the form of convoluted profiles which are discharged as an acellular lining layer on the air surface of squamous respiratory cells. CONCLUSIONS: Granular cells are analogous to the type II cells of mammalian pulmonary alveolus. Squamous atrial and respiratory cells, of a common embryonic origin, do not meet any counterpart in epithelial cell populations of lung terminal airways in vertebrates. The specific trilaminar substance--lipoproteinaceous in nature--is a constant compound of atria and air capillaries.

Anchoring and support system of pulmonary gas-exchange tissue in four bird species.

Avian air capillaries are delicate structures compared to the mammalian pulmonary alveolus. A transmission and scanning electron microscopic study was carried out on several species of birds with the aim of determining the support structures of the avian gas-exchange mantle. Lung tissue of two bird species belonging to strong flying birds (pigeon and barn owl) and two relatively flightless species (domestic fowl and quail) was subjected to standard processing for transmission and scanning electron microscopy after intratracheal inflation. Twisted profiles of lipoproteinaceous trilaminar substance as specific secretory product of avian squamous respiratory cells can be seen in the cell body and cytoplasmic extensions that are wedged between the blood capillaries, partly surrounding them. The intracytoplasmatically located trilaminar complexes form a three-dimensional intricate spiderweb-like system between the blood capillaries and air capillaries, which presumably function as an anchoring and support structure of the gas-exchange tissue. This system is strengthened by retinacula--pairs of attenuated parallel processes of squamous respiratory cells that project to the airway lumen--expanding and bridging the opposite side of air capillaries. The trilaminar substance is discharged in the form of a 15-nm-thick acellular lining layer which is uniquely adapted to the extremely thin respiratory epithelium. The trilaminar substance arises in the cytoplasm of squamous respiratory cells from profiles of granular and smooth endoplasmic reticulum. The integrity and stability of the gas-exchange tissue is likely to be guaranteed by a specific arrangement of the squamous respiratory cells, in which the trilaminar substance plays a paramount role. This general pattern can be observed in strong flying bird species as in the relatively flightless birds.

Pulmonary macrophages in birds (barn owl, Tyto tyto alba), domestic fowl (Gallus gallus f. domestica), quail (Coturnix coturnix), and pigeons (Columbia livia).

BACKGROUND: Birds have a limited number of resident macrophages in the normal steady-state respiratory tract. The discovery of phagocytes in lavages of lung from birds contrasts with findings that phagocytes are seldom seen in investigations in situ. An electron microscopic study was performed in the respiratory units, the parabronchi, and air capillaries in particular in several adult bird species to localize the seat of respiratory macrophages. METHODS: Lung tissue of barn owl, domestic fowl, quail, and town and homing pigeons was subjected to standard processing for light and electron microscopy after immersion fixation, intratracheal instillation, and intravascular perfusion. RESULTS: Clusters of macrophages were predominantly housed in the loose connective tissue at the floor of atria at the entrance to the infundibula and gas-exchange tissue proper. Scattered solitary phagocytes were also found in connective tissue of air sacs, interatrial septa, and adventitia of inter- and intraparabronchial arteries and veins and in peribronchial lymphoid tissue. Phagocytized foreign particulate material mostly consists of hard, dense, crystalline formations surrounded by a limiting membrane. The transport of small airborne particles occurs via the squamous atrial epithelium to the underlying macrophages. The macrophages are often accompanied by mast cells. CONCLUSIONS: The present results demonstrate that avian respiratory macrophages are predominantly located in atrial connective tissue compartments and do not seem to migrate to the airway surfaces.

Projections of neurochemically specified neurons in the porcine colon.

