Localisation of substance P-, somatostatin-, vasoactive intestinal polypeptide and met-enkephalin-immunoreactive nerves in the peripheral and central nervous systems of the leech (Hirudo medicinalis)

Summary The distribution of substance P (SP)-, somatostatin (SOM)-, vasoactive intestinal polypeptide (VIP)- and met-enkephalin (mENK)-immunoreactive nerve fibres and cell bodies has been studied in the gastrointestinal tract, lateral blood vessel (heart) and segmental ganglia of the leech (Hirudo medicinalis). In the crop and intestine, there was a sparse distribution of VIP-, SP-, SOM- and mENK-immunoreactive nerves, while in the intestine, a dense network of SP-, a moderate network of SOM-, and a sparse distribution of mENK- and VIP-immunoreactive nerve fibres was seen. SP-, SOM- and VIP-immunoreactive nerve cell bodies were found in all the gut regions studied, the greatest number being in the intestine. No mENK-containing cell bodies were seen in any region of the gastrointestinal tract. The heart contained a few SP-, SOM-, and VIP-immunoreactive nerve fibres, but no nerve cell bodies were found. Immunoreactive nerve cell bodies were also present in the segmentai ganglia. A typical midbody ganglion contained up to seven pairs of SP-containing neurones, four pairs of SOM-containing neurones, two pairs of VIP-containing neurones and one to three pairs of mENK-immunoreactive nerve cell bodies. The lateral pair of large SOM-immunoreactive nerve cell bodies is of similar size and correct position to the lateral N cells. One of the pairs of large SP-immunoreactive nerve cell bodies is probably identical to the Leydig cells. A tentative identification of other immunofluorescent nerve cells is attempted. Immunoreactive nerve fibres to all four peptides were distributed throughout the neuropil, those to SP being the most numerous.     

Light and electron microscopic localization of GABA-like immunoreactivity in myenteric plexus of chicken

Summary Whole-mounts of 1-day-old chicken midgut were incubated with an antiserum against GABA-glutaraldehyde-BSA conjugate. The immunoreaction was visualized by using the peroxidase-antiperoxidase method, and processed for consecutive light and electronmicroscopic observation. GABA was selectively localized in some of the varicose and nonvaricose nerve fibres of the myenteric plexus. The varicose fibres formed dense networks within the myenteric ganglia, some of which — mainly in duodenum — also contained immunopositive nerve cell bodies. Some of the varicose fibres projected out from the myenteric plexus into the circular muscle layer. At the electronmicroscopic level, labelled axon terminals formed synaptic contact with unlabelled perikarya and vica versa. At the same time, no labelled terminals were found on immunostained cells. In a few cases, axon terminals with GABA positivity were situated close to the smooth muscle cells in the circular muscle layer, suggesting a prejunctional GABA effect on the neighbouring nerve terminals on the release of their transmitters.     

The origin and possible significance of substance P immunoreactive networks in the prevertebral ganglia and related structures in the guinea-pig

The distribution and origin of substance P immunoreactive nerve elements have been studied in the guinea-pig prevertebral ganglia by the indirect immunohistochemical technique, using a monoclonal antibody to substance P. Non-varicose substance P immunoreactive nerve fibres enter or leave the ganglia in all nerves associated with them, traversing the ganglia in larger or smaller bundles. Networks, mainly single-stranded, of varicose substance P immunoreactive nerve fibres also permeate the ganglia, forming a loose meshwork among the neurons. Similar networks are present in the lumbar paravertebral ganglia. In all these ganglia, neuronal somata do not in general show substance P immunoreactivity. The various nerves connected with the inferior mesenteric ganglion have been cut, in single categories and in various combinations, and the ganglion examined, after intervals of up to six days. Cutting the colonic or hypogastric nerves, which connect the ganglion with the hindgut and pelvic organs, leads to accumulation of substance P immunoreactive material in their ganglionic stumps, extending retrogradely to intraganglionic non-varicose fibres traceable through into the intermesenteric and lumbar splanchnic nerves. There is some local depletion of intraganglionic varicose networks. Cutting the intermesenteric nerve, which connects the coeliac-superior mesenteric ganglion complex with the ganglion, leads to accumulation of substance P immunoreactive material in its cranial stump and depletion of its distal stump; a minimal depletion is detectable in the inferior mesenteric ganglion itself. Cutting the lumbar splanchnic nerves, which connect the ganglion with the upper lumbar spinal cord and dorsal root ganglia, leads to accumulation of substance P immunoreactive material in their proximal stumps and total depletion of their distal, ganglionic stumps; in the ganglion there is subtotal loss of non-varicose substance P immunoreactive fibres and of varicose nerve networks, and the few surviving non-varicose fibres are traceable across the ganglion from the intermesenteric nerve to the colonic and hypogastric nerves. Cutting the intermesenteric and lumbar splanchnic nerves virtually abolishes substance P immunoreactive elements from the ganglion within three days postoperatively. It is concluded that these arise centrally to the ganglion.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ontogeny of the expression of some catecholamine synthesising enzymes in the female porcine preoptic area

