Histochemistry (1988) 88:155-163 Histochemistry 9 Springer-Verlag 1988 Neuropeptides in the fish gut An immunohistochemical study of evolutionary patterns C. Bjenning* and S. Hoimgren Comparative Neuroscienee Unit, Department of Zoophysiology, University of G6teborg, Box 250 59, S-400 31 G6teborg, Sweden Accepted September 14, 1987 Summary. The presence and distribution of bombesin-, en- kephalin-, gastrin/cholecystokinin-, neuropeptide Y-, neu- rotensin-, somatostatin-, substance P-, and VIP-like im- munoreactivities in gut nerves of representatives of nineteen cyclostome, elasmobranch and teleost species have been studied. The results have been correlated to results from previous studies in other species. Nerve plexuses showing bombesin- like, substance P-like and VIP-like immunoreactivity are commonly occurring, while other neuropeptides may have a more varied distribution. Tentative evolutionary patterns, and the possible func- tion and importance of each peptide is discussed. Introduction The autonomic nervous system of the gut forms a separate functioning unit - the enteric nervous system - influenced by, but not dependent on, the activity in the sympathetic and parasympathetic systems (Langley 1898, 1921). The en- teric nervous system of fish follows that of other vertebrates in its general organization, with only minor variations, such as a lower number of ganglion cells in the plexuses (Kirt- isinghe 1940; Nicol 1952). A ganglionated myenteric plexus is situated between the circular and longitudinal muscle layers, and sends fibres to the sparsely innervated longitudi- nal layer and the richly innervated circular layer. A submu- cous plexus, also ganglionated, sends fibres to the circular muscle, structures of the submucosa, the muscularis mucosa and the mucosa. Immunohistochemical studies have now shown that, as in mammals, a number of neuropeptides may be present in the gastrointestinal nerves in fish. However, hitherto pub- lished material gives scattered information, which may not be representative of the whole and complex group of more than 20000 fish species. It has been our ambition within this study to look for innervation patterns within families of fishes, as well as between different families. We have for that purpose investigated representatives from several different families, including as many gadides and labrides as possible, and have attempted to correlate these studies to already published material. * To whom offprint requests should be sent Materials and methods The studies have been performed on stretch preparations of differ- ent parts of the gut, treated according to Costa et al. (1980), and on sections of tissues fixed in parabenso-quinone according to Coons (1956) (see Holmgren et al. 1985a). The species of fish stud- ied are indicated in Table 1. Fish were obtained live at the Kristine- berg Marine Biological Station or from local hatcheries and fisher- men. The antisera used in the present study are presented in Table 2. Incubation with the primary antiserum was made at room tempera- ture in a moist chamber for 18 h. Rinses were made with phosphate buffered saline, pH 7.3. Localization of reaction sites was obtained by a secondary incubation with swine-anti-rabbit IgG antiserum conjugated with FITC (Dako, 1:10), with subsequent viewing in a Leitz Ortholux fluorescence microscope. Specificity of positive reactions has been tested by incubation with positive antisera prein- cubated with the original hapten (10 nmol/ml). Results and discussion The information from immunohistochemical studies on neuropeptide occurrence in gastrointestinal nerves of differ- ent fish species is summarized in Table 1. In the cyclostome, Myxine glutinosa, no immunoreactive nerves were found with any of the antisera used, although repeated experi- ments were performed. In elasmobranch and teleost fish species, however, several neuropeptides are present. More detailed descriptions of the distribution of the investigated peptides are given below, along with a discussion of their possible function and importance. Bombesin The tetradecapeptide bombesin, first isolated from amphib- ian skin (Anastasi et al. 1971), has subsequently been dem- onstrated also in endocrine cells and nerves of the gut of several non-mammalian species. Although exogenous bom- besin is active in mammals, it is considered that the natural- ly occurring mammalian bombesin-like peptide is the so called gastrin releasing peptide (GRP), with a location only in nerves (McDonald et al. 1978; M/irki et al. 1981). In mammals, bombesin-like peptides have several functions, comprising vascular and cardiac effects, a stimulatory effect on gut motility and release of hormones such as gastrin, cholecystokinin (CCK) and pancreatic polypeptide (Ers- pamer and Melchiorri 1975; Bayorh and Feuerstein 1985).
