Paul Karila

Galanin-like immunoreactivity in extrinsic and intrinsic nerves to the gut of the Atlantic cod,Gadus morhua, and the effect of galanin on the smooth muscle of the gut

The presence of galanin-like immunoreactivity in nerves to the stomach of the Atlantic cod has been investigated by immunohistochemistry. The distribution of ganglion cells showing galanin-like immunoreactivity was compared with the total distribution in nerves and ganglia. Projection studies were made to determine the origin of the galanin neurons. The effect of galanin was studied in smooth muscle strip preparations of the gut wall and arteries. Galanin-like immunoreactive ganglion cells frequently occurred along the vagal branches to the stomach. Most of them projected cranially. Immunoreactive nerve fibres were present in all layers of the gut and around arterial branches on the surface of the stomach. Ligations of the vagus and splanchnic nerves produced accumulations of immunoreactive material on both sides of the ligature. Galanin produced weak contractile effects unaffected by tetrodotoxin on the gut wall and on gut arteries. It is concluded that a population of the ganglion cells along the vagus nerve in the Atlantic cod contains a galanin-like peptide. Some of these cells may be parts of autonomic parasympathetic pathways innervating the gut of the Atlantic cod, having direct excitatory effects on the smooth muscles of the gut wall and gut arteries.

Coexistence of NADPH-diaphorase and vasoactive intestinal polypeptide in the enteric nervous system of the Atlantic cod (Gadus morhua) and the spiny dogfish (Squalus acanthias)

The distribution of NADPH (nicotinamide adenine dinucleotide phosphate)-diaphorase in nerve cells in the gastrointestinal tract has been investigated and compared in three fish species representing different evolutionary branches. In mammals, NADPH-diphorase is identical to nitric oxide synthase (NOS) and can, in the presence of NADPH, reduce the dye nitroblue tetrazolium, resulting in a blue product. Using this method, we have found numerous NADPH-diaphorase-containing nerve cells in the myenteric plexus of the Atlantic cod (Gadus morhua) and the spiny dogfish (Squalus acanthias) but none in the hagfish (Myxine glutinosa). In the cod, nerve fibres were sparsely stained, whereas in the dogfish, they formed a dense pattern of fibre bundles. Double-staining for NADPH-diaphorase and the neuropolypeptides VIP (vasoactive intestinal polypeptide) and PACAP (pituitary adenylate cyclase activating peptide) revealed three separate populations designated VIP/NADPH, VIP/- and NADPH/-. The majority but not all of the NADPH-diaphorase-positive cells also showed VIP or PACAP immunoreactivity and vice versa. The presence of NADPH-diaphorase in neurons and the distribution of these neurons in the gastrointestinal tract of the two species indicate a physiological role for nitric oxide in the control of gut motility.

Neuropeptide immunoreactivity and co-existence in cardiovascular nerves and autonomic ganglia of the estuarine crocodile, Crocodylus porosus, and cardiovascular effects of neuropeptides

The two aortas of the crocodile are in open connection at two sites, the foramen of Panizzae immediately outside the ventricles, and the arterial anastomosis at the level of the gut. The present study was performed to elucidate the innervation of the cardiovascular structures of the crocodile, in part to provide a further basis for the assumption that the apertures of the foramen and the anastomosis may be altered, possibly leading to changes in the flow profiles of the central vessels. The presence of smooth muscle arranged at the circumference of the foramen and in the walls of the anastomosis was demonstrated. The cardiovascular structures were innervated by nerves containing co-existing tyrosine hydroxylase, NPY and somatostatin immunoreactivities, which also occurred in neurons of the sympathetic ganglia. CGRP and substance P immunoreactive material co-existed in cardiovascular nerves, and in the nodose ganglion. In addition, bombesin, VIP and galanin immunoreactive nerves were found. Effects of neuropeptides on blood flows and blood pressures were studied in vivo. Substance P increased all blood flows measured, NPY increased the flow through the arterial anastomosis while neurotensin caused an initial decrease in the flow through the arterial anastomosis. In conclusion, there is a rich innervation of the heart and major vessels of the estuarine crocodile, including the foramen of Panizza and the arterial anastomosis. These nerves possibly regulate the distribution of blood in the cardiovascular system, which is further suggested by the results of the injection of neuropeptides.

