Evidence for 5-HT-containing intrinsic neurons in the teleost intestine

Cell Tissue Res (1983) 230:377-386 Cell and Tissue Research �9 Springer-Verlag 1983

Evidence for 5-HT-containing intrinsic neurons in the teleost intestine

Colin Anderson Department of Zoology, University of Melbourne, Parkville, Victoria, Australia

Summary. The stomach and small intestine of seven species of teleost fish were examined with both fluorescence and ultrastructural histochemical techniques for demonstrating monoamines. Fluorescence histochemistry demonstrated large numbers of yellow fluorescent neurons in the myenteric plexus of the intestine, but not the stomach, of four species. Varicose fibres arising from these neurons ran in the myenteric plexus, circular muscle layer and submucosa.

Ultrastructural histochemistry of tissue fixed with a modified chromaffin reaction showed that some neurons in the myenteric plexus contained biogenic amines in vesicles of 40-100 nm diameter. It is concluded that 5-HT-containing neurons are present in the intestine of some teleost species.

Key words: Intestine - Teleosts - 5-HT - Enteric neurons

Throughout the vertebrate series certain enteric neurons are capable of taking up exogenous 5-hydroxytryptamine (5-HT) (e.g. Goodrich et al. 1980; Epstein et al. 1980; Gershon and Altman 1971). It has been claimed that the neurons are tryptaminergic, that is, they utilize 5-HT as a neu- rotransmitter (Goodrich et al. 1980). However, no enteric neurons in higher vertebrates have a native 5-HT content that can be shown by fluorescence histochemical procedures, although recently some guinea-pig enteric neurons were shown to exhibit 5-HT-like immunoreactivity (Costa et al. 1982).

Of the lower vertebrates, only in cyclostomes has 5-HT been localized to both axons and enteric neurons (Honma 1970; Baumgarten et al. 1973;

Send offprint requests to: C. Anderson, Dept. of Zoology, University of Melbourne, Parkville, Victoria, 3052, Australia

Acknowledgements. This work was supported by the Australian Research Grants Committee. I would like to thank Professor Graeme Campbell and Dr. Ian Gibbins for valuable discussion and criticism of the manuscript. Support from production of the colour plate was given by the University of Melbourne.

378 C. Anderson

Goodrich et al. 1980). In the intestine of teleost fish 5-HT has been visualized only in neural processes (Baumgarten 1967; Watson 1979) and not in cell bodies. Watson (1979) concluded that in fish, 5-HT-containing axons must arise from extrinsic cell bodies. However, no 5-HT-containing neurons have been found in the teleostean sympathetic chain (Nilsson 1976), coeliac ganglion or splanchnic nerves (Campbell and Gannon 1976; Watson 1980).

This study was undertaken to determine if the 5-HT-containing axons previously reported in teleost intestine might actually be of intrinsic origin. Sites of 5-HT storage were identified by fluorescence histochemistry (Falck et al. 1962) and, ultrastructurally, using the modified chromaffin reaction for biogenic amines (Tranzer and Richards 1976).

Materials and methods

Seven species of fish were examined in this study: yellow-eye mullet, Aldrichettaforsteri (Mugili- dae); sand flathead, Platycephalus bassensis (Platycephalidae); brown trout, Salmo trutta (Sal- monidae); Australian short-finned eel, Anguilla australis (Anguillidae); green-back flounder, Rhornbosolea tapirina (Pleuronectidae); smooth toadfish, Torquigener glaber (Tetraodontidae); goldfish, Carassius auratus (Cyprinidae).

In those species with stomachs (Aldrichetta, Platycephalus, Salmo and Anguilla) the intestine immediately posterior to the pyloric sphincter was examined; sections of both cardiac and pyloric divisions of the stomach were also taken for fluorescence histochemistry. In the species of fish without stomachs (Carassius, Rhombosolea and Torquigener) tissue from midway along the length of the alimentary tract was used.

