Immunohistochemical study of 5-HT-containing neurons in the teleost intestine: relationship to the...

Cell Tissue Res (1988) 254:553 559

andTt ue R e s e a r c h �9 Springer-Verlag 1988

Immunohistochemical study of 5-HT-containing neurons in the teleost intestine: relationship to the presence of enterochromaffin cells

Colin Anderson* and Graeme Campbell Department of Zoology, University of Melbourne, Melbourne, Australia

Summary. The formaldehyde-induced fluorescence technique had shown 5-hydroxytryptamine-conta ining enteric neurons in the intestine of the teleost Platycephalus bassensis, but did not reveal such neurons in the intestine of Te- tractenos glaber or Anguilla australis. Re-examinat ion of these animals with 5-hydroxytryptamine immunohis to- chemistry showed immunoreact ive enteric neurons in the intestine of all three teleost species. The 5-hydroxytryptamine-containing enteric neurons showed essentially the same morphology in all species examined: the somata were si tuated in the myenteric plexus, extending down into the circular muscle layer, but none were found in the submucosa; processes were found in the myenteric plexus, the circular muscle layer and the lamina propria. It was con- cluded that the neurons may innervate the muscle layers or the mucosal epithelium, but were unlikely to be interneurons. In a range of teleosts, enterochromaff in cells were found in the intestine of only those species in which the formaldehyde technique did not visualize neuronal 5-hydroxytryptamine. Avai lable evidence suggests that, in vertebrates, 5-HT-containing enterochromaff in cells are lacking only where there is an innervat ion of the gut mucosa by nerve fibres containing high concentrat ions of 5-HT.

Key words: Enterochromaff in cells - Immunohis tochemis- try - Intestine - Neurons Serotonin Anguilla australis, Platycephalus bassensis, Tetractenos glaber (Teleostei)

The formaldehyde-induced fluorescence histochemical pro- cedure has shown that the intestine of some but not all teleost fish contains neurons that store 5-hydroxytryptamine (5-HT: Baumgarten 1967; Watson 1979; Anderson 1983). It is not clear whether the failure to locate 5-HT- containing enteric neurons in some teleosts reflects the absence of such neurons or whether it is due to a l imitat ion of the histochemical technique used. Previous studies on mammal ian 5-HT-containing enteric neurons suppor t the lat ter suggestion: numerous studies using formaldehyde-induced fluorescence failed to provide any evidence for enteric

* Present address: Baker Medical Research Institute, Melbourne, Australia

Send offprint requests" to: Prof. G. Campbell, Department of Zoolo- gy, University of Melbourne, Parkville, Vic. 3052, Australia

neurons containing 5-HT (Costa and Furness 1971; Ahl- man et al. 1973; Baumgarten et al. 1970; Ah lman and Ener- brick 1974; Dubois and Jacobowitz 1974; Furness and Costa 1974, 1978; Diab et al. 1976), but later studies, using immunohistochemistry, have found 5-HT-containing neurons in the intestine of a variety of mammals (Costa et al. 1982; Kur ian et al. 1983; Griffi th and Burnstock 1983; Dahls t rom and Ahlman 1983; Legay et al. 1984). These results seem to show that immunohis tochemist ry is more sensitive than formaldehyde-induced fluorescence in reveal- ing neuronal 5-HT. It was therefore decided to carry out an immunohistochemical study of the intestines of selected teleosts.

Immunohis tochemis t ry directed against 5-HT was applied to the intestines of two teleosts, Anguilla australis and Tetractenos glaber, in which no evidence of intrinsic 5-HT- containing neurons had been obta ined in a previous formaldehyde-induced fluorescence study (Anderson 1983). The intestine of another teleost, Platycephalus bassensis, re- por ted in the same study to have 5-HT-containing neurons, was also examined. In addit ion, the intestines of a variety of teleosts were examined with formaldehyde-induced fluorescence to determine the presence or absence of mucosal enterochromaff in cells.