The intramural projections of nerve cells containing serotonin (5-HT), calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP) and nitric oxide synthase or reduced nicotinamide adenine dinucleotide phosphate diaphorase (NOS/NADPHd) were studied in the ascending colon of 5- to 6-week-old pigs by means of immunocytochemistry and histochemistry in combination with myectomy experiments. In control tissue of untreated animals, positive nerve cells and fibres were common in the myenteric and outer submucous plexus and, except for 5-HT-positive perikarya, immunoreactive cell bodies and fibres were also observed in the inner submucous plexus. VIP- and NOS/NADPHd-positive nerve fibres occurred in the ciruclar muscle layer while VIP was also abundant in nerve fibres of the mucosal layer. 5-HT- and CGRP-positive nerve fibres were virtually absent from the aganglionic nerve networks. In the submucosal layer, numerous paravascular CGRP-immunoreactive (IR) nerve fibres were encountered. Myectomy studies revealed that 5-HT-, CGRP-, VIP- and NOS/NADPHd-positive myenteric neurons all displayed anal projections within the myenteric plexus. In addition, some of the serotonergic myenteric neurons projected anally to the outer submucous plexus, whereas a great number of the VIP-ergic and nitrergic myenteric neurons send their axons towards the circular muscle layer. The possible function of these nerve cells in descending nerve pathways in the porcine colon is discussed in relation to the distribution pattern of their perikarya and processes and some of their morphological characteristics.

Immunocytochemical survey of the neuroepithelial endocrine system in the respiratory tract of the Tokyo salamander, Hynobius nebulosus tokyoensis TAgo.

The epithelial lining of the respiratory tract of urodeles has been shown to harbor an innervated system of neuroepithelial endocrine (NEE) cells. Even between phylogenetically closely related species, large differences have been reported in the appearance and chemical coding of the NEE system. Although urodeles are well suited for the purpose, none of the prior studies have provided an immunocytochemical survey of the NEE system in all parts of the respiratory tract. In the present study, many bioactive substances and a general marker were immunocytochemically demonstrated in serial sections of the entire respiratory tract of the Tokyo salamander, Hynobius nebulosus tokyoensis, a species in which neuroepithelial bodies (NEBs) were previously characterized at the electron microscopic level. In the current study, serotonin-immunoreactive solitary NEE cells were observed in variable numbers in the larynx, in all parts of the trachea, and in areas of the lungs covered with ciliomucous epithelium. Serotonin-containing NEBs, however, were detected in small cranial areas of the lung only. Solitary NEE cells were seen in the trachea and lungs of H. nebulosus tokyoensis by immunocytochemical staining for somatostatin, calcitonin, calcitonin gene-related peptide, and bombesin, but the number, localization, and appearance of the labeled NEE cells differed considerably. Only calcitonin-like immunoreactivity was also noted in some NEB-like cell clusters in the cranial parts of the lungs. Unlike many other vertebrates, neuron specific enolase was found to be a poor marker for the NEE system in the salamander species used in this investigation. It may be concluded that the NEE system of H. nebulosus tokyoensis contains at least five different bioactive substances.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Neuroepithelial endocrine and nervous system in the respiratory tract of Cynops pyrrhogaster with special reference to the distribution of nitric oxide synthase and serotonin.

The respiratory tract of urodeles harbours an intramural nerve network comprising an innervated system of neuroepithelial endocrine (NEE) cells. However, striking differences have been noted between phylogenetically closely related species. Zamboni- or formaldehyde-fixed whole-mount preparations and sections of the saclike lungs of a Japanese salamander, Cynops salamander, Cynops pyrrhogaster, have been investigated for the immunocytochemical detection of nitric oxide synthase (NOS), serotonin (5-HT), VIP, somatostatin, calcitonin, and bombesin; for the enzyme-cytochemical demonstration of NADPH diaphorase (NADPHd); and for formaldehyde-induced fluorescence. In addition, the ultrastructural morphology has been examined by using glutaraldehyde/osmium tetroxide fixed lung tissues. Ovoid 5-HT-immunoreactive (IR) NEE cells occur singly or grouped in the ciliomucous epithelium of the trachea and lungs of Cynops, and a few somatostatin-, calcitonin-, and bombesin-like IR NEE cells are also observed. These cells exhibit a characteristic neuroendocrine morphology as seen with the electron microscope. In addition, large numbers of 5-HT-IR interstitial cells, with round to oval cell bodies and two or three long, slender, sometimes branching processes, are located preferentially along large blood vessels in the connective tissue capsule of the lung and trachea. Immunoelectronmicroscopy shows that 5-HT is localized over large dense granules in the cell bodies and processes of these interstitial cells. NOS-like immunoreactivity occurs in a nerve plexus composed of thick nerve bundles and nerve cells, and in a fine varicose nerve network that originates at least partly from intrapulmonary NOS-containing nerve cells. VIP-like immunoreactivity appears to be colocalized with NOS in the latter network. All NOS-positive nerve fibres in the lungs of Cynops pyrrhogaster and Ambystoma mexicanum stain for NADPHd. It is concluded that the pulmonary NEE cells observed in Cynops pyrrhogaster are similar to those described in other vertebrate species and that the 5-HT-IR interstitial cells resemble mast cells. In addition, nitric oxide is likely to be a bioactive substance involved in nonadrenergic, noncholinergic inhibitory neurotransmission in the pulmonary nervous system of urodeles, where it may be colocalized with VIP.