Some tissue was examined after treatment in vitro or in vivo with exogenous 5-HT creati- nine sulphate (Sigma) or with ct-methyl-noradrenaline (Sterling-Winthrop). In vitro treatment consisted of incubating tissue in the appropriate physiological solution containing one or both drugs, each at a concentration of 10 - 6 g/ml, for 45 min followed by a wash in drug-free solution for 15 min. The physiological solutions used were: Salmo: Cortland fish saline (Wolf 1963); Rhornbosolea: see Grove and Campbell (1979); Anguilla: modified Cortland fish saline (composition in mmole/l: NaC1, 124; KC1, 5.1; CaCI2, 1.62; MgSO4.7H20, 1.1; Na2PO4" 2H20, 2.6; NaHCO3, 12.0; glucose, 5.6); all other species: cod physiological solution (Holm- gren and Nilsson 1976). In addition, some fish were pretreated in vivo by injection of 5-HT or ct-methyl-noradrenaline (1-10 mg/kg i.p.) and were killed 30~0 min later.

Some fish were also pretreated with 6-hydroxydopamine (6-OHDA, Labkemi A.B.). A single dose of 100 mg/kg 6-OHDA was injected i.p. 48 h prior to the normal in vitro or in vivo treatment with 5-HT and ~t-methyl-noradrenaline.

Formaldehyde-induced fluorescence

Some tissue was treated using the aluminium-formaldehyde (ALFA) histofluorescence technique (Ajelis et al. 1979). This technique showed no advantage over the following more conven- tional technique which was more often used. Small pieces of gut were quenched in liquid propane chilled in liquid nitrogen and stored in liquid nitrogen until transferred to a freeze dryer (Dynavac FDC/H). The tissue was maintained below 10 -3 Torr and below - 3 5 ~ in the presence of phosphorus pentoxide (Sicapent, Merck) for 24 h, following which it was exposed to formaldehyde vapour at 70% humidity and 80 ~ C for I h. Following embedding in Paraplast Plus (Sherwood, Missouri), sections 10 ~tm thick were cut and mounted in paraffin oil, coverslipped, and examined with a Leitz Dialux 20 microscope fitted with a Ploemopak 2.3 incident light fluorescence system. Photomicrographs were taken with an Orthomat W camera using Kodak Ektachrome film, 400 ASA.

5-HT neurons in fish gut 379

Electron microscopy

The histochemical method for the ultrastructural localization of biogenic amines, modified from Tranzer and Richards (1976) was used. This technique has proven to give very good preservation of ultrastructural fine detail (e.g. Gibbins and Hailer 1979) as well as localizing biogenic amines.

The tissue was immersion fixed in a primary fixative of 1.75% formaldehyde (Merck) and 2.5% glutaraldehyde (Merck) in a 0.1 M chromate/dichromate buffer, pH 7.2, for 30 min. It was incubated in a 0.2 M chromate/dichromate buffer, pH 6.0, for 18-24 h and then osmi- cated in 2% OsO4 in 0.1 M chromate/dichromate buffer at pH 7.2. All procedures to this point were at 0--4~ C with 5% sucrose added. The tissue was then dehydrated in alcohol and embedded in Spurr's resin (Agar Aids). Ultrathin sections were stained with saturated uranyl acetate followed by alkaline lead citrate. Some sections were stained with alkaline lead citrate only, for better demonstration of the chromaffin reaction product.

Results

Fluorescence histochemistry

The fluorescence histochemical observations showed differences both between species and between stomach (where present) and intestine in the same species. The fluorescent neural structures observed comprised: yellow fluorescent neurons and their processes, confined to the intestine; blue-green fluorescent fibres innervating non-vascular structures; and blue-green fluorescent fibres innervating vascular structures. The regional and species distri- butions of these structures are shown in Table 1.

Yellow fluorescent structures

In the intestine of the four species with yellow fluorescent structures, the myenteric plexus contained large numbers of neurons showing yellow, fast- fading fluorescence (Figs. I, 2). The neurons were not aggregated into dis- crete ganglia but were dispersed singly or in groups of two or three throughout the plexus. Most neurons were unipolar, although some appeared to be multipolar. A smaller number of yellow fluorescent neurons was also found in the circular muscle layer some distance away from the myenteric plexus: the neurons were elongate, either unipolar or bipolar, and lay parallel to the smooth muscle cells. No fluorescent neurons were found in the submucosa or in the longitudinal muscle layer.