Materials and methods

Sand flathead (Platycephalus bassensis, Platycephalidae, n = 6), were caught by handline in Port Phillip Bay, Victoria. Yellow eye mullet (Aldrichetta forsteri, Mullidae, n=3), smooth toadfish (Tetractenos glaber, Tetraodont idae , n = 6) and green-backed f lounder (Rhombosolea tapirina, Pleuron- ectidae, n = 3) were caught by beach seining in Westernpor t Bay, Victoria. Aust ra l ian short-finned eels (Anguilla australis occidentalis, Anguill idae, n = 6), were obtained from Eels Pty Ltd, Skipton Victoria. Brown trout (Salmo trutta, Sal- monidae, n=6) and goldfish (Carassius auratus, Cyprini- dae, n = 3) were obtained from local commercial suppliers. Fish were mainta ined and anaesthetised as described by Anderson (1983).

Physiological solutions

Dissected tissues were held and incubated with drugs in the following physiological solutions:

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(a) P. bassensis and T. glaber: cod physiological solution (Holmgren and Nilsson 1976). Composition in raM: NaC1, 148.9; KC1, 5.1; NaHzPO4, 2.8; MgSO4, 1.8; CaCI2, 3.0; glucose, 5.6.

(b) A. australis: a modification of WoWs (1963) Cor- tland fish saline. Composition in raM: NaC1, 124.1; KCI, 5.1; NaH2PO4., 2.6; NaHCO3, 11.9; MgSO4, 0.9; CaC12, 1.6; glucose, 5.6.

All solutions were gassed with 95% 02:5% CO/. The solutions were chosen because they were designed for mar- ine and freshwater teleost tissues, respectively. Nerve-me- diated responses of cardiac and smooth muscle preparations from these species persist for many hours in the respective solutions (Donald and Campbell 1983, and unpublished observations). Cortland saline was modified to match Na + and K + concentrations as measured in A. australis plasma (n=2).

Pretreatments

Catecholamine-containing nerves were destroyed in vivo with 6-hydroxydopamine HC1 (6-OHDA) dissolved in a small volume of antioxidant solution (composition in mM : NaC1, 111.2; KC1, 2.0; ascorbic acid, 11.4). Fish were in- jected with 100 mg kg-1 6-OHDA i.p. four days prior to use. Alternatively, segments of gut were incubated in vitro for 3 h in the appropriate physiological solution containing 400 gM 6-OHDA (Costa et al. 1982). Some preparations were incubated in vitro for 1 h with 5-HT creatinine sul- phate (25 gM) or 5,7-dihydroxytryptamine creatinine sul- phate (5,7-DHT, 250 gM), either alone or added to the 6-OHDA incubation medium for the final hour. No antioxidant was used. The tissue was rinsed briefly in drug-free physiological solution before fixation. All drugs were obtained from Sigma, St. Louis, Missouri, USA.

Immunohistochemistry

The intestines from P. bassensis, T. glaber and A. australis were processed as wholemounts for immunofluorescence as follows (Costa et al. 1982). Lengths of intestine were cut open along the mesenteric edge and pinned, serosal side up, on pieces of dental wax. The mounted tissue was im- mersed for 2 h in 4% formaldehyde in 0.1 M phosphate buffer, pH 7.2, at 4 ~ C. The fixed tissue was rinsed 9 • 10 min in 80% ethyl alcohol and stored in phosphate- buffered saline (PBS, composition in mM: NaCI, 145.4; Na2HPO4, 1.07; NaH2PO~, 2.5). Prior to incubation with antibodies the pieces were sometimes split into two layers, consisting of the muscularis externa and the submucosa, respectively. The mucosa was scraped off prior to splitting the tissue. Rat monoclonal anti-5-HT antibodies (Sera-Lab, Crawtey Down, England) diluted 1 : 200 in PBS were applied and left for 18 to 24 h at room temperature. Following 3 rinses in PBS the tissue was incubated in fluorescein isoth- iocyanate-labelled sheep anti-rat antibodies (Wellcome Laboratories, Beckenham, England) for 1 h at room temperature, rinsed in 3 changes of PBS and mounted in 50% glycerol/50% bicarbonate buffer, pH 8.6. The resulting wholemounts were examined on a Leitz Dialux 20 fluorescence microscope fitted with a Ploemopak 2.3 incident light system and filter block I2. Photomicrographs were taken with a Leitz Orthomat camera on Kodak Tri-X or Ilford FP4 film, both rated at ISO 400.