Ontogeny of the catecholaminergic system of the preoptic area (PA) was studied in various animal species including mice, rats, cats and lower vertebrates. Until now, there has been no data about development of catecholaminergic structures in the porcine PA. To study this problem, hypothalami from six groups of animals were collected. Three groups of foetuses (70, 84 and 112 days old) and three groups of female pigs (1 day, 10 weeks and 7-8 months old) were used. Nerve structures immunoreactive for the studied substances: tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DbetaH) and phenylethanoloamine-N-metylthransferase (PNMT) were observed in different periods. In PA, TH-IR (immunoreactive) structures appeared before 70th day of foetal life, DbetaH-IR between 70th and 84th day of foetal live and PNMT-IR only in 10-week old and adult animals. In the PA of 70-day old foetuses, single smooth and varicose nerve fibres immunoreactive only to TH were found. In PA of 84-day old foetuses, additionally, single nerve cell bodies immunoreactive to TH were shown and some of them also contained immunoreactivity to DbetaH. In PA of 1-day old piglets, moderate numbers of nerve fibres immunoreactive to TH and only single TH/DbetaH-IR nerve terminals were observed. TH-IR nerve cell bodies were also moderate in number and many of them contained simultaneously immunoreactivity to DbetaH. In PA of 10-week old pigs, a moderate number of immunopositive nerve fibres was observed. They contained mainly TH, but part of them stained also for TH/DbetaH. Only very few nerve fibres containing exclusively DbetaH were found. These nerve terminals were observed in a close vicinity of blood vessels. In PA, moderate numbers of TH-IR nerve cell bodies were found, some of them contained also immunoreactivity to DH but never to PNMT. Perikarya containing PNMT were TH-negative. In the PA of sexually mature sows, additional, single, large nerve cell bodies (about 35 microm in a diameter) containing TH only were found. In many cases, TH- and DbetaH-IR "basket-like" structures surrounding nerve cell bodies were seen, suggesting an influence of those fibres on the neuronal activity.     

Distribution of GABA-like immunoreactivity in myenteric plexus of carp, frog and chicken

The distribution of GABA-like immunoreactivity was studied by means of indirect immunocytochemical methods in some lower vertebrate species (carp, frog, chicken). An immunoreactive network was revealed in the myenteric plexus of the alimentary canal of carp. GABA-positive nerve cells were attached closely to the fibres in the stomach. In other gut regions immunostained neurons were less frequent. Immunoreactive fibres often formed baskets on the surfaces of immunonegative neurons along the whole length of the alimentary canal. The number of immunopositive nerve fibres and pericellular baskets seemed to be lower in the mid- and hingut than in the foregut region. A similar distribution of GABA-immunoreactivity was revealed in the frog myenteric plexus. The ganglionated foregut region possessed a relatively dense GABAergic innervation. This part of the gut contained immunostained nerve cells and fibres, while the mid- and hindgut possessed only a scanty fibre system. Chicken exhibited an extensive immunoreactive plexus for GABA, although the GABA-stained perikarya were restricted mainly to the duodenum. Further regions of the small intestine were poor in immunoreactive cell bodies, which suggests a segmental origin and arrangement of GABAergic innervation within the plexus. In all three species studied, GABA-positive fibres run into the circular muscle layer. The varicosity suggests their influence on the movement of the smooth muscles through modifying the transmitter release of neighbouring terminals.     