Nitric oxide synthase-containing neurons in the pig large intestine: topography, morphology, and viscerofugal projections.

The distribution of neurons that are capable of synthesizing nitric oxide (NO) has been demonstrated in the porcine large intestine by means of NO synthase (NOS) immunocytochemistry and nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry. An overall colocalization of NOS immunoreactivity and NADPHd staining was observed. Nitrergic neurons were abundant in the myenteric and outer submucous plexus of the caecum, colon, and rectum. Only a few nitrergic perikarya were seen in the inner submucous plexus of the colon and caecum, whereas a substantially larger number was observed in the rectum. Nitrergic nerve fibers were present in the three ganglionic nerve plexuses. Contrary to the outer longitudinal muscle layer and the mucosal region, the circular muscle layer received a dense nitrergic innervation. The nitrergic nerve cells were variable in size and shape, and several displayed vasoactive intestinal polypeptide (VIP) immunoreactivity (IR). Retrograde tracing studies revealed the existence of nitrergic neurons that project to the caudal (inferior) mesenteric ganglion. They were observed in the myenteric and outer submucous plexus of the transverse and descending colon and the rectum. These observations strongly suggest that several subpopulations of NO-synthesizing neurons, namely, motor neurons and interneurons, should be distinguished in the porcine large intestine, thereby emphasizing the importance of NO as a biologically active mediator.

Stages in the development of the rat lung: morphometric, light and electron microscopic studies.

Ontogenic changes of the pulmonary epithelium of the rat, ranging from fetal day 15 to 1 hour after birth (21st day), were observed using light and electron microscopy as well as morphometric analysis on the area occupied by terminal segments of epithelial tubes or by alveolar sacs (ATETAS) in the terminal region of the lung. The development of the lung was classified into four stages. In the pseudoglandular period (fetal days 15 and 16), epithelial tubes lined by columnar epithelium were not yet associated with blood capillaries. The percentages occupied by ATETASs in the terminal region of the lung were 15.3% and 15.7%, respectively, on fetal days 15 and 16. In the precanalicular period (fetal days 17 and 18), blood capillaries began to appose to epithelial tubes lined by cuboidal epithelium. Small osmiophilic lamellar bodies (OLBs) emerged in cuboidal epithelial cells (type II cells) on day 18. The percentages occupied by ATETAS were 24.9% and 25.5%, respectively, on days 17 and 18. In canalicular period (fetal days 19 and 20), sac-like end segments showed progressive thinning of the epithelial linings, and the amount of interstital tissues markedly decreased. The epithelial cells differentiated into squamous, or type I cells, and type II cells containing OLBs. Extrusion of OLBs was recognized on day 20. Most of the capillaries were located close to the epithelial linings. The thickness of the blood-air barrier on day 20 was 10 times that of the adult. The percentages occupied by ATETAS were 35.8% and 38.5%, respectively, on days 19 and 20. In the terminal sac period (in neonate), the wall of terminal sacs showed a thin epithelial lining. Blood capillaries protruded close to the air-way surfaces. The thickness of the blood-air barrier was 3.5 times as thick as that of adult. The percentage occupied by ATETAS was 53.1% of the total terminal region of the lung. When the area occupied by ATETAS at a given day was compared to the area of the adult, the percentage on day 20 was approximately 60% of the adult value, whereas the percentage in neonates was 85%. This suggested that a remarkable transformation took place in ATETAS between day 20 and neonate. In addition to the four periods mentioned above, we also discussed the embryonic and alveolar periods in the development of the rat lung.