The specificity of antibody staining was tested by staining with antisera preabsorbed with 0.5 M 5-HT for 6 h at room temperature.

Formaldehyde-induced fluorescence

Pieces of intestine from A. forsteri, A. australis, C. auratus, P. bassensis, R. tapirina, S. gairdneri and T. glaber were dissected out and processed for formaldehyde-induced fluorescence as described by Anderson (1983). The resulting sections were examined on a Leitz Dialux 20 microscope fitted with a Ploemopak 2.3 illumination system and filter block D.

Results

Immunohistochemistry

5-HT-like immunoreactivity (5-HT-IR) was detected in enteric nerve cell bodies and processes in the intestine of P. bassensis, A. australis and T. glaber. The 5-HT-IR of neurons in untreated tissue was weak, particularly in A. australis and T. glaber. The nerve cell bodies were usually more intensely fluorescent than the processes in all untreated fish. Treatment with exogenous 5-HT or 5,7-DHT resulted in a marked increase in the immunoreactivity of nerve cell bodies and particularly of nerve processes in all three species studied, but did not alter the distribution of 5-HT-IR. Pretreatment of the intestine with 6-OHDA either in vitro or in vivo did not affect the distribution of the 5-HT-IR.

P. bassensis had 5-HT-IR neurons (Fig. 1 A, B) throughout the length of the intestine. The 5-HT-IR neurons were oval with the long axis of the cell averaging 20.7+7.1 ~tm (n=46; all figures are given as mean +SEM) in length. The 5-HT-IR nerve cell bodies had a generally smooth sur- face and a prominent, long varicose process extending from one pole of the cell (Fig. 1 B); no other major process could be seen by moving the focal plane through wholemounts, so that most cells appeared to be unipolar. Some cells, however, had an additional short, blunt process extending from the end of the cell opposite the long varicose process. A smaller number of cells had more than one short, blunt process. Most 5-HT-IR nerve cell bodies were located in the myenteric plexus, between the longitudinal and circular muscle layers, but a few were deep in the circular muscle layer. Immunoreactive nerve cell bodies were abundant in the myenteric plexus, with an average density of 366_+ 175 cells cm 2. The density of cells did not appear to vary systematically along the length of the intestine. The neurons were not aggregated into ganglia but were often concen- trated around the periphery of large, longitudinally orientated nerve trunks in the myenteric plexus (Fig. 1 A). In the nerve trunks it was apparent that the 5-HT-IR nerve cell bodies were not in any particularly close association with the 5-HT-IR fibres running in the nerve trunk. Many nerve cell bodies, whether associated with longitudinal nerve trunks or situated in the myenteric plexus between the nerve trunks, were oriented with their long axis parallel to the longitudinal smooth muscle cells.

The 5-HT-IR nerve cell bodies in the circular muscle layer of P. bassensis occurred at about 1/6 the density of those in the myenteric plexus. These nerve cell bodies were very elongated and were aligned parallel to the surrounding smooth muscle cells. The nerve cell bodies appeared to be

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Fig. 1, 5-HT-IR in wholemounts of intestine, pretreated with 5-HT in vitro. Low and high magnification micrographs of P. bassensis (A, B), A. australis (C, D) and 7". glaber (E, F) small intestine, showing nerve cell bodies and processes in the myenteric plexus. The arrow in A lies on the line of a primary strand of the myenteric plexus. Scale bars=100 lam (A, C, E: x85), or 50 lam (B, D, F: x 220)

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unipolar or, at most, bipolar. No nerve cell bodies were ever seen in the submucosa.