Leech Retzius cells and 5-hydroxytryptamine

A pair of giant Retzius (R) cells in each segmental ganglion of the leech contain 5-hydroxytryptamine (5-HT). They are the only 5-HT-containing neurones in the central nervous system to send branches to the periphery, yet many peripheral tissues (e.g. body wall muscles, heart, reproductive organs, nephridia and gut) possess 5-HT-like immunoreactive nerve fibres. 5-HT and/or R cell stimulation relax basal tension of body wall muscles and reduce their relaxation times following contraction, enhance pharyngeal movements and salivary gland secretion but inhibit muscle movements of the posterior gut regions and of the reproductive tract. It is suggested that R cells are multifunction neurones modulating activity of many tissues so that feeding behaviour of the leech is carried out as efficiently as possible.     

Light and electron microscopic localization of GABA-like immunoreactivity in myenteric plexus of chicken

Whole-mounts of 1-day-old chicken midgut were incubated with an antiserum against GABA-glutaraldehyde-BSA conjugate. The immunoreaction was visualized by using the peroxidase-antiperoxidase method, and processed for consecutive light and electronmicroscopic observation. GABA was selectively localized in some of the varicose and nonvaricose nerve fibres of the myenteric plexus. The varicose fibres formed dense networks within the myenteric ganglia, some of which--mainly in duodenum--also contained immunopositive nerve cell bodies. Some of the varicose fibres projected out from the myenteric plexus into the circular muscle layer. At the electronmicroscopic level, labelled axon terminals formed synaptic contact with unlabelled perikarya and vica versa. At the same time, no labelled terminals were found on immunostained cells. In a few cases, axon terminals with GABA positivity were situated close to the smooth muscle cells in the circular muscle layer, suggesting a prejunctional GABA effect on the neighbouring nerve terminals on the release of their transmitters.     

Distribution of GABA-like immunoreactivity in myenteric plexus of carp,frog and chicken

Summary The distribution of GABA-like immunoreactivity was studied by means of indirect immunocytochemical methods in some lower vertebrate species (carp, frog, chicken). An immunoreactive network was revealed in the myenteric plexus of the alimentary canal of carp. GABA-positive nerve cells were attached closely to the fibres in the stomach. In other gut regions immunostained neurons were less frequent. Immunoreactive fibres often formed baskets on the surfaces of immunonegative neurons along the whole length of the alimentary canal. The number of immunopositive nerve fibres and pericellular baskets seemed to be lower in the mid- and hindgut than in the foregut region. A similar distribution of GABA-immunoreactivity was revealed in the frog myenteric plexus. The ganglionated foregut region possessed a relatively dense GABAergic innervation. This part of the gut contained immunostained nerve cells and fibres, while the mid- and hindgut possessed only a scanty fibre system. Chicken exhibited an extensive immunoreactive plexus for GABA, although the GABA-stained perikarya were restricted mainly to the duodenum. Further regions of the small intestine were poor in immunoreactive cell bodies, which suggests a segmental origin and arrangement of GABAergic innervation within the plexus. In all three species studied, GABA-positive fibres run into the circular muscle layer. The varicosity suggests their influence on the movement of the smooth muscles through modifying the transmitter release of neighbouring terminals.     

Distribution of Eisenia tetradecapeptide immunoreactive neurons in the nervous system of earthworms

A detailed mapping of Eisenia-tetradecapeptide-immunoreactive neurons in the central and peripheral nervous system combined with quantitative morphological measurements was performed in Eisenia fetida and Lumbricus terrestris. In Eisenia, most labelled neurons were observed in the ganglia of the ventral cord (20.38% of the total cell number of the ganglion) and 15.67% immunoreactive cells occurred in the brain, while 6% of the neurons could be shown in the subesophageal ganglion. In the case of Lumbricus, most immunoreactive cells were found in the subesophageal ganglion (16.17%) and in the ventral ganglia (12.54%). The brain contained 122 ETP-immunoreactive cells (5.6%). The size of the immunoreactive cells varied between 35-75 microm. A small number of Eisenia-tetradecapeptide immunoreactive fibres were seen to leave the ventral ganglia via segmental nerves, and labelled processes could also be observed in the stomatogastric system and the body wall. Labelled axon branches originating from the segmental nerves formed an immunoreactive plexus both between the circular and longitudinal muscle layer and on the inner surface of the longitudinal muscle layer. This inner plexus was especially rich in the setal sac. Among the superficial epithelial cells the body wall contained a significant number of immunoreactive cells. Only a few Eisenia-tetradecapeptide immunoreactive neurons and fibres occurred in the stomatogastric ganglia. In the enteric plexus the number of immunoreactive neurons and fibres decreased along the cranio-caudal axis of the alimentary tract. Eisenia-tetradecapeptide immunoreactive cells were also present among the epithelial cells in the alimentary canal. Some of these cells resembled sensory neurons in the foregut, while others showed typical secretory cell morphology in the midgut and hindgut.     