Neuropeptides in the fish gut An immunohistochemical study of evolutionary patterns
C. Bjenning* and S. Hoimgren Comparative Neuroscienee Unit, Department of Zoophysiology, University of G6teborg, Box 250 59, S-400 31 G6teborg, Sweden
Accepted September 14, 1987
Summary. The presence and distribution of bombesin-, en- kephalin-, gastrin/cholecystokinin-, neuropeptide Y-, neu- rotensin-, somatostatin-, substance P-, and VIP-like im- munoreactivities in gut nerves o f representatives of nineteen cyclostome, elasmobranch and teleost species have been studied.
The results have been correlated to results from previous studies in other species. Nerve plexuses showing bombesin- like, substance P-like and VIP-like immunoreactivity are commonly occurring, while other neuropeptides may have a more varied distribution.
Tentative evolutionary patterns, and the possible func- tion and importance of each peptide is discussed.
Introduction
The autonomic nervous system of the gut forms a separate functioning unit - the enteric nervous system - influenced by, but not dependent on, the activity in the sympathetic and parasympathetic systems (Langley 1898, 1921). The en- teric nervous system of fish follows that of other vertebrates in its general organization, with only minor variations, such as a lower number of ganglion cells in the plexuses (Kirt- isinghe 1940; Nicol 1952). A ganglionated myenteric plexus is situated between the circular and longitudinal muscle layers, and sends fibres to the sparsely innervated longitudi- nal layer and the richly innervated circular layer. A submu- cous plexus, also ganglionated, sends fibres to the circular muscle, structures of the submucosa, the muscularis mucosa and the mucosa.
Immunohistochemical studies have now shown that, as in mammals, a number o f neuropeptides may be present in the gastrointestinal nerves in fish. However, hitherto pub- lished material gives scattered information, which may not be representative of the whole and complex group of more than 20000 fish species. It has been our ambition within this study to look for innervation patterns within families of fishes, as well as between different families. We have for that purpose investigated representatives from several different families, including as many gadides and labrides as possible, and have attempted to correlate these studies to already published material.
* To whom offprint requests should be sent
Materials and methods
The studies have been performed on stretch preparations of differ- ent parts of the gut, treated according to Costa et al. (1980), and on sections of tissues fixed in parabenso-quinone according to Coons (1956) (see Holmgren et al. 1985a). The species of fish stud- ied are indicated in Table 1. Fish were obtained live at the Kristine- berg Marine Biological Station or from local hatcheries and fisher- men.
The antisera used in the present study are presented in Table 2. Incubation with the primary antiserum was made at room tempera- ture in a moist chamber for 18 h. Rinses were made with phosphate buffered saline, pH 7.3. Localization of reaction sites was obtained by a secondary incubation with swine-anti-rabbit IgG antiserum conjugated with FITC (Dako, 1:10), with subsequent viewing in a Leitz Ortholux fluorescence microscope. Specificity of positive reactions has been tested by incubation with positive antisera prein- cubated with the original hapten (10 nmol/ml).
Results and discussion
The information from immunohistochemical studies on neuropeptide occurrence in gastrointestinal nerves of differ- ent fish species is summarized in Table 1. In the cyclostome, Myxine glutinosa, no immunoreactive nerves were found with any of the antisera used, although repeated experi- ments were performed. In elasmobranch and teleost fish species, however, several neuropeptides are present. More detailed descriptions of the distribution of the investigated peptides are given below, along with a discussion of their possible function and importance.
Bombesin
The tetradecapeptide bombesin, first isolated from amphib- ian skin (Anastasi et al. 1971), has subsequently been dem- onstrated also in endocrine cells and nerves o f the gut of several non-mammalian species. Although exogenous bom- besin is active in mammals, it is considered that the natural- ly occurring mammalian bombesin-like peptide is the so called gastrin releasing peptide (GRP), with a location only in nerves (McDonald et al. 1978; M/irki et al. 1981). In mammals, bombesin-like peptides have several functions, comprising vascular and cardiac effects, a stimulatory effect on gut motility and release o f hormones such as gastrin, cholecystokinin (CCK) and pancreatic polypeptide (Ers- pamer and Melchiorri 1975; Bayorh and Feuerstein 1985).
