Neurotransmitters, neuromodulators, and neurotrophin receptors in the gut of pantex, a hybrid sparid fish (Pagrus major x Dentex dentex). Localizations in the enteric nervous and endocrine systems

The gut of Pantex, a sparid hybrid fish (Pagrus major x Dentex dentex) with a great potential importance for the Italian aquaculture, was histochemically and immunohistochemically investigated in order to evidence components of the intramural nervous and diffuse endocrine systems. The general structural aspects of the intramural nervous system were shown by the Nissl-thionin staining. As in most other fish, it was only organized in the myenteric plexus. Acetylcholinesterase (AChE) activity was observed in both nerve cell bodies and terminals all along the gut. The NADPH-diaphorase reactivity too, possibly linked to the synthesis and release of nitric oxide, was present in nerve cell bodies and nerve terminals of the oesophagus, stomach and intestine. In addition, the intramural nervous system was shown to contain Trk (tyrosinekinase) receptors for neurotrophin, as evidenced by Trk A-, Trk B- and Trk C-like immunoreactivities, thus suggesting an involvement of neurotrophin in the function of this system. Trk B- and Trk C-like immunoreactivities were detected in epithelial endocrine cells, too. The additional presence of serotonin- and metenkephalin-like immunoreactivities in numerous endocrine cells in the epithelial layers of the stomach and intestine was showed.     

Outgrowth of sympathetic adrenergic neurons in mice treated with a nerve-growth factor (NGF)

Summary Various tissues from mice treated with a nerve-growth factor (NGF) were studied with the histochemical technique ofFalck andHillarp, which visualizes the adrenergic transmitter in the sympathetic postganglionic neurons. Growth stimulation was detectable in all parts of the sympathetic adrenergic neurons. An increased density of the adrenergic ground plexus was observed in e.g. the iris, submaxillary and parotid glands, blood vessels and intramural ganglionic plexuses of the intestinal tract. Normally non-innervated tissues were also found to contain a considerable number of adrenergic terminals. Of special interest is the striking increase in number of adrenergic terminals in various types of autonomic ganglia, in all probability with an inhibitory effect on ganglionic transmission.This investigation was supported by research grants from the Swedish Medical Research Council (B67-12x-714-02), Magnus Bergwalls stiftelse and Stiftelsen Therese och Johan Anderssons Minne. The skillful technical assistance of MissBarbro Riese is gratefully acknowledged.     

Histo- and immunocytochemical characterization of the neurons of the mucosal plexus in the rat colon.

The mucosal plexus of the rat colon descendens is constituted of a network of nerves that, in contrast to most other segments of the digestive tract, contains also ganglia. The ganglia, consisting of neurons and glial cells, are located in the basal part of the lamina propria at distances between 100 and 1,200 microns. They are not vascularized. The neurons in these ganglia were characterized by means of: (1) the histochemical demonstration of acetylcholinesterase (AChE) activity, (2) the immunocytochemical identification of neurofilament proteins (NFP; 200 kD) and (3) their ultrastructure. The glial cells, which were AChE negative, could be distinguished from the neurons by differences in size and chromatin pattern. All neurons of the mucosal plexus reveal AChE activity in the perikaryon, but only parts of the axons are AChE positive. NFP-like immunoreactivity was detected in the perikarya but only in a minor part of the axons. These findings confirm previous light-microscopical observations and add new evidence for the existence of neurons (ganglia) in the mucosal plexus of the rat colon.     

Reflex pressure response to the electric stimulation of different parts of the rat stomach

The role of the autonomic nervous system in the pressor response to the electrical stimulation of different gastric zones has been studied in rats. The stimulus was applied before and after the following interventions: bilateral vagotomy, ganglionic blockade, alpha-adrenergic receptor blockade and beta-adrenergic receptor blockade. After the ganglionic blockade no pressor responses to the electrical stimulus were observed. After the alpha-adrenergic blockade a lower pressor response was observed. A hypertensive response can be induced by mechanical, chemical or electrical stimulation of gastric receptors. It is concluded that the pressor reflex following the application of an electrical stimulus on different zones of the digestive tract is mediated by the sympathetic nervous system and that the efferent pathways are mainly alpha-adrenergic ones.     