The pulmonary neuroepithelial endocrine system in the quail, Coturnix coturnix. Light- and electron-microscopical immunocytochemistry and morphology.

Despite extensive knowledge of the neuroepithelial endocrine (NEE) system in the lungs of species of various vertebrate classes, data on avians are limited. The present investigation deals with the light- and electron-microscopical immunocytochemistry and morphology of pulmonary NEE cells in the quail, Coturnix coturnix. Light-microscopically, serotonin immunoreactivity was detected in numerous solitary and clustered NEE cells located in the cilio-mucous epithelium of primary and secondary bronchi in adult as well as in newly hatched quails. Only in newly hatched quails could a small number of bombesin- and somatostatin-like immunoreactive NEE cells be demonstrated. Electron-microscopical morphology revealed that NEE cells contained dense-cored vesicles of a wide range of diameters and electron densities. Nearly all of the NEE cells were seen to rest on the basement membrane of the cilio-mucous epithelium, lacking direct contact with the luminal surface. Nerve varicosities or nerve endings, of both afferent and efferent morphological appearance, were found directly apposed to the basal portion of NEE cells, invaginating between NEE cells or between NEE cells and adjacent epithelial cells. Often, synaptic specializations could be recognized between NEE cells and nerve terminals. Electron-microscopical immunocytochemistry confirmed that the intraepithelial serotonin-containing cells correspond to the cells with NEE characteristics. Moreover, two types of NEE cells could be distinguished in newly hatched quail lungs. Both types showed serotonin immunoreactivity selectively distributed over the dense-cored vesicles, but somatostatin- and bombesin-like immunoreactivities were only noted in one of the NEE cell types and were never seen colocalized. Thus, the avian NEE system too, harbors at least three different bioactive substances and has a morphology comparable to that of mammals, reptiles and amphibians.

Nitric oxide synthase immunoreactivity in the enteric nervous system of the developing human digestive tract.

We have investigated indirectly the presence of nitric oxide in the enteric nervous system of the digestive tract of human fetuses and newborns by nitric oxide synthase (NOS) immunocytochemistry and nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry. In the stomach, NOS immunoactivity was confined to the myenteric plexus and nerve fibres in the outer smooth musculature; few immunoreactive nerve cell bodies were found in ganglia of the outer submucous plexus. In the pyloric region, a few nitrergic perikarya were seen in the inner submucous plexus and some immunoreactive fibers were found in the muscularis mucosae. In the small intestine, nitrergic neurons clustered just underneath or above the topographical plane formed by the primary nerve strands of the myenteric plexus up to the 26th week of gestation, after which stage, they occurred throughout the ganglia. Many of their processes contributed to the dense fine-meshed tertiary nerve network of the myenteric plexus and the circular smooth muscle layer. NOS-immunoreactive fibres directed to the circular smooth muscle layer originated from a few NOS-containing perikarya located in the outer submucous plexus. In the colon, caecum and rectum, labelled nerve cells and fibres were numerous in the myenteric plexus; they were also found in the outer submucous plexus. The circular muscle layer had a much denser NOS-immunoreactive innervation than the longitudinally oriented taenia. The marked morphological differences observed between nitrergic neurons within the developing human gastrointestinal tract, together with the typical innervation pattern in the ganglionic and aganglionic nerve networks, support the existence of distinct subpopulations of NOS-containing enterice neurons acting as interneurons or (inhibitory) motor neurons.

Topographical distribution and immunocytochemical features of colonic neurons that project to the cranial mesenteric ganglion in the pig.

Using the retrograde neuronal tracers Fast blue and Fluorogold, the topographical distribution and morphological features of porcine colonic neurons projecting to the cranial (superior) mesenteric ganglion have been investigated. Two to four weeks after injection of the tracer into the cranial mesenteric ganglion of immature pigs, labelled neurons were found throughout the colon. In the myenteric and outer submucous plexuses, they were present in ganglia situated to the side of the mesenteric attachment. The highest density of labelled neurons was observed at the end of the ascending colon, which in the pig represents 78-80% of the total colon length. The viscerofugal neurons had a multidendritic appearance and part of them were immunoreactive for calcitonin gene-related peptide or serotonin. This study has revealed similarities but also significant differences in the colono-sympathico-colonic pathways between the pig and small laboratory animals such as the guinea-pig.