Glutamate-like immunoreactivity in the leech central nervous system

Summary Using a monoclonal antibody for glutamate the distribution was determined of glutamate-like immunoreactive neurons in the leech central nervous system (CNS). Glutamate-like immunoreactive neurons (GINs) were found to be localized to the anterior portion of the leech CNS: in the first segmental ganglion and in the subesophageal ganglion. Exactly five pairs of GINs consistently reacted with the glutamate antibody. Two medial pairs of GINs were located in the subesophageal ganglion and shared several morphological characteristics with two medial pairs of GINs in the first segmental ganglion. An additional lateral pair of GINs was also located in segmental ganglion 1. A pair of glutamate-like immunoreactive neurons, which are potential homologs of the lateral pair of GINs in segmental ganglion 1, were occasionally observed in more posterior segmental ganglia along with a selective group of neuronal processes. Thus only a small, localized population of neurons in the leech CNS appears to use glutamate as their neurotransmitter.     

Distribution of FMRFamide-like immunoreactivity in the nervous system of the slug Limax maximus

Summary The distribution of FMRFamide-like immunoreactive neurons in the nervous system of the slug Limax maximus was studied using immunohistochemical methods. Approximately one thousand FMRFamide-like immunoreactive cell bodies were found in the central nervous system. Ranging between 15 m and 200 m in diameter, they were found in all 11 ganglia of the central nervous system. FMRFamide-like immunoreactive cell bodies were also found at peripheral locations on buccal nerve roots. FMRFamide-like immunoreactive nerve fibres were present in peripheral nerve roots and were distributed extensively throughout the neuropil and cell body regions of the central ganglia. They were also present in the connective tissue of the perineurium, forming an extensive network of varicose fibres. The large number, extensive distribution and great range in size of FMRFamide-like immunoreactive cell bodies and the wide distribution of immunoreactive fibres suggest that FMRFamide-like peptides might serve several different functions in the nervous system of the slug.     

Glutamate-like immunoreactivity in the leech central nervous system.

Using a monoclonal antibody for glutamate the distribution was determined of glutamate-like immunoreactive neurons in the leech central nervous system (CNS). Glutamate-like immunoreactive neurons (GINs) were found to be localized to the anterior portion of the leech CNS: in the first segmental ganglion and in the subesophageal ganglion. Exactly five pairs of GINs consistently reacted with the glutamate antibody. Two medial pairs of GINs were located in the subesophageal ganglion and shared several morphological characteristics with two medial pairs of GINs in the first segmental ganglion. An additional lateral pair of GINs was also located in segmental ganglion 1. A pair of glutamate-like immunoreactive neurons, which are potential homologs of the lateral pair of GINs in segmental ganglion 1, were occasionally observed in more posterior segmental ganglia along with a selective group of neuronal processes. Thus only a small, localized population of neurons in the leech CNS appears to use glutamate as their neurotransmitter.     

Monoamines in the nervous system of the tube-wormChaetopterus variopedatus (Polychaeta): Biochemical detection and serotonin immunoreactivity

Summary The presence and distribution of biogenic monoamines in the tube-wormChaetopterus variopedatus were investigated by a radioenzymatic method and HPLC with electrochemical detection, and the cellular localization of serotonin by peroxidase-antiperoxidase (PAP) immunohistochemistry with an antibody against serotonin-formaldehyde-protein conjugate. Dopamine, norepinephrine, epinephrine, serotonin (5-HT) and some of their metabolites were detectable, dopamine and norepinephrine being present in substantially larger amounts than 5-HT and epinephrine. With few exceptions, the largest amounts of amines were localized in the most nerve-rich tissues such as tentacles, and those containing cerebral ganglia and the ventral nerve cord. Serotonin-immunoreactive unipolar neurons were widely distributed in the dorso-lateral cerebral ganglia, the neurosecretory pharyngeal ganglion and the segmental ganglia of the anterior (dorsolateral) and posterior (medioventral) nerve cords. Some nerve-fiber tracts stained in the cerebral ganglia, but the neuropiles of segmental ganglia were the most intensely reactive CNS structures. Numerous reactive fibers were also present in connectives, commissures and segmental nerves. All peripheral sensory structures included serotonin-immunoreactive cells and neurites, especially the parapodial cirri and the bristle receptors of the setae. Trunk and parapodial muscles contained reactive varicose fibers and neuronal somata. These results suggest that monoamines are abundant and widespread in these worms and that 5-HT appears to have a key sensory role.     