The vascularization of the submucous external plexus (Schabadasch) and the submucous internal plexus (Meissner) in the small intestine of swine and cat

Each ganglion of the plexus Schabadasch is supplied by a specific periganglionic capillary network. Within the plexus Meissner, several ganglia are connected to circulation areas. The communicating branches of the plexus Schabadasch have their own capillary system; as to the plexus Meissner, this is valid for the cat only. The results allow to conclude that the centres of the intramural nervous system of the intestinal wall are equipped with a preferred and self-acting vascularization.     

大鼠肠道内NOS与AChE、VIP阳性神经元的分布关系研究

应用一氧化氮合酶 (NOS)、乙酰胆碱酯酶 (ACh E)组织化学及血管活性肠肽 (VIP)免疫组织化学方法 ,光镜下比较观察大鼠肠道内 NOS、ACh E、VIP阳性神经元的形态学特征。结果显示 ,肠肌间丛 NOS阳性神经元胞体大小不等 ,形态不一 ,NOS、ACh E和 VIP阳性神经元的分布密度为 ACh E>NOS>VIP,在不同的肠段和层次分布密度有差异 ,NOS与 ACh E存在共染。在肌间丛和粘膜下丛 ,少数 VIP与 NOS共染。在粘膜下丛 ,三种阳性神经元的分布密度为 ACh E>VIP>NOS。在肌间丛和粘膜下丛 ,可见 VIP阳性末梢环抱 NOS阳性神经元胞体 ,两者呈终扣样接触。上述结果提示 NOS阳性神经元与 ACh E、 VIP阳性神经元有密切的形态学联系。在消化道功能调节上 ,它们可能起协调作用。     

Enkephalin-like immunoreactivity in ganglionic cells in the larynx and superior cervical ganglion of the rat.

The distribution of enkephalin-like immunoreactivity (ENK-LI) in the larynx, the superior cervical ganglion (SCG) and the nodose ganglion of adult rats was examined in the present study. A substantial number of the local acetylcholinesterase (AChE)-positive, presumably parasympathetic, ganglionic cells in the larynx displayed ENK-LI. These cells also exhibited neuropeptide Y (NPY)- and vasoactive intestinal polypeptide (VIP)-LI. Varicose nerve fibers showing ENK-LI were observed close to the acini and ducts of the glands, in the perichondrium and in the lamina propria. The varicosities exhibiting ENK-LI frequently displayed NPY- and VIP-LI. The ENK-LI was detected in a subpopulation of AChE-positive nerve fibers in the laryngeal tissue. In the SCG, only a small number of the ganglionic cells displayed ENK-LI. These cells, in contrast to other ganglionic cells of the SCG, did not show NPY-LI. None of the ganglionic cells of the nodose ganglion exhibited ENK-LI. Sympathectomy and vagotomy affected neither the number nor the distribution of fibers showing ENK-LI in the larynx. In conclusion, ENK appears to be present together with NPY and VIP in the parasympathetic innervation of the larynx and in a very limited number of the ganglionic cells of a sympathetic ganglion, the SCG, of the adult rat.     

The effect of leucine-enkephalin on the peristaltic reflex of the isolated guinea-pig ileum

Leucine (leu)-enkephalin depresses or inhibits the peristaltic reflex of the isolated guinea-pig ileum. Opiate antagonists (naloxone and nalorphine), choline esters (acetylcholine, methacholine and carbachol), cholinomimetics (muscarine and arecoline) and polypeptides which stimulate peristalsis (eledoisin and angiotensin) antagonize the peristaltic block caused by leu-enkephalin. On the other hand, nicotinic ganglionic stimulants (nicotine and dimethylphenylpiperazine) as well as muscarinic ganglionic stimulants (McN-A-343 and AHR-602) do not restore the peristaltic reflex abolished by leu-enkephalin. Thus the inhibitory effect of leu-enkephalin is due mainly to an action on myenteric ganglia as well as on axon terminals of the myenteric plexus subserving the peristaltic reflex. The inhibitory action of leu-enkephalin may be ascribed to the opiate as well as to the cholinoceptive sites in the nervous elements in the myenteric plexus. The blocking action of leu-enkephalin is not associated with ganglionic muscarinic M-1 receptors as well as with ganglionic nicotinic receptors in the myenteric plexus of the guinea-pig isolated ileum.     