Table 1. Summary of the available information from immunohistochemical studies on the presence of neuropeptides in gastrointestinal nerves of different fish species. 1, 2, and 3 indicate increasing densities of immunoreactive nerve fibres, 0 indicates absence of visible immunoreactive material, + indicates that no attempt has been made to quantify the amount of immunoreactivity present and - indicates that no investigation has been performed
BM=bombesin , ENK=enkephalin, G/CCK=gastrin/cholecystokinin, NPY=neuropeptide Y, NT=neurotensin, SP=substance P, VIP=vasoactive intestinal polypeptide, S=s tomach , /=intes t ine*, R=rectum. * In stomachless fish, ' I ' denotes the proximal 2/3 of the intestine
References." I. Present study; 2. Reinecke et al. (1981); 3. Holmgren and Nilsson (1983a); 4. El Salhy (1984) 5. Holmgren and Nilsson (1983b) 6. Van Noorden and Falkmer (1980); 7. Holmgren et al. (1982); 8. Thorndyke et al. (1984); 9. Langer et al. (1979); 10. Rombout and Reinecke (1984); 11. Burkhardt and Hohngren, to be published; 12. Van Noorden and Patent (1980); 13. Holmgren and Grove, unpublished; 14. Jensen and Holmgren (1985); 15. J6nsson et al. (1987); 16. Holmgren and J6nsson (1987); 17. Holmgren (1985)
Table 2. Details of antisera used in this study
Antigen Code Working dilution Source Region specificity Ref.
Bombesin G 07 1:100 Own bombesin/GRP 1 L 90 1:200 G. Dockray 2
CCK 4 G 03 1:100 Own gastrin/CCK4 3
Met-enkephalin L 146 1 : 100 G. Dockray free met-enk 4
NPY * Ab 22 1 : 200 CRB 9801 1 : 200 Peninsula
Neurotensin NtAb 1:100 G. Dockray C-terminus 2
Substance P G l0 1:200 Own C-terminus 5
VIP* 18-28 L 85 1:250 G. Dockray C-terminal 6
157
* =raised against synthetic porcine antigen, CCK=cholecystokinin, GRF= gastrin releasing peptide, NPY= neuropeptide Y, VIP= vasoactive intestinal polypeptide. References: [1] Holmgren and J6nsson (1987); [2] G. Dockray, pers. commun.; [3] J6nsson et al. (1987); [4] Bu'lock et al. (1983); [51 Jensen et al. (1987); [6] Dimaline et al. (1980)
The immunohistochemical studies indicate that bombe- sin-like peptides have a wide distribution in nerves in fish, occurring in the gut of representatives of several fish fami- lies. In elasmobranchs, some of the immunoreactive fibres innervate submucosal blood vessels (Holmgren and Nilsson 1983a; present study, Fig. 1). In some species, e.g. Squalus acanthias (Holmgren and Nilsson 1983 a) and Myoxocepha- lus scorpius (present study, Fig. 2) immunoreactive ganglion cells are present especially in the plexuses of the stomach.
We have found that, especially amongst members of the family Gadidae, bombesin-like immunoreactivity is prominent in the stomach, producing an intense reaction with the used antisera. However, using the same antisera we have found that the bombesin-like material in other parts of the gadid gut as well as that in the whole gut of some other species (e.g. the rainbow trout) shows a much weaker immunoreaction. This may indicate the presence of more than one bombesin-like neuropeptide in fish (see also Holmgren and J6nsson 1987).
The physiological actions of bombesin-like peptides in fish have been investigated to some extent. Gastric acid secretion is increased in the cod, Gadus morhua (Holstein and Humphrey 1980), and a stimulatory effect on stomach motility, which may involve a potentiation of an acetylcho- line effect, has been demonstrated in several teleost species (Holmgren 1983; Thorndyke etal. 1984; Holmgren and J6nsson 1987). Rectal preparations from the elasmobranch, Squalus acanthias, are stimulated by bombesin (Lundin et al. 1984) while intestinal preparations from the cod are inhibited by the bombesin-related peptide litorin (Holmgren and J6nsson 1987).