Localization of a substance P-like material in the central and peripheral nervous system of the snail Helix aspersa

Summary A monoclonal antibody against substance P was used for immunocytochemical staining of the central ganglia of the snail Helix aspersa and several peripheral tissues including the gut, reproductive system, cardiovascular system, tentacle and other muscles.Within the central ganglia many neurones, and many fibres in the neuropile and the nerves entering the ganglia, were stained for the SP-like material. The largest numbers of reactive cell bodies were in the pleural ganglia and on the dorsal surfaces of the pedal ganglia. A group of cells was also found, surrounding the right pedal-cerebral connective, that did not fluoresce, but were enveloped by reactive processes terminating directly onto the neurone somata.Specific staining was observed in all peripheral tissues examined and always appeared to be concentrated in nerve terminals. Most particularly these occurred in the heart and aorta, the pharyngeal retractor muscle and the tentacle. Although mostly present in muscular tissues, some fluorescence was also observed in the nervous layer surrounding the retina. The tentacular ganglion also contained immunoreactive cell bodies.     

Localization of a substance P-like material in the central and peripheral nervous system of the snail Helix aspersa

A monoclonal antibody against substance P was used for immunocytochemical staining of the central ganglia of the snail Helix aspersa and several peripheral tissues including the gut, reproductive system, cardiovascular system, tentacle and other muscles. Within the central ganglia many neurons, and many fibres in the neuropile and the nerves entering the ganglia, were stained for the SP-like material. The largest numbers of reactive cell bodies were in the pleural ganglia and on the dorsal surfaces of the pedal ganglia. A group of cells was also found, surrounding the right pedal-cerebral connective, that did not fluoresce, but were enveloped by reactive processes terminating directly onto the neurone somata. Specific staining was observed in all peripheral tissues examined and always appeared to be concentrated in nerve terminals. Most particularly these occurred in the heart and aorta, the pharyngeal retractor muscle and the tentacle. Although mostly present in muscular tissues, some fluorescence was also observed in the nervous layer surrounding the retina. The tentacular ganglion also contained immunoreactive cell bodies.     

The distribution and colocalization of neuropeptides in perivascular nerves innervating the large arteries and veins of the snake,Elaphe obsoleta

Summary Single- and dual-labelling immunohistochemistry were used to determine the distribution and coexistence of neuropeptides in perivascular nerves of the large arteries and veins of the snake, Elaphe obsoleta, using antibodies for vasoactive intestinal polypeptide, substance P, calcitonin gene-related peptide, neuropeptide Y, galanin, somatostatin, and leu-enkephalin. Blood vessels were sampled from four regions along the body of the snake: region 1, arteries and veins anterior to the heart; region 2, central vasculature 5 cm anterior and 10 cm posterior to the heart; region 3, arteries and veins in a 30-cm region posterior to the liver; and region 4, dorsal aorta and renal arteries, renal and intestinal veins, 5–30 cm cephalad of the vent. A moderate to dense distribution of vasoactive intestinal polypeptide-like immunoreactive fibres was found in most arteries and veins of regions 1–3, but fibres were absent from the vessels of region 4. The majority of vasoactive intestinal polypeptide-like immunoreactive fibres contained colocalized substance P-like immunoreactivity, and these fibres were unaffected by either capsaicin or 6-hydroxydopamine (6-OHDA) pretreatment. In the anterior section of the snake, the vagal trunks contained many cell bodies with colocalized vasoactive intestinal polypeptide and substance P-like immunoreactivity. It is suggested that the vasoactive intestinal polypeptide/substance P-like immunoreactive cell bodies and fibres are parasympathetic postganglionic nerves. Neuropeptide Y-like immunoreactive fibres were observed in all arteries and veins, being most dense in regions 3 and 4. The majority of these fibres also contained colocalized galanin-like immunoreactivity, and were absent in tissues from 6-OHDA pretreated snakes, suggesting that neuropeptide Y and galanin are colocalized in adrenergic nerves. A small number of neuropeptide Y-like immunoreactive fibres contained vasoactive intestinal polypeptide but not galanin, and were unaffected by 6-OHDA treatment. All calcitonin gene-related peptide-like immunoreactive fibres contained colocalized substance P-like immunoreactivity, and these fibres were observed in all vessels, being particularly dense in the carotid artery and jugular veins. All calcitonin gene-related peptide/substance P-like immunoreactive fibres appeared damaged after capsaicin treatment suggesting they represent fibres from afferent sensory neurons. A sparse plexus of somatostatin-like immunoreactive fibres was observed in the vessels only from region 4. No enkephalin-like immunoreactive fibres were found in any blood vessels from any region. This study provides morphological evidence to suggest that there is considerable functional specialization within the components of the rat snake peripheral autonomic system controlling the circulation, in particular the regulation of venous capacitance.     