Cholinergic, nitrergic and peptidergic (Substance P- and CGRP-utilizing) innervation of the horse intestine. A histochemical and immunohistochemical study

The small and large intestine of adult horses were histochemically and immunohistochemically investigated in order to evidence components of the intramural nervous system. The general structural organization of the intramural nervous system was examined by using Nissl-thionin staining as well as the anti-neurofilament 200 (NF200) immunoreaction, which demonstrated the presence of neurons in the submucous as well as myenteric plexuses. The additional presence of subserosal ganglia was shown in the large intestine. Acetylcholinesterase (AChEase) activity was observed in both the submucous and myenteric plexuses. Localization of acetylcholine-utilizing neurons was also evidenced by immunohistochemical reactions for choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT). With both histochemistry and immunohistochemistry possible cholinergic nerve fibres were detected in the inner musculature. The two possible cholinergic co-mediators Calcitonin Gene-Related Peptide (CGRP) and Substance P (SP) have been investigated by an immunohistochemical approach. CGRP immunoreactivity was detected in roundish nerve cell bodies as well as in nerve fibres of the submucous plexus, whereas SP immunoreactivity was evidenced in nerve fibres of the tunica mucosa, in nerve cell bodies and fibres of the submucous plexus and in nerve fibres of the myenteric plexus. NADPH-diaphorase reactivity, which is linked to the synthesis and release of nitric oxide, was detected in nerve cell bodies and nerve fibres of both the submucous and myenteric plexuses as well as in a subserosal localization of the large intestine. The nitrergic components were confirmed by the anti-NOS (nitric oxide synthase) immunoreaction. Results are compared with those of other mammals and related to the complex intestinal horse physiology and pathophysiology.     

Cytophotometric study of the AChE-containing nerve cells and fibers of the rabbit heart after electrostimulation of the area hypothalamica posterior

By means of cytophotometry and a computer in the cardiac intramural neural plexus of 17 experimental and 17 control rabbits acetilcholine esterase (AChE) activity (revealed by Karnovsky--Roots' method) was investigated. It was stated that a prolonged electrostimulation of the area hypothalamica posterior (AHP) produced statistically significant 2-fold decrease of AChE activity in the cardiac neural cells and fibres. When the stimulation of AHP was stopped, the decreased enzymatic activity was observed for the first two weeks, then by the end of the 4th week it again reached its control level. The results of the work were discussed in view of possible disorders of regulating vegetative-trophic effects of the posterior hypothalamus on innervative and executive tissue elements of the heart.     

Proteomics of the rat gut: analysis of the myenteric plexus-longitudinal muscle preparation

The enteric nervous system (ENS)--present all along the gastrointestinal tract - is the largest and most complicated division of the peripheral nervous system that can function independently of the brain. The peripheral nerve cells are organized in two separate but interconnected meshworks, called the myenteric and submucous plexus. The nervous control of intestinal motility is primarily governed by the myenteric plexus (MP), which lies in-between the longitudinal- (LM) and circular-muscle layers and regulates their functions. To determine whether the proteomic technology is adapted to the analysis of specific gut tissues, we dissected the MP-LM layers from the jejunum, ileum, and colon of Long Evans rats, homogenized them, and separated the proteins using two-dimensional gel electrophoresis. A subset of all the visualized protein spots, covering the entire range of molecular weights and isoelectric points, was then selected and further analyzed by matrix-assisted laser desorption/ionization-time of flight and liquid chromatography mass spectrometry. We identified around 80 proteins in each gut segment, and among those, five were segment-specific. Most of the proteins identified were derived from muscle cells, but we also detected some neuron-specific proteins. This study represents, to our knowledge, the first extensive protein catalog of a neuromuscular layer of the rat intestine and it may constitute the basis to understand pathophysiological mechanisms related to the ENS.     

L-arginine immunoreactive enteric glial cells in the enteric nervous system of rat ileum

L-arginine is a precursor of nitric oxide (NO) that may be involved in neuronal activity in the gastrointestinal tract. It is known that NO is formed from L-arginine by NO synthase which is localized in neurons in the enteric nervous system. The present study demonstrated that significant L-arginine immunoreactivity was present in the enteric ganglia. Ultrastructural examination showed that L-arginine immunoreactivity was present in the ganglionic glial cells but not in neurons. These findings suggest that enteric glial cells may represent the main reservoir of L-arginine, which may possibly be transferred to neurons when used.     