The single, varicose process arising from each 5-HT-IR nerve cell body could be traced for various distances from the cell or origin. Some varicose processes coursed through the myenteric plexus with no preferred orientation. How- ever, most fibres ran in well-defined, longitudinally-directed axon bundles in the myenteric plexus. 5-HT-IR axons did not terminate around 5-HT-IR nerve cell bodies nor did they appear to terminate around non-immunoreactive nerve cell bodies. Some 5-HT-IR axons were seen to turn sharply into the circular muscle layer after their origin from the nerve cell body. Such processes ran deep into the circular muscle layer before turning again to run as single varicose axons parallel with the smooth muscle cells (Fig. 2A). The fibres did not appear to be abundant but it is likely that some fibres had been damaged when the submucosa was peeled off. 5-HT-IR processes running in the myenteric plexus and in the circular muscle layer were never seen to branch. The overall impression gained was that the 5-HT-IR fibres seen in the myenteric plexus and in the circular muscle were easily accounted for by the large number of 5-HT-IR nerve cell bodies.

In P. bassensis, 5-HT-IR fibres were seen to pass from the circular muscle layer into the submucosa. At the base of the circular muscle layer, single varicose fibres running parallel to the circular muscle layer and fibres running verti- cally through the muscle layer collected into large nerve trunks. The nerve trunks entered the submucosa and ran through the intervening connective tissue to the lamina propria (Fig. 2 E). Here the nerve trunks broke up into a dense plexus of very fine, varicose axons that appeared to be closely applied to the base of the mucosa (Fig. 2F). The plexus of fibres extended into the core of each villus.

No staining was seen in cell bodies or processes when antiserum preabsorbed with 5-HT was used.

A. australis resembled P. bassensis in the morphology and distribution of 5-HT-IR nerve cell bodies and their processes. The 5-HT-IR nerve cell bodies of A. australis (Fig. 1 C, D) were similar in shape and size to those of P. bassensis, averaging 19.6 + 6.3 gm in length (n=24), but occurred at a slightly higher density of 432_ 57.3 per cm 2. One other difference was that the 5-HT-IR axons in the myenteric plexus of A. australis were not markedly aggregated into longitudinally orientated nerve trunks and thus the 5-HT-IR nerve cell bodies tended to lie in a diffuse network of 5-HT-IR axons (Fig. 1 C). In all other respects, including the presence of 5-HT-IR nerve cell bodies in the circular muscle layer (Fig. 2C, D) and the presence of a dense plexus of fine, varicose fibres in the lamina propria, the distribution of 5-HT-IR nerve cell bodies and processes in A. australis resembled that in P. bassensis.

The 5-HT-IR enteric neurons of T. glaber differed sub- stantially for those of P. bassensis and A. australis. The cell bodies were larger, an average 37.1 _+ 10.5 p,m (n=21) in length (Fig. 1E, F), and although the density was not determined, the neurons seemed less abundant than those in the other species. The nerve cell bodies tended to be circular rather than elongated in outline. A single long, varicose process arose from the soma, but numerous short, tapering processes also arose from the remainder of the cell body. The myenteric plexus, in keeping with the smaller number of cell bodies present compared to the other two species, had fewer 5-HT-IR fibres. The plexus often showed

loosely aggregated bundles of 5-HT-IR processes running longitudinally. No nerve cell bodies were found in the circular muscle or submucosa. There was a dense innervation of the circular muscle layer by 5-HT-IR fibres (Fig. 2B). These fibres stained only faintly, even after 5,7-DHT or 5-HT loading, and had very small varicosities, while inter- varicose regions were only rarely stained. As in the other two species, large immunoreactive nerve trunks ran through the submucosa to disperse over the epithelium as a dense plexus of fine fibres.

Enterochromaffin cells

5-HT-IR cells were seen in the intestinal mucosa of A. australis and T. glaber. They were absent from the intestine of P. bassensis. These cells resembled classical enterochromaffin (EC) cells in that they occurred singly in the mucosal epithelium and were typically elongated elements with a centrally located nucleus and a tapering process at each end that extended towards, and often reached the lumenal and ablumenal sides of the mucosal epithelium.