Localization in the gastrointestinal tract of immunoreactive prosomatostatin

Antisera against 5 different regions of the entire prosomatostatin molecule were used for immunohistochemical mapping of prosomatostatin-containing structures in the pig gastrointestinal tract, and for radioimmunological and chromatographical analysis of the products of prosomatostatin in extracts of ileal mucosa. The latter showed that the antisera were capable of identifying components containing N-terminal as well as C-terminal parts of prosomatostatin. Endocrine cells were identified with all antisera in most parts of the gastrointestinal tract, and varicose nerve fibres were observed in all parts of the small intestine but not in the stomach and the colon. The colon contained very few immunoreactive structures. Immunoreactive nerve cell bodies were found in the submucous plexus of the small intestine. All immunoreactive endocrine cells in the stomach and the duodenum and all immunoreactive nerves were stained by all 5 antisera whereas the small intestinal endocrine cells did not stain for the most N-terminal region of prosomatostatin. The results suggest that all gastrointestinal somatostatin is derived from the same precursor molecule, which, however, in the small intestinal endocrine cells is processed differently from that of the other tissues.     

Control of leech swimming activity by the cephalic ganglia

We investigated the role played by the cephalic nervous system in the control of swimming activity in the leech, Hirudo medicinalis, by comparing swimming activity in isolated leech nerve cords that included the head ganglia (supra- and subesophageal ganglia) with swimming activity in nerve cords from which these ganglia were removed. We found that the presence of these cephalic ganglia had an inhibitory influence on the reliability with which stimulation of peripheral (DP) nerves and intracellular stimulation of swim-initiating neurons initiated and maintained swimming activity. In addition, swimming activity recorded from both oscillator and motor neurons in preparations that included head ganglia frequently exhibited irregular bursting patterns consisting of missed, weak, or sustained bursts. Removal of the two head ganglia as well as the first segmental ganglion eliminated this irregular activity pattern. We also identified a pair of rhythmically active interneurons, SRN1, in the subesophageal ganglion that, when depolarized, could reset the swimming rhythm. Thus the cephalic ganglia and first segmental ganglion of the leech nerve cord are capable of exerting a tonic inhibitory influence as well as a modulatory effect on swimming activity in the segmental nerve cord.     

Immunohistochemical study on the distribution of vasoactive intestinal polypeptide (VIP) containing nerve fibers in the chicken pancreas

The distribution of vasoactive intestinal polypeptide (VIP) containing nervous elements in the chicken pancreas was immunohistochemically investigated by light microscopy. Strongly VIP immunoreactive ganglia existed in the interlobular connective tissue. Ganglion containing both VIP immunoreactive and non-immunoreactive nerve cells was occasionally observed in the connective tissue. Almost all the ganglion cells also showed acetylcholinesterase (AChE) activity. No extrapancreatic nerve bundles containing VIP immunoreactive nerve fibres were detected. VIP immunoreactive nerve fibres formed plexuses in the subepithelial layer of secretory ducts and the muscle layer of small arteries. The distribution pattern of VIP immunoreactive nerve fibers was similar to that of AChE-positive nerve fibers on adjacent sections. The exocrine pancreas received a rich supply of varicose nerve fibers showing VIP immunoreactivity. B-islets also were richly innervated by VIP immunoreactive varicose nerve fibers, whereas A-islets, only poorly. These observations suggest that VIP containing nerves in the chicken pancreas have an intrinsic origin, are probably derived from VIP immunoreactive, intrapancreatic ganglion cells and innervate secretory ducts, arteries, acinar cells and B-islets, and that VIP must coexist with acetylcholine in the nervous elements.     

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 fibres 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 existenc of distinct subpopulations of NOS-containing enterice neurons acting as interneurons or (inhibitory) motor neurons.