Distribution and morphological features of nitrergic neurons in the porcine large intestine.

The distribution of nitric oxide synthase (NOS), an enzyme involved in the synthesis of the presumed non-adrenergic noncholinergic inhibitory neurotransmitter nitric oxide (NO), was demonstrated in the enteric nervous system of the porcine caecum, colon and rectum. Techniques used were NOS-immunocytochemistry and nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd)-histochemistry. Throughout the entire large intestine, NOS-immunoreactive (IR) and NADPHd-positive neurons were abundant in the myenteric and outer submucous plexus. In the inner submucous plexus, only a small number of positive neurons were found in the caecum and colon, while a moderate number was observed in the rectum. The nitrergic neurons in the porcine enteric nerve plexuses were of a range of sizes and shapes, with a small proportion showing immunostaining for vasoactive intestinal polypeptide. Varicose and non-varicose NOS-IR and NADPHd-positive nerve fibres were present in the ganglia and connecting strands of all three plexuses. Nerve fibres were also numerous in the circular muscle layer, scarce in the longitudinal muscle coat and negligible in the mucosal region. The abundance of NOS/NADPHd in the intrinsic innervation of the caecum, colon and rectum of the pig implicates NO as an important neuronal messenger in these regions of the gastrointestinal tract.

Occurrence, distribution and neurochemical features of small intestinal neurons projecting to the cranial mesenteric ganglion in the pig.

The small intestine of the pig has been investigated for its topographical distribution of enteric neurons projecting to the cranial mesenteric ganglion, by using Fast Blue or Fluorogold as a retrogradely transported neuronal tracer. Contrary to the situation in small laboratory animals such as rat and guinea-pig, the intestinofugally projecting neurons in the porcine small intestine were not restricted to the myenteric plexus, but were observed in greater numbers in ganglia of the outer submucous plexus. The inner submucous plexus was devoid of labelled neurons. Retrogradely labelled neurons were mostly found, either singly or in small aggregates, in ganglia located within a narrow border on either side of the mesenteric attachment. For both nerve networks, their number increased from duodenum to ileum. All the retrogradely labelled neurons exhibited a multidendritic uniaxonal appearance. Some of them displayed type-III morphology and stained for serotonin. This study indicates that, in the pig, not only the myenteric plexus but also one submucous nerve network is involved in the afferent component of intestino-sympathico-intestinal reflex pathways. The finding that some of the morphologically defined type-III neurons participate in these reflexes is in accord with the earlier proposal that type-III neurons are supposed to fulfill an interneuronal role, whether intra- or extramurally.

Microspectrofluorimetric study of monoamines in the hypothalamus of Scyliorhinus stellaris L.

In a combined fluorescence histochemical and microspectrofluorimetrical study, the distribution, morphology and amino content of the hypothalamic circumventricular organs of an elasmobranch (Scyliorhinus stellaris) have been investigated. In the walls of the third ventricle, two aminergic circumventricular organs, viz. the preoptic recess organ and organon vasculosum hypothalami, were found to display an intense blue-green fluorescence. The emission maximum was registered at about 490 nm and the excitation spectra, conducted from 240 nm to 460 nm at neutral pH, displayed a main peak at 410 nm, with additional peaks at about 320 nm and 260 nm, values that are indicative of catecholamines. In neutral state, the quotient of the excitation peak ratio values 410/260 nm exceeded 1, thus determining the differentiation between noradrenaline and adrenaline, excluding the latter as a secondary monoamine. Recordings of hydrochloric acid (HCl)-treated sections showed a hypsochromic shift of the main excitation peak from 410 nm to 370 nm and a concurrent increase of the peak at 320 nm, with the mean peak ratio 370/320 nm being 0.98, pointing to the presence of dopamine. The fading curves indicated a photodecomposition of less than 20%, both in neutral and acidified sections, which is typical of dopamine. It can therefore be deduced that the blue-green fluorescent cells in the hypothalamic circumventricular organs of the dogfish contained mainly dopamine. Some cells expressed a yellowish fluorescence, showing rapid photodecomposition characteristic of indolethylamine fluorophores, pointing to the presence of 5-hydroxytryptamine (5-HT).