Projections and actions of tachykininergic, cholinergic, and serotonergic neurones in the intestine of the Atlantic cod

Abstract The native tachykinins cod neurokinin A and cod substance P, serotonin and acetylcholine have excitatory effects on the circular smooth muscle of the cod intestine. Furthermore, immunoreactivities to the cod tachykinins, serotonin and two markers for cholinergic neurones, viz. choline acetyltransferase and vesicular acetylcholine transporter, have been demonstrated in myenteric neurones of the cod intestine. In order to elucidate whether the neurones containing these substances project orally and thus might be involved in the ascending excitatory reflex of peristalsis, myotomy operations have been performed on the cod intestine. The immunoreactive areas of the myenteric plexus immediately oral and anal to the myotomy operations have been measured by using confocal laser scanning microscopy. Large accumulations of immunoreactivity to the tachykinins are found on the anal side of the myotomies, indicating oral projections of tachykininergic neurones. The areas immunoreactive to serotonin and choline acetyltransferase are of equal size on the oral and anal sides. Since the tachykinin containing neurones of the intestine project orally, and since cod neurokinin A and cod substance P have excitatory effects on circular smooth muscle, we conclude that tachykininergic neurones are involved in the ascending excitatory reflex of peristalsis in the cod intestine.

Primary structure, distribution, and effects on motility of CGRP in the intestine of the cod Gadus morhua

Calcitonin gene-related peptide (CGRP) was isolated from an extract of the intestine of the cod Gadus morhua. The primary structure of this 37-amino acid peptide was established as follows: ACNTA TCVTH RLADF LSRSG GIGNS NFVPT NVGSK AF-NH2. The peptide shows close structural similarities to other nonmammalian (3-4 amino acid substitutions) and mammalian (5-8 amino acid substitutions) CGRPs, and it contains the two residues Asp14 and Phe15 that seem to be characteristic for CGRP in nonmammalian vertebrates. Cod CGRP (10(-9)-10(-7) M) inhibited the motility of spontaneously active ring preparations from the cod intestine and was significantly (P < 0.05) more potent than rat alpha-CGRP. Neither prostaglandins nor nitric oxide is involved in the inhibitory response produced by cod CGRP, and the lack of effect of tetrodotoxin suggests an action of CGRP on receptors on the intestinal smooth muscle cells. The competitive CGRP antagonist human alpha-CGRP-(8-37) significantly (P < 0.05) reduced the response to cod CGRP. Immunohistochemistry demonstrated CGRP-immunoreactive neurons intrinsic to the intestine, and a dense innervation with immunoreactive nerve fibers was observed in the myenteric plexus and the circular muscle layer. Myotomy studies show that CGRP-containing nerves project orally and anally in the myenteric plexus, whereas nerve fibers in the circular muscle layer project mainly anally, indicating a role for CGRP in descending inhibitory pathways of the cod intestine.Calcitonin gene-related peptide (CGRP) was isolated from an extract of the intestine of the cod Gadus morhua. The primary structure of this 37-amino acid peptide was established as follows: ACNTA TCVTH RLADF LSRSG GIGNS NFVPT NVGSK AF-NH2. The peptide shows close structural similarities to other nonmammalian (3-4 amino acid substitutions) and mammalian (5-8 amino acid substitutions) CGRPs, and it contains the two residues Asp14 and Phe15 that seem to be characteristic for CGRP in nonmammalian vertebrates. Cod CGRP (10-9-10-7M) inhibited the motility of spontaneously active ring preparations from the cod intestine and was significantly ( P < 0.05) more potent than rat α-CGRP. Neither prostaglandins nor nitric oxide is involved in the inhibitory response produced by cod CGRP, and the lack of effect of tetrodotoxin suggests an action of CGRP on receptors on the intestinal smooth muscle cells. The competitive CGRP antagonist human α-CGRP-(8-37) significantly ( P < 0.05) reduced the response to cod CGRP. Immunohistochemistry demonstrated CGRP-immunoreactive neurons intrinsic to the intestine, and a dense innervation with immunoreactive nerve fibers was observed in the myenteric plexus and the circular muscle layer. Myotomy studies show that CGRP-containing nerves project orally and anally in the myenteric plexus, whereas nerve fibers in the circular muscle layer project mainly anally, indicating a role for CGRP in descending inhibitory pathways of the cod intestine.