Yellow fluorescent nerve fibres were found in the myenteric plexus and could be seen to arise from both multipolar and unipolar fluorescent neurons. None of these fibres penetrated the longitudinal muscle layer. In contrast, the circular muscle layer was richly provided with yellow fluorescent fibres, both as individual varicose axons and as axon bundles, running parallel to the smooth muscle cells (Fig. 3). Some of the processes in the circular muscle layer could be traced back to the yellow cell bodies in the myenteric plexus. Probably others of the processes arose from the neurons lying in the muscle layer, although there were relatively few of those neurons.

380 C. Anderson

Table 1. A table showing species studied and the biogenic amines present in nerves of the stomach, intestine and perivascular plexuses of the enteric blood vessels. 5-HT 5-hydroxytryptamine; CA catecholamine

Species Types of amine containing axon(s) present in:

Stomach * Intestine * Perivascular nerves (both regions)

Anguillidae : Anguilla australis CA CA CA

Salmonidae: Salmo trutta CA 5-HT + CA CA

Cyprinidae: Carassius auratus No stomach 5-HT + CA CA

Platycephalidae: Platyeephalus bassensis CA 5-HT CA

Mugilidae: AIdrichetta forsteri CA 5-HT + CA CA

Tetraodontidae: Torquigener glaber No stomach CA CA

Pleuronectidae: Rhombosolea tapirina No stomach CA CA

* = not including perivascular nerves

Platycephalus exhibited the apparent innervation of the circular muscle described above. In addition, Platycephalus had large bundles of yellow axons running directly from the myenteric plexus to the submucosa, where they divided into smaller bundles. The large axon bundles ran at right angles to the circular muscle layer.

In all four species showing yellow fluorescent neurons there was a rich plexus of fine yellow fibres in the submucosa (Fig. 4).

The yellow fluorescent neurons and processes were clearly visible in Platycephalus and Aldrichetta without previous exposure to any exogenous drugs. In Salmo and Carassius the yellow fluorescence was less strong. Loading with 5-HT, in vitro or in vivo, increased both the fluorescence intensity and the persistence of the yellow fluorescence without changing the observable pattern of innervation. In the intestine of the three species that showed no native yellow fluorescence and in all stomachs, incubation with 5-HT did not reveal any yellow fluorescent neurons or processes.

Blue-green fluorescent axons

In the intestine and stomach (where present) of all species studied, blood vessels in the longitudinal and circular muscle layers, in the myenteric plexus and in the submucosa were surrounded by a dense plexus of blue-green fluorescent nerve fibres. Furthermore, nerve fibres with similar fluorescence


Fig. 1. Section through the myenteric plexus of Aldrichetta, cut tangential to the serosa. Yellow fluorescent neurons lie in a plexus of blue-green fluorescent axons. Formaldehyde-induced fluorescence. Calibration bar, 100 lam

Fig. 2. Two yellow fluorescent neurons in the myenteric plexus of Aldrichetta. Each neuron sends a process into nerve bundles containing blue-green fluorescent axons (arrow). Formalde- hyde-induced fluorescence. Calibration bar, 50 lam

Fig. 3. The circular muscle (era) of Aldriehetta contains many yellow fluorescent axons. Blue- green fluorescent axons can be seen around blood vessels in the submucosa (sm) and in the circular muscle. Formaldehyde-induced fluorescence. Calibration bar, 100 ~tm

Fig. 4. A dense plexus of yellow fluorescent axons in the submucosa of Platycephalus extending down to the epithelial cells of the villi (V). Formaldehyde-induced fluorescence. Calibration bar, 50 I~m

characteristics innervated non-vascular structures in all stomachs and in the intestine of all species except Platycephalus. The blue-green fluorescent nerve fibres formed a dense network in the myenteric plexus, where they ran in large longitudinal bundles cross-connected by smaller bundles. There were no obvious pericellular baskets of blue-green fibres around neurons, whether non-fluorescent or showing yellow fluorescence. Blue-green fibres left the myenteric plexus and entered the circular muscle layer where they could be seen running as small bundles or as individual varicose axons. Only occasional fibres were found in the longitudinal muscle layer, and then only in the stomach. The only blue-green fibres that could be detected in the submucosa seemed to be associated with blood vessels.