Physiological experiments, immunohistochemistry and radioimmunoassay studies thus indicate the physiological importance of one or several bombesin-like neuropeptides in the fish gut.
Enkephalin
The enkephalins are pentapeptides, with effects on the mammalian alimentary canal that are varying and not clearly understood. Both excitatory and inhibitory effects on gut secretory activity and on gut motility are reported (Corder and Rees 1981 ; Konturek 1981). Also in fish, little is known of the function and physiological importance of the enkephalins. An excitation of stomach preparations
from the rainbow trout is reported, while the effects on the intestine of the cod are variable (Holmgren 1983 ; Jensen and Holmgren 1985).
In the present study, met-enkephalin-like immunoreac- tivity was found in nerves of the anterior part of the alimen- tary canal (stomach and duodenum) in all teleosts investi- gated, except the rainbow trout. An especially dense inner- vation was found in the family Labridae, the perch, Perca fluviatilis, and the eel, Anguilla anguilla, where the immuno- reactive nerves also extended into the rectum, and where numerous immunoreactive nerve cell bodies were present in the plexuses (Figs. 4-6). Previous studies report the pres- ence of immunoreactivity in nerves as well as in endocrine cells in a number of teleost species (Langer et al. 1979; Van Noorden and Falkmer 1980; Van Noorden and Patent t980; Rombout and Reinecke 1984). Thus, gastrointestinal nerves containing enkephalin-like material seem to be of frequent occurrence amongst teleosts, while there are no reports of such a presence in cyclostomes or elasmobranchs. Virtually nothing is, however, known of their function in teleosts.
Gastrin/CCK
The gastrins, the cholecystokinins and the amphibian pep- tide caerulein share the same C-terminal pentapeptide; most immunohistochemical studies in fish have been performed with antisera directed against this common C-terminal, thus not differentiating between these peptides.
From studies in mammals it has been suggested that gastrin is present in endocrine cells in the stomach while CCK is found in both endocrine cells and nerves of the intestine (see Dockray and Dimaline 1985).
In fish, there are several reports of the occurrence of gastrin/CCK-like peptides in endocrine cells (see Holmgren et al. 1986), while only two previous studies report the pres- ence of immunoreactive material confined to nerves in the fish gut. In the spiny dogfish, Squalus acanthias, nerves of the myenteric plexus especially of the rectum are immu- noreactive and, in the cod, immunoreactive material has been characterized both by immunohistochemistry and ra- dioimmunoassay (Holmgren and Nilsson 1983 a; J6nsson et al. 1987). The present study further confirms the presence of nerves containing gastrin/CCK-like material, with a con-
158
Figs. 1-8. Bombesin (BM)-like immunoreactivity (IR) (Figs. 1-3), enkephalin (ENK)-like IR (Figs. 4-6), gastrin/cholecystokinin (G/CCK)- like IR (Fig. 7) and neurotensin (NT)-[ike IR (Fig. 8) in enteric nerves of different fish species. Fig. 1. is of a section, Figs. 2--8 are of whole mounts. Fig. 1. Nerve fibres innervating a blood vessel in the rectal submucosa of Raja naevus, x 300. Fig. 2. Ganglion cells in the myenteric plexus (MEP) of the cardiac stomach of Myoxoeephalus scorpius, x 300. Fig. 3. Fibres with large varicosities in the MEP of the cardiac stomach of Gadus morhua, x 275. Fig. 4. Bundle of nerve fibres in the rectum of Anguilla anguilla, x 300. Fig. 5. Nerve cells in the rectum of Anguilla anguilla, x 480. Fig. 6. Nerve fibres and nerve cells in the MEP of the pyloric stomach of Perca fluviatilis, x 280. Fig. 7. Varicose fibres in the longitudinal muscle of the pyloric stomach of Gadus morhua, x 250. Fig, 8. Nerve fibres in the cardiac stomach of Percafluviatilis. x 300
159
centration to the stomach, in gadid species (Fig. 7). Apart from that we find nerve-bound gastrin/CCK-like immuno- reactivity only in occasional species. Unless further studies are performed, it is difficult to conclude whether the pres- ence of gastrin/CCK in gastrointestinal nerves is character- istic of certain families, and what the relation between these families is. The distribution of gastrin/CCK in the spiny dogfish and the occurrence in the stomachless carp, Cyprin- us carpio, however, argues against a general limitation of the fish gastrin/CCK-like peptide to stomach nerves, al- though this may be the case within the Gadidae family.