When processed for formaldehyde-induced fluorescence, EC cells showing the fast-fading yellow fluorescence typical of 5-HT were found in the intestinal mucosa of A. australis. R, tapirina and T. glaber. None were found in the intestine of A. forsteri, C. auratus, P. bassensis or S. gairdneri.

Discussion

Distribution of 5-HT-eontaining neurons

5-HT-immunohistochemistry confirmed the presence of 5-HT-containing neurons in the intestine of P. bassensis, showing neurons with exactly the same morphology and distribution as previously reported with formaldehyde-induced fluorescence. In contrast, it now appears that at least two of the species reported by Anderson (1983) to lack 5-HT-containing enteric neurons do in fact have such neurons, detectable with immunohistochemistry: the intestines of A. australis and T. glaber contained 5-HT-IR neurons, which had a distribution essentially the same as that seen in P. bassensis. It is apparent that the neurons in A. australis and T. glaber normally contain 5-HT, for 5-HT-IR was visualised even in tissue fixed immediately after dissection, without incubation with 5-HT. However, the level of 5-HT in the neurons is apparently too low to be detected by formaldehyde-induced fluorescence. Furthermore, the low level of 5-HT in the neurons of A. australis and T. glaber appears to reflect a small storage capacity for amine: although they took up 5-HT and 5,7-DHT, as shown by a marked increase in immunfluorescence, loading with 5-HT did not render them visible with formaldehyde-induced fluorescence (Anderson 1983).

Intestinal 5-HT-containing neurons have now been found in a range of teleostean species, including: (i) fish with (e.g.S. gairdneri) and without (e.g.R. tapirina) a stomach; (ii) herbivores like C. auratus and carnivores like P. bassensis; (iii) species inhabiting fresh water (e.g., Tinca tinca) or sea water (e.g., P. platessa); and (iv) species from primitve groups such as the Anguillidae (A. australis) and from advanced groups such as the Tetraodontidae (7". glaber) (Baumgarten 1967; Watson 1979; Anderson 1983" present work). It therefore seems likely that all teleosts have

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Fig. 2. 5-HT-IR in wholemounts of intestine, pretreated with 5-HT in vitro. A. Fibres in circular muscle of P. bassensis, x 280. B. Fibres in circular muscle of T. glaber, x 180. C, D. Low ( x 80) and high ( x 200) magnification micrographs of nerve cell bodies in circular muscle of A. australis. E. Nerve bundles in the submucosa of P. bassensis, x 180. F. A plexus of varicose axons in the lamina propria of intestinal villi from P. bassensis, x 180. Scale bars=50 ~tm, except 100 p.m in C

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5-HT-containing enteric neurons. However, in some species the neurons are 5-HT-rich and can be visualised by the formaldehyde technique, while in other species they store little 5-HT and can be visualised only by immunohistochemistry. In this context, apparently there can be variation along the length of the gut within a species: in S. gairdneri, the intestinal 5-HT-containing neurons are 5-HT-rich (An- derson 1983), whereas the neurons in the stomach contain little 5-HT and can be visualised only by means of immunohistochemistry (Holmgren et al. 1985) but not by the formaldehyde technique (Anderson 1983).

The 5-HT-containing enteric neurons in teleosts seem to have a uniform morphology, irrespective of the level of 5-HT that they contain. In the species examined in this study, like other species examined previously (Watson 1979; Anderson 1983), the neurons have a strong projection to the lamina propria. Axons also project to the circular muscle in all the species we have examined, with T. glaber showing the densest innervation, although Watson (1979) did not describe this projection in the species he examined. In no case were axons seen to form pericellular baskets around nerve cell bodies in the myenteric plexus. The 5-HT-containing neurons are therefore unlikely to be interneurons. They might be motoneurons regulating the circular muscle and the mucosal epithelium.