Intramural ganglia of the human gallbladder: morphological and functional observations]

The purpose of this study was the reinvestigation of the intrinsic innervation of human gall bladder with an immunohistochemical technique named peroxidase anti-peroxidase (PAP). The antigen demonstrated was the S100 protein normally present in the surface of glial cells, Schwann cells and satellite cells in ganglia. The tissues used were taken from 20 human gall bladders, fixed after surgery. This technique is not specific to demonstrate adrenergic or cholinergic innervation but it reveals just myelinated fibers. The current study was undertaken in order to study the organization and the function of plexus of nerves and ganglia present in the wall of the gall bladder. The neck of the gall bladder was the region in which the higher number of nerve cells and nervous fibers was present. The technique used has demonstrated ganglionated plexus and nerves in submucosal layer, fibromuscular and adventitial layer according to the enteric nervous system. All ganglia are postganglionic stations related with preganglionic cholinergic fibers. These results confirm that the intramural ganglia of the gall bladder are analogous to those of the enteric nervous system according to their common origin.     

GABA, Glutamic Acid Decarboxylase, and GABA Transaminase Levels in the Myenteric Plexus in the Intestine of Humans and Other Mammals

Abstract: Regional distribution of endogenous γ- aminobutyric acid (GABA), its synthesizing enzyme, glutamic acid decarboxylase (GAD), and metabolic enzyme, GABA transaminase (GABA-T), were determined in the intestinal tract of guinea pigs and cats and the findings compared with the number of ganglion cells in Auerbach's plexus. There were positive correlations among the GABA contents and the numbers of neural cells of the plexus. The precise localization of GABA and GAD in individual layers (mucosa, circular and longitudinal muscles, and Auerbach's plexus) in the human and cat colon was also determined. The endogenous GABA contents and GAD activity were the highest in Auerbach's plexus in tissues of both species. These results indicate that GABA is synthesized and localized in Auerbach's plexus and probably plays a significant role in the enteric nervous system.     

Morphofunctional stateof the cholinergic nervous apparatus of the normal duodenum and the duodenum after subdiaphragmatic vagotomy

Histochemical study of the intramural nervous apparatus of the duodenum was carried out under normal conditions and following bilateral subphrenic vagotomy. Morphometric and microspectrofluorimetric methods gave informations on the reduction of the number of the cholinergic nervous fibers and of the acetylcholinesterase activity in them after a brief increase of these indices during the first 24 hours after vagotomy, with their subsequent return to the initial values.     

Neurotransmitters and putative neuromodulators in the gut of Anguilla anguilla (L.). Localizations in the enteric nervous and endocrine systems

The gut of silver eels (Anguilla anguilla L.) was investigated in order to describe both the cholinergic and adrenergic intramural innervations, and the localization of possible accessory neuromediators. Histochemical reactions for the demonstration of nicotinamide adenine dinucleotide phosphate, reduced form-(NADPH-)diaphorase and acetylcholinesterase (AChEase) were performed, as well as the immunohistochemical testing of tyrosine hydroxylase, met-enkephalin, substance P, calcitonin gene-related peptide (CGRP), bombesin, vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), somatostatin, cholecystokinin-octapeptide (CCK-8), serotonin, cholineacetyl transferase. The results evidenced a different pattern in comparison with other vertebrates, namely mammals, and with other fish. Both NADPH-diaphorase and AChEase activities were histochemically detected all along the gut in the myenteric plexus, the inner musculature and the propria-submucosa. Tyrosine hydroxylase immunoreactivity was observed in the intestinal tract only, both in the myenteric plexus and in the inner musculature. Several neuropeptides (metenkephalin, CGRP, bombesin, substance P, VIP, NPY, somatostatin) were, in addition, detected in the intramural innervation; some of them also in epithelial cells of the diffuse endocrine system (met-enkephalin, substance P, NPY, somatostatin). Serotonin was only present in endocrine cells. Tyrosine hydroxylase immunoreactivity was present in localizations similar to those of NADPH-diaphorase-reactivity, and in the same nerve bundles in which substance P- and CGRP-like-immunoreactivities were detectable in the intestinal tract. In addition, NADPH-diaphorase-reactive neurons showed an anatomical relationship with AChEase-reactive nerve terminals, and a similar relationship existed between the latter and substance P-like immunoreactivity.     