Porcine gastrin stimulates gastric acid secretion in a dog- fish (Vigna 1983) while, in a study of the cod, gastrin, CCK and caerulein all inhibited the basal gastric acid secretion (Holstein 1982). It is, however, probable that the endoge- nous peptide involved in the secretory mechanism is con- tained in those endocrine cells of the mueosa, reported e.g. in the cod to show gastrin/CCK-like immunoreactivity (J6nsson et al. 1987), rather than in the immunoreactive nerves, which are mainly located in the myenteric plexus. These nerves may instead be involved in motility mecha- nisms, and it has recently been reported that gastrin/CCK- like peptides, and especially caerulein, stimulate the motility of the cod gut smooth muscle (J6nsson et al. 1987). Some reports also show the stimulatory action of gastrin/CCKs on motility of the fish gallbladder (Vigna and Gorbman 1977; Aldman and Holmgren 1987).
In conclusion, a peptide, which may be closely related to CCK or caerulein, is contained in gastrointestinal nerves of at least some fish species, and may be involved in the control of gut motility.
Neuropeptide Y (NP Y)
NPY is a recently-discovered 36 amino acid neuropeptide, which is present in extensive neuronal pathways of the cen- tral nervous system and the peripheral nervous system of mammals (Andrews et al. 1985). The presence of NPY in teleost material has not previously been reported, but in avian, mammalian and amphibian material an NPY-like peptide is present in perivascular nerves. NPY seems to be co-stored with noradrenaline (mammals) or adrenaline (amphibians) in the perivascular nerves, and on release causes a more long-lasting vasoconstriction than the adren- ergic transmitter (Gray and Morley 1986; Gibbins et al. 1987).
In the present study we have found NPY-like material in nerves of the stomach in all investigated teleost species possessing a stomach, with the exception of the eel. Only occasionally are nerves found in the intestine or rectum of a teleost, as in the carp, Cyprinus carpio, or the cod, Gadus morhua (Fig. 10). In four species of Rajidae, how- ever, we found an even distribution of such nerves through- out the gut (Fig. 11).
The density of the innervation is usually moderate or low. The immunoreactive nerves are present not only around small vessels (Fig. 9), but also in bundles of a few fibres running in the plexuses (Fig. 11) and along the muscle fibres (Fig. 10). 'Non-vascular ' gastro-intestinal NPY- fibres have also been reported in mammals (Furness et al. 1983 ; Sundler et al. 1983), and NPY has been demonstrated to have an inhibitory effect on the guinea-pig ileal motility (Garz6n et al. 1986). Nothing is known of the effect of NPY in the fish gut.
Neurotensin
The tri-decapeptide neurotensin, with a wide distribution in vertebrates (Carraway et al. 1982), has been associated with fat metabolism in mammals, where it causes inhibition of gastrointestinal motility and vasodilation, thereby en- hancing the absorption of fat (Hammer and Leeman 1981).
Amongst fish, neurotensin-like immunoreactivity has been found in endocrine cells of both elasmobranchs and teleosts (Reinecke etal. 1980; Holmgren and Nilsson 1983a; E1 Salhy 1984; Rombout and Reinecke 1984). So far, no elasmobranchs have been found to show nerve- bound neurotensin-immunoreactive material, while an in- nervation of the whole gut is reported in the holostean fish Lepisosteus (Holmgren and Nilsson 1983b) as well as in the perch (Fig. 8), the labrides, and the sculpin, Myoxoce- phalus scorpius (present study). Several other species con- tain immunoreactive nerves in parts of the alimentary canal and it may be concluded that, with few exceptions, all inves- tigated teleosts have a neurotensin-like peptide present in the enteric nervous system.
There is only one report on the effect of neurotensin in fish. In this study, an excitatory effect of exogenous neu- rotensin in high concentrations is demonstrated on prepara- tions from the rainbow trout stomach (Holmgren 1983).