382 C. Anderson

No blue-green fluorescent nerve cell bodies were found in the stomach or intestine of any fish. This finding is in contrast to the study of Campbell and Gannon (1976). They found adrenergic nerve cell bodies in the stomach of Salmo near the points of entry of major branches of the splanchnic nerve. Similar points of entry of major nerve trunks were not specifically examined in this study and so the presence of adrenergic cell bodies in the stomach of this species cannot be ruled out. The natural intensity of the blue-green fluorescence was low, but the fluorescence did not fade during irradiation. Pretreatment in vivo or in vitro with e-methyl noradrenaline increased the intensity of fluorescence without changing the pattern of its distribution.

Effects of 6-OHDA

The effect of treatment of fish with 6-OHDA was investigated in Aldrichetta, Salmo and Platycephalus, which have intestinal yellow fluorescent neurons, and of Anguilla, which does not. 6 -OHDA (100 mg/kg) injected i.p. 48 h prior to processing the tissue for fluorescence histochemistry eliminated all blue-green fluorescent fibres found in the stomach and intestine. The yellow fluorescent neurons and processes were still visible and were apparently unaffected by 6 -OHDA pretreatment.

Electron microscopy

It was possible to identify, using the chromaffin fixation technique, amine containing neurons in the intestine of Salmo, Aldrichetta and Platycephalus. Small neurons, 10-20 ~tm in length with a centrally located nucleus, were found which were characterized by the presence of variable numbers of vesicles containing an electron dense core of chromaffin reaction product (Fig. 5). This strongly suggests the presence of a biogenic amine within the vesicles. Vesicles ranged from 40 nm to 100 nm in diameter. In all three species, chromaffin positive vesicles were found in tissue that had not previously been loaded with amine. Neurons containing chromaffin-positive vesicles were found in the myenteric plexus and a small number of similar neurons was found in the circular muscle layer. They were not found in the longitudinal muscle layer or in the submucosa. Examination of tissue treated with 6 -OHDA showed that the neurons were not affected. Neurons with the above characteristics thus corresponded in morphology, distribution and apparent presence of an amine with the yellow fluorescent neurons seen with fluorescent histochemistry.

The nuclei of the neurons with chromaffin-positive vesicles contained little dense staining heterochromatin and often had a prominent nucleolus. The cytoplasm of the neurons contained many small, oval or elongate mito- chondria, 200-750 nm in length. There were numerous free ribosomes and smooth endoplasmic reticulum was common. Golgi complexes were often present near the nucleus. The cytoplasm also contained numerous microtu- bules and neurofilaments.


Fig. 5. A presumed 5-HT-containing neuron seen with the electron microscope following fixation with the modified chromaffin method. A large number of vesicles showing the dense, black, chromaffin reaction product indicate the presence of a biogenic amine. This neuron lies in the circular muscle layer of Aldrichetta small intestine. The neuron forms a close contact (arrow) with a smooth muscle cell. Lead citrate stained. Calibration bar, 2 gm. Inset: a higher magnification micrograph of vesicles in the above neuron showing chromaffin reactive cores (arrows). Lead citrate stained. Calibration bar; 250 nm

Discussion

The amine-containing enteric innervation of the teleosts examined can be divided, on the basis of fluorescence histochemistry, into three separate components. The first component comprises yellow fluorescent neurons and

384 C. Anderson

their processes running in the intestinal myenteric plexus, circular muscle layer and in the submucosa. These neurons and processes were present in Aldrichetta, Carassius, Platycephalus and Salmo, but could not be found in Anguilla, Rhombosolea or Torquigener. The second component of the innervation, present in all fish studied except Platycephalus, consists of blue- green axons running in the myenteric plexus and circular muscle layer. The third component of the innervation, present in all fish, consists of blue-green axons confined to the walls of blood vessels.

The yellow fluorescent neurons have not previously been described in teleosts. The yellow, fast fading fluorescence of these neurons is typical of 5-HT (Falck et al. 1962). The intensity of the yellow fluorescence could be increased by exposure to exogenous 5-HT, either in vivo or in vitro. The fluorescence was not affected by a dose of 6-OHDA that completely eliminated any blue-green fluorescent fibres present, which presumably contained a catecholamine. Neurons which matched in size and distribution to the yellow neurons seen with fluorescence histochemistry were located by electron microscopy in the intestine of Aldrichetta, Salmo and Platycepha- lus. The amine-containing nature of these neurons was confirmed by ultrastructural histochemistry: the neurons characteristically contained numerous vesicles filled with dense chromaffin reaction product, indicating the presence of a biogenic amine, presumably 5-HT. The presence of 5-HT in the intestine has been shown chromatographically in Myoxoeephalus and Pleuronectes (Watson 1979) and in Platycephalus (J.L. Morris, unpubl, obs.).