Cholinergic innervation of the gastric wall of the cat.

The inbuilt intrinsic cholinergic nervous apparatus of the gastric wall of the cat was studied by using two thiocholine methods for mapping the acetylcholinesterase-positive nerves and nerve cells. A rich distribution of acetylcholinesterase-positive nerves was observed in all layers of the gastric wall, except the superficial half of the lamina propria (with the epithelium), which was completely devoid of acetylcholinesterase activity, and the submucosa, in which a scarce distribution of large nerve fascicles and nerve trunks was observed. Acetylcholinesterase-positive ganglia were observed both in the subserous layer and in the myenteric plexus of Auerbach, whereas none were recognized in the submucous plexus of Meissner. This obviously fits well to the results of some electrophysiological experiments which indicate that the submucous plexus of Meissner includes an important intramural pathway from the extrinsic vagus nerves to the antrum region; so the submucous plexus of Meissner seems to be mainly involved in direct rapid conduction of nerve impulses without integrative activities, like a cable. Certain clear differences exist in the pattern of organization of the cholinergic intrinsic nervous apparatus within the different layers of the gastric wall in the fundic and pyloric regions. These differences seem to correspond quite logically to the different types of motor, secretory and neurohumoral activities of these main regions of the stomach. The activity of the non-specific cholinesterases was localized both in the neural elements and the smooth muscle, as well as in some epithelial cells.     

Comparative observations on AChE distribution in pancreas of some amphibians,reptiles and birds,with special reference to the islets of langerhans

Summary AChE activity in pancreas of some amphibians, reptiles and birds was investigated with light microscope using Karnovsky's technique.A nervous and an extranervous localization of the enzyme showing considerable qualitative and quantitative differences depending on the systematic group, could be discerned. Pancreatic peripheral cholinergic autonomic system comprises nerve fibers and perikarya. The latter seem to occur in newt and frogs as Cajal's interstitial cells (CIC) and are undoubtedly present in birds as CIC and as ganglionic cells. In lizard and snakes they were not present.The extranervous localization of AChE activity was observed in islet cells in frogs, lizards and snakes, also in the walls of blood vessels of all investigated poikilotherms.The findings are compared with results previously obtained in mammalian pancreas or in other organs. Some hypotheses on the physiological significance of enzyme presence in endocrine cells, as well as their belonging to APUD cell series are discussed.This work was supported by a fellowship of the Alexander von Humboldt-Stiftung, Bonn-Bad Godesberg and by the Deutsche Forschungsgemeinschaft (La 229-3). The author expresses his thanks to Prof. Dr. R. H. Lange for scientific advice.     

The intramural innervation of the rabbit upper urinary tract

The role of the autonomic innervation of the upper urinary tract for pyeloureteral motility is not completely understood. It is still debatable if the autonomic nervous system might play a modulating role on the ureteral peristalsis. The aim of this study was to investigate the distribution and regional variation of the intramural innervation using whole-mount preparations of the rabbit upper urinary tract. Whole-mount preparation was performed at upper urinary tracts of healthy rabbits. Immunohistochemistry was employed using Neurofilament (NF), Tyrosine Hydroxylase (TH), Choline Acetyltransferase (ChAT) and Substance P (SP) antibodies. NADPH-diaphorase and Acetylcholinesterase (AChE) histochemistry was carried out at the specimens. The stains were evaluated using brightfield, fluorescence and laser confocal microscopy. NF-, TH-, ChAT- and SP-immunoreactive (-IR) nerves formed distinct neuronal plexuses at the submucosal and muscle layers. Perivascular TH-, ChAT- and SP-IR fibres were demonstrated. AChE positive nerves were revealed in all layers, but only moderate NADPH-diaphorase positive innervation was found. Renal pelvis, upper and lower ureter showed enriched intrinsic innervation. Ganglia were found at the ureteropelvic border and the distal ureter. Whole-mount preparation technique revealed detailed informations about morphology and regional variation of the intramural innervation of the rabbit upper urinary tract.