Somatostatin
The occurrence of somatostatin in teleost nerves has not been investigated in the present study but weakly immuno- reactive material has been found in enteric nerves of Raja species. A few previous studies report somatostatin-like ma- terial in enteric nerves of the spiny dogfish (Holmgren and Nilsson 1983a) and the cyprinid, Barbus conchonius, (Rom- bout and Reinecke 1984). The primary structure of an elas- mobranchian somatostatin has been determined by Conlon and Thim (I 985).
Substance P
Substance P and related members of the tachykinin group are probably the most investigated of the neuropeptides discussed today, and also in fish we have some knowledge of the function and mechanisms of action, as well as the identity of the tachykinins present.
Two tachykinins, named scyliorhinin I and II, have been isolated from the gut of the common dogfish Scyliorhinus caniculus, and sequenced. One crossreacts with substance P antisera, while the other crossreacts with antisera raised against the C-terminal of neurokinin A (Conlon et al. 1986). No teleost tachykinin has so far been sequenced, but cir- cumstantial evidence indicate that the substance P-like pep- tide present in the cod is more closely related to substance P than to a series of other tachykinins tested (Jensen et al. 1987).
The immunohistochemical studies indicate a wide and dense distribution of nerves showing substance P-like im- munoreactivity in the spiny dogfish (Holmgren 1983) and in almost all teleosts investigated (Figs. 12 and 13). An ex- citatory effect on the gut motility of substance P has been shown in the spiny dogfish as well as in the rainbow trout and the cod (Holmgren 1983, 1985; Jensen and Holmgren 1985).
It is interesting that the mechanisms of action for the
160
Figs. 9-16. Neuropeptide Y (NPY)-like immunoreactivity (IR) (Figs. 9-11), substance P (SP)-like IR (Figs. 12-13) and vasoactive intestinal polypeptide (VIP)-like IR (Figs. 14-16) in enteric nerves of the gut of different fish species. Figs. 9 and i l are of sections, Figs. 10 and 12-16 are whole-mounts. Fig. 9. Nerve fibres (NF) around small blood vessel running in the rectal submucosa of Raja montagui. x 300. Fig. 10. Small bundles of NF running along the circular muscle fibres in the proximal intestine of Gadus morhua, x 240. Fig. 11. Bundle of varicose fibres in the rectal myenteric plexus (MEP) of Raja microocellata, x 240. Fig. 12. NF forming a dense network in the MEP of the proximal part of the alimentary canal of Cyprinus carpio, x 170. Fig. 13. Interconnecting NF in the cardiac stomach of Ciliata mustela, x 120. Fig. 14. Dense network of NF in the MEP of the middle part of the intestine of Anguilla anguilla, x 280. Fig. 15. Varicose fibres in the MEP of the cardiac stomach of Anguilla anguilla, x 300. Fig. 16. Dense network of NF in the MEP of the rectum of Gadus morhua, x 190
tachykinins seem to vary between species. Thus, in the spiny dogfish, the effect of endogenous substance P on gut motili- ty seems to be exclusively caused by a direct effect on the smooth muscle cells (Holmgren 1983) while in the rainbow
trout stomach both a direct effect as well as an indirect effect via release of 5-hydroxytryptamine from serotonergic neurons seem to be responsible for the excitatory action of substance P (Holmgren et al. 1985 b). In the cod intestine,
161
three pathways of action have been suggested; one direct, one via the release of 5-HT and one via the release of acetyl- choline (Jensen et al. 1987). Too few fish species have been studied to allow evolutionary speculations, but the cod, which is considered to be an "advanced" teleost, shows the cholinergic link which has been described in mammals.
In mammals, at least two types of substance P receptors have been described (Iversen et al. 1982). The SP-P receptor is approximately equally sensitive to the three tachykinins substance P, physalaemin and eledoisin, while the SP-E re- ceptor is more sensitive to eledoisin than to the other ta- chykinins. In fish, studies in the spiny dogfish, the rainbow trout and in the cod have indicated that in these three repre- sentatives of fish, the substance P receptors if anything are more closely related to the SP-P receptor (Holmgren 1983; Holmgren et al. 1985b; Jensen et al. 1987).