Enterochromaffin cells

EC cells are found in protochordates (Lison 1933; Gerzeli 1961) and throughout the vertebrates (see Erspamer 1954). EC cells have never been found in cyclostomes, and it was initially thought that they were not present in teleosts: the chromaffin method and other early techniques did not reveal EC cells in nine teleost genera (Vialli and Erspamer 1933; Uggeri 1938). Use of the formaldehyde-induced fluorescence technique has since shown 5-HT-containing EC cells in some but not all teleosts: Read and Burnstock (1968) found EC cells in the stomach and intestine of A. australis, and in the stomach but not the intestine of S. gairdneri; Watson (1979) found EC cells in the stomach but not the anterior intestine of Clupea harengus, Myoxoce- phalus scorpius and Pleuronectes platessa. We have confirmed the observations on the intestine o f A. australis and S. gairdneri and, in addition, have found intestinal EC cells in T. glaber but not in A. forsteri, C. auratus or P. bassensis.

Our results reveal an interesting correlation between intestinal EC cells and 5-HT-containing neurons that might offer an explanation for the sporadic occurrence of EC cells in fish. All of the fish examined had 5-HT-IR fibres inner- vating the intestinal mucosa. In each species that lacked intestinal EC cells, the neurons were 5-HT-rich and could be visualised by formaldehyde-induced fluorescence (An- derson 1983). Conversely, intestinal EC cells were present only in those species where formaldehyde-induced fluorescence did not reveal the 5-HT in the neurons (Andersons 1983). Thus, EC cells seem to be absent only in teleosts displaying an abundant innervation of the mucosa by 5-HT- rich neurons. The observations of Watson (1979) on M. scorpius and P. platessa, although perhaps not those on C. harengus, are consistent with this view.

The correlation seems to hold for other parts of the gut in teleosts and for other vertebrate groups. Although there are 5-HT-IR neurons in the stomach of S. gairdneri (Holmgren et al. 1985), they are not revealed by formalde-

hyde-induced fluorescence and EC cells are present in the mucosa; in contrast, the intestine of S. gairdneri has 5-HT- rich neurons and lacks EC cells (Read and Burnstock 1968; Anderson 1983). In the intestine of the cyclostome Lampe- tra fluviatilis, 5-HT-rich axons form a mucosal plexus and 5-HT-containing EC cells are absent (Baumgarten et al. 1973). While there are also 5-HT-rich nerve fibres in the intestine of the chondrostean fish Acipenser baeri and A. stellatus, the fibres do not distribute to the mucosa and EC cells are abundant (Salimova and Feh6r 1982). Elasmo- branch fish possess EC cells (Vialli and Erspamer 1933; Uggeri 1938) and, at least in Squalus acanthias, there is no 5-HT-IR in enteric nerve cell bodies and reactivity is seen in only very occasional nerve fibres (Holmgren and Nilsson 1983). Finally, EC cells are found throughout the gut in all tetrapods, while 5-HT-containing neurons are either absent (reptiles, birds, prototherian mammals: Adam- son and Campbell 1988) or, when present, are not distrib- uted to the mucosa (amphibians: Anderson and Campbell 1984; eutherian mammals: Costa et al. 1982).

If there is truly an inverse correlation between the presence of a 5-HT-rich mucosal innervation and the absence of EC cells, it could be argued that: (a) 5-HT plays a role in the regulation of the mucosa; (b) 5-HT can be provided equally effectively from EC cells or from 5-HT-rich neurons; (c) the relationship is mutually exclusive in that a species expresses a high concentration of 5-HT in only one of the possible control structures in any part of the gut. It is plausible that neurons have taken over the role of the EC cells in the lamprey and some teleosts, rather than the reverse, since it is the EC cells that have been preserved in most other vertebrates. This may relate to Erspamer's (1954) view that the non-chromaffin 'pre-enterochromaffin argentophil ' cells present in the gastrointestinal mucosa of teleosts and cyclostomes (Uggeri 1938) are EC cells that have lost their 5-HT in the course of evolution.

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Accepted May 27, 1988

Immunohistochemical study of 5-HT-containing neurons in the teleost intestine: relationship to the...

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