evidence for 5-hydroxytryptamine in neurones in the gut of the toad, bufo marinus

Cell Tissue Res (1984) 238:313-317

a n d R e s e a r c h �9 Springer-Verlag 1984

Evidence for 5-hydroxytryptamine in neurones in the gut of the toad, Bufo marinus

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

Summary. The stomach, small intestine and large intestine of the toad, Bufo mar&us, were processed for formaldehyde-induced fluorescence histochemistry. After extrinsic denervation or pretreatment with 6-hydroxydopamine to remove catecholamine fluorescence, yellow fluorescence typical of 5-hydroxytryptamine was observed in neurones in the small intestine only. The cell bodies and their processes were confined to the myenteric plexus. Additional pretreatment with 5-hydroxytryptamine enhanced the fluorescence of neurones in the small intestine and revealed yellow- fluorescent nerve fibres, but not cell bodies, in the longitudinal and circular muscle layers and myenteric plexus of the large intestine. No fluorescent neurones were observed in the stomach. Following reserpine treatment, which removed native yellow fluorescence in the small intestine, exposure to 5-hydroxytryptophan produced yellow fluorescence in axons in both small and large intestine; exposure to tryptophan never restored fluorescence. The neurotoxin, 5,7-dihydroxytryptamine had no effect on the distribution of yellow-fluorescent neurones in the small and large intestine. No 5-HT-containing mast cells were present in either the small or large intestine. Thin layer chromatography with three different mobile phases showed a 5-hydroxytryptamine-like compound in extracts of mucosa-free small and large intestine but not of stomach.

Key words: Serotonin (5-HT) - Stomach - Intestine, small and large - Myenteric ganglia - 5-HT fluorescence histochemistry - Bufo marinus

Goodrich et al. (1980) have suggested that enteric neurones that use 5-hydroxytryptamine (5-HT) as a transmitter sub- stance are to be found at all levels of vertebrate organisa- tion. Goodrich et al. (1980) showed uptake of 3H-5-HT into the myenteric plexus and circular muscle of the intestine of Rana catesbeiana. It might be expected that the formaldehyde-induced fluorescence (FIF) technique would be able to confirm the presence of neuronal stores of 5-HT in amphibian enteric neurones. To date, only one FIF study has suggested that neuronal 5-HT is present in the gut of an amphibian (Wong et al. 1971). Other studies on the adrenergic innervation of the amphibian gut have used FIF techniques that should have shown a tryptaminergic inner-

Send offprint requests to." Colin Anderson, Department of Zoology, University of Melbourne, Parkville, Victoria, 3052, Australia.

vation if one were present (Read and Burnstock 1968a, b; Gillard and Read 1971). None of these studies revealed tryptaminergic neurones. This study seeks to confirm the presence of 5-HT in enteric nerves in the toad, Bufo marinus, using FIF techniques and thin-layer chromatography.

Materials and methods

Toads (Bufo marinus) of 50-150 g weight were captured from wild populations in Northern Queensland and maintained at 25~ on a dark/light cycle consistent with the site of capture. They were kept without feeding for up to four weeks and anaesthetised for dissection in tricaine meth- ane sulphonate solution (0.5% MS 222, Rural Chemical Industries, Australia). Numbers of animals examined, see below.

Formaldehyde-induced fluorescence

Small pieces of gut were quenched in liquid propane chilled in liquid nitrogen, and stored in liquid nitrogen until transfer 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 1 h. Following em- bedding in Paraplast Plus (Sherwood Medical, Athy, Ire- land), sections 10 lam thick were cut and mounted in paraf- fin 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 Tri-X film, 400 ASA.

It was possible to prepare freeze-dried whole-mount preparations of large intestine. The large intestine was pinned out on dental wax and the submucosa and mucosa stripped off. The muscularis externa was then stretched out on a glass side and frozen in liquid propane. This prepara- tion was processed for formaldehyde-induced fluorescence as described above.