Vasoactive intestinal polypeptide ( VIP)
The 28 amino acid peptide VIP was first described as a vasodilatory agent, but has been found to have several addi- tional functions, including effects on smooth muscle tonus, gut secretions and metabolism in mammals (cf. Said 1981), possibly due to a stimulation of adenylate-cyclase at the receptor level (Fahrenkrug 1981). VIP-containing gastro- intestinal nerves are suggested to be involved in the gastric receptive relaxation and in the relaxation of the pyloric sphincter allowing passage of food (Said 1981).
As is evident from Table 1 and Figs. 14 16, VIP-like immunoreactivity is present in extensive nerve plexuses throughout the gut in almost all species studied. The nerves follow and surround blood vessels to some extent but the main distribution of fibres is in small bundles parallel to the smooth muscle fibres, especially in the circular muscle layer, and in bundles of different sizes forming plexuses between the muscle layers (the myenteric plexus) and in the submucosa.
The physiological function of VIP in fish has not been satisfactorily established. Porcine VIP reduces gastric acid secretion in the cod (Holstein and Humphrey 1980) but has inconsistent effects on the gastrointestinal motility in both the rainbow trout and the cod (Holmgren 1983; Jensen and Holmgren 1985). This may depend on differences in structure in the naturally-occurring fish VIP (Dimaline and Thorndyke 1986) and the porcine VIP used for exogenous administration, or even on the possible function of VIP as a neuromodulator or cotransmitter rather than a trans- mitter functioning on its own (cf. Lundberg 1981). The mo- tility of the gut of the spiny dogfish, however, is consistently inhibited by VIP in low concentrations (Lundin et al. 1984 and unpublished), and, in the cod, the muscularis mucosae from the swimbladder (a derivative from the gut) is relaxed by VIP (Lundin and Holmgren 1984).
Little is known of the vasoactive function of VIP in fish, but it has been reported that porcine VIP increases the flow through the swimbladder of the cod (Lundin and Holmgren 1984).
Conclusions
We find in this study that some neuropeptides have a more general distribution between species than others. The most frequently occurring peptide appears to be a VIP-like pep- fide, which shows a wide distribution in nerves in all layers
throughout the gut in almost all species studied. An excep- tion is the four investigated Raja species. This may point to a true species difference, but may also depend on inability of the C-terminally directed antiserum to recognize the C- terminal part of the Raja VIP peptide. The C-terminal end of the Raja VIP may in analogy with the situation in elas- mobranchs vary considerably compared to the porcine counterpart, which is the hapten for the antibody used in our studies (Dimaline and Thorndyke 1986).
Frequently present are also bombesin-like and sub- stance P-like peptides; substance P again with the exception of Raja species. Other authors report the absence of sub- stance P-like immunoreactivity in different species, while we, with our own antiserum, find substance P-like immuno- reactivity in almost all the species we have studied.
An NPY-like neuropeptide shows an even distribution in the gut in the elasmobranch Raja species, but appear to be concentrated mainly to the stomach in teleosts.
Enkephalins and neurotensin have so far not been re- ported in elasmobranch nerves. Both Perca fluviatilis and the Labrids show a dense innervation of nerves containing enkephalin-like and neurotensin-like material, while the oc- currence varies more between other teleosts. Gastr in/CCK- like material is found only in a few species and then with a clear regional distribution.
It appears that members of the same fish family usually have a similar distribution pattern of a certain peptide while, with the still comparatively small material available, it is difficult to find any clear evolutionary trends from 'o lder ' to ' younger ' fish families or groups.
Acknowledgements. We thank Mrs. C. Hagstr6m, Mrs. I. Holm- qvist and Ms. B. L6fnertz for skitful technical assistance and Dr. D.J. Grove and Professor S. Nilsson for critical reading of the manuscript. The gifts of antisera from Professor G. Dockray, Liverpool and Doc A-C J6nsson (own laboratory) are gratefully acknowledged. The study was supported by grants from Magn. Bergvalls Stiftelse, the Anna Ahrenberg Foundation and the Swedish Natural Science Research Council.
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