Nitric oxide synthase- and neuropeptide Y-containing subpopulations of sympathetic neurons in the coeliac ganglion of the Atlantic cod, Gadus morhua, revealed by immunohistochemistry and retrograde tracing from the stomach

In this study retrograde tracing was used to locate sympathetic ganglion cells innervating the stomach of a teleost fish, Gadus morhua. A subpopulation of small neurons in the coeliac ganglion was retrogradely labelled after Fast Blue injection in the stomach wall. Neurons projecting to the myenteric plexus and muscle layers contained tyrosine hydroxylase immunoreactivity, and neurons projecting to submucosal layers and blood vessels contained neuropeptide Y immunoreactivity in addition to being tyrosine hydroxylase immunoreactive. A population of nitric oxide synthase containing tyrosine hydroxylase immunoreactive neurons was also found in the coeliac ganglion. These neurons were not frequently labelled after injection in any layer of the stomach. The presence of entero-enteric pathways was also surveyed, but too few enteric neurons were labelled with Fast Blue after injection in the coeliac ganglion to indicate a presence of an entero-enteric reflex. We conclude that in teleost fish, as previously reported in a variety of mammals, a pattern of target specific chemical coding of sympathetic neurons exists, but that all reflex systems of mammalian vertebrates are perhaps not present in fish.

Anally projecting neurons exhibiting immunoreactivity to galanin, nitric oxide synthase and vasoactive intestinal peptide, detected by confocal laser scanning microscopy, in the intestine of the Atlantic cod, Gadus morhua.

Abstract.The projections of enteric neurons showing immunoreactivity for vasoactive intestinal peptide (VIP), nitric oxide synthase (NOS) and galanin were investigated in the myenteric plexus of the intestine of the Atlantic cod, Gadus morhua. Quantification of immunoreactive material on the proximal and distal side of a myotomy was performed by means of confocal laser scanning microscopy. NOS immunoreactivity was reduced anal to the myotomy, whereas there was an accumulation of immunoreactivity for VIP and for galanin oral to the cut. These results suggest the presence of VIP, NOS and galanin in neurons with oral–to–anal projections along the intestine of the cod. Since descending neurons in the myenteric plexus of many other vertebrates also contain these substances, we conclude that the oral–to–anal projections of neurons containing VIP, NOS and galanin are highly conserved features and important for the descending phase of intestinal peristalsis on an evolutionary basis.

Effects of substance P and distribution of substance P-like immunoreactivity in nerves supplying the stomach of the cod, Gadus morhua

The innervation of the cod stomach by neurons showing substance P-like immunoreactivity (SPLI), and the effect and mechanism of action of substance P (SP) on the vascularly perfused cod stomach and on isolated muscle strip preparations from the pyloric sphincter have been investigated.Infusion of SP produced a contraction of the stomach wall, which could not be blocked by tetrodotoxin, atropine or methysergide, indicating a direct effect on the stomach smooth muscle. Similarly, the contraction produced by SP on preparations from the pyloric sphincter was unaffected by tetrodotoxin.Nerves showing SPLI were frequent in the myenteric plexus of the whole stomach, and in the submucosa and mucosa of the pyloric part of the stomach. SPLI was also observed in fibres in the intestinal branch of the vagus and occasionally in the splanchnic nerves. Ligation of the nerves showed an accumulation of SPLI above as well as below the ligature, being more prominent proximal to the ligature in the vagus and distal to the ligature in the splanchnic nerve. In the vagus nerve, descending and ascending SPLI-fibres were seen surrounding non-reactive cell bodies. No reduction in intensity of the immunoreaction of the neurons in the stomach wall was observed after ligation or sectioning of the vagosympathetic trunk or the splanchnic nerves, nor were SP-levels measured by radioimmunoassay reduced. After denervation of vagal branches close to the stomach wall an insignificant decrease of immunoreactivity was observed in the myenteric plexus. Capsaicin treatment had no conclusive effect on the distribution of SPLI.It is concluded that the innervation showing SPLI may be of intrinsic as well as extrinsic origin, with pathways in both vagal and splanchnic branches. Only a direct effect of SP on the smooth muscle could be demonstrated.

Dendritic morphology of neurons in sympathetic ganglia of the goldfish, Carassius auratus ☆

We used intracellular dye injection to examine the dendritic morphology of postganglionic neurons in the coeliac ganglion of goldfish. About 80% of the neurons had at least one dendrite, with the mean number of dendrites per cell being 7.8 +/- 5.5 (+/- SD, n = 37 cells). Dendrites varied in length from a few microns to more than 400 microns. Around 37% of the neurons possessed axon collateral in addition to dendrites. These results show that postganglionic sympathetic neurons of goldfish can have a complex morphology, more like the sympathetic neurons of small mammals than those of amphibians. This raises the possibility that at least some sympathetic ganglion cells of teleost fish receive multiple convergent preganglionic inputs, suggesting a hitherto unsuspected level of complexity in these pathways.