Fluorescence histochemical studies of monoamines in the teleost intestine can now be shown to have produced results of three types. Three studies have shown only an adrenergic innervation of the intestine, in Salmo and Anguilla (Read and Burnstock 1968), Carassius (Saito 1973) and Pleuron- ectes (Fange and Grove 1979). Three studies have shown, in addition, 5-HT- containing nerve fibres in the intestine of Tinca (Baumgarten 1967), Pleuron- ectes and Myoxocephalus (Watson 1979) and Aldrichetta, Carassius, Platyce- phalus and Salmo (this paper). Finally, only the present study has demonstrated intrinsic 5-HT-containing neurons. It appears that some of the reports which did not reveal 5-HT-containing axons, specifically the earlier studies of Carassius, Pleuronectes and Salmo, were in error, either because 5-HT was not preserved by the procedures used or because the optics did not permit distinction of the fluorophores of catechol- and indoleamines. The observation of 5-HT-containing axons without corresponding intrinsic neurons (Baumgarten 1967; Watson 1979) is harder to explain. Since there has been no demonstration of peripheral 5-HT-containing neurons extrinsic to the gut it would seem that the axons must arise from intrinsic neurons, as proved to be the case in the present study. It might be thought that 5-HT is more concentrated and therefore more readily observed in the axons than in the cell bodies. However, no marked difference in fluorescence intensity between neurones and processes was found in this study, nor were any fish found in which only 5-HT-containing axons could be seen. The only major technical difference between this paper and the earlier ones is that epi-illumination was used, but this does not obviously explain the difference


in results. A final point to be made is that some fish apparently do not possess the 5-HT-containing system at all: three of the species studied here did not show yellow fluorescent structures, even after pretreatment with 5-HT which clearly enhanced the yellow fluorescence of 5-HT-containing neurons in other species.

Watson (1979) did not find a 5-HT-containing innervation of the circular muscle in the fish he examined. He reported that yellow fluorescent axon bundles only passed through the muscle layer to the submucosa. However the present study showed individual, varicose, yellow axons in the circular muscle, suggesting that the circular muscle is innervated by these axons. Goodrich et al. (1980) found an uptake of 3H-5-HT into axons in the circular muscle layer of Carassius intestine which supports the present work. However, they found no 3H-5-HT uptake into the submucosa, whereas a dense innervation of the submucosa by yellow fluorescent axons was found both by Watson (1979) and in the present study.

The presence of tryptaminergic neurons in the gut of mammals now seems well documented (see Gershon 1982). The present study demonstrates that tryptaminergic neurons are also present in some teleosts. However, the teleost tryptaminergic neurons exhibit several features not seen in mammalian tryptaminergic neurons. They have 5-HT in quantifies easily demon- strable by histofluorescence. In addition, their distribution to the circular muscle and submucosa does not match what is known about mammalian tryptaminergic neurons, which seem to be solely interneurons. Given these differences it seems premature to suggest that all tryptaminergic neurons will be largely similar in characteristics. It may also be premature to suggest that tryptaminergic neurons are " a general feature of the vertebrate bowel" (Goodrich et al. 1980) given that some fish seem to lack tryptaminergic neurons. It might be thought that those teleost species that apparently lack tryptaminergic neurons in fact have tryptaminergic neurons similar to those found in mammals. In this case, they would be impossible to demonstrate with fluorescence histochemistry.

The blue-green fluorescence seen in axons in the myenteric plexus and circular muscle layer and in axons around the blood vessels in the gut is characteristic of catecholamines. Surprisingly, Platycephalus appears to lack an adrenergic innervation of the intestinal myenteric plexus and circular muscle. While it may be possible that an adrenergic innervation is present but was not demonstrated by the techniques used, it was easy to demonstrate an adrenergic innervation of the stomach and of blood vessels in the intestine of the flathead. If the flathead does, in fact, lack an adrenergic innervafion of the intestinal musculature or myenteric plexus, it is the first such vertebrate reported to date.

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Accepted November 24, 1982

Evidence for 5-HT-containing intrinsic neurons in the teleost intestine

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