Thin-layer chromatography

Tissues were kept in ice-cold toad physiological solution, pH7.4 (composition in raM: NaC1, 115; KC1, 3.2; NaHCO3, 20; NaHzPO~, 3.1; MgSO4, 1.4; CaCI/, 1.3; D- glucose, 16.7). The mucosa was removed from stomach by dissection and from the small and large intestine by scraping. A single stomach provided 1.0-1.4 g of mucosa-free

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tissue for extraction. Small or large intestines from five toads were pooled to provide 1.0-1.4 g of mucosa-free tissue.

Tissue was homogenised in ice-cold 0.1 M perchloric acid (10 ml acid per 1 g tissue) with an Ultra Turrax T18/10 homogeniser. The homogenate was shaken at room temper- ature for 30 min and cemrifuged at 8000 g for 20 min at 0-4 ~ C. The supernatant was brought to pH 6.0 with 1 M KOH, passed through a 0.45-i.tm filter and run through a Sep Pak C18 cartridge (Waters Associates) pretreated with 5 ml of methanol and 5 ml of 0.02 M phosphate buffer, pH 6. The cartridge was washed with 2.5 ml H20 and the indoleamines were eluted off the cartridge with 2 ml of methanol (Tonelli et al. 1982). The eluate was dried in va- cuo. The sample was re-suspended in 50 ~tl methanol and applied to TLC plates (Silica Gel 60, 0.25 mm layer thick- ness). The plates were developed in: acetone, isopropanol, water, acetic acid (50:40:7:3); chloroform, acetic acid, methanol, water (65 : 20:10: 5); or isopropanol, methylace- tate, ammonium hydroxide (35:45:20). After drying for 30 min at 60 ~ C the plates were sprayed with o-phthaldialdehyde (0.4% w/v in equal parts methanol, concentrated HC1) and heated at 100~ for 15 min. The indoleamines were then visible under UV light of 254 nm or 366 nm. To confirm that scraping off the mucosa removed all enterochromaffin cells, pieces of small and large intestine from three toads were processed as whole mounts for formaldehyde-induced fluorescence as described above.

Fluorescence histochemistry

Initially the small and large intestines of two untreated toads were processed for fluorescence histochemistry. It soon became apparent that adrenergic nerves in the gut would obscure any other types of fluorescent nerve present, and so twelve animals were subjected to chemical adrenergic denervation by a single subdermal injection of 6-hydroxydopamine hydrochloride (6-OHDA, 50 mg/kg, 4 days before use) dissolved in an antioxidant solution (NaC1, 0.65%; KC1, 0.014%; ascorbic acid, 0.02%: Gillard and Read 1971). Tissue from six 6-OHDA-treated toads was incubated in vitro with a range of drugs as follows: Rings of small intestine, 1 cm long, and large intestine, 0.5 cm long, were removed from anaesthetised animals and incubated at 25 ~ C in toad physiological solution (for composition, see above): bubbled with 5% CO2, 95% 02, for a total of I h. Pargyline hydrochloride (500 nM) was present in the incubation solution for 15 min before one of the following drugs was added : 5-hydroxytryptamine creatinine sulphate (5-HT, 25 nM, n=3), 5-hydroxytryptophan (5-HTP, 45 nM, n=2), L-tryptophan (500 nM, n= 3), L- dopa (500 nM, n= 1), dopamine hydrochloride (525 nM, n = l ) , noradrenaline hydrochloride (500 nM, n= l ) . The fluorescent histochemical appearance of tissue treated as above was compared to control tissue from the same animal incubated for 1 h in drug-free solution (n=6) or solution containing only pargyline (500 nM, n = 6).

The remaining six 6-OHDA pre-treated animals were given a single dose of reserpine (Serpasil, 20 mg/kg) 2 days prior to using the toad. Tissue from the reserpine treated animals was treated in vitro as described above, with the following drugs: 5-HT (n=6), 5-HTP (n=6), tryptophan (n = 3), 5,7-dihydroxytryptamine (5,7-DHT, 250 nM; n = 2), L-dopa (n=2), dopamine (n=2), noradrenaline (n=3).

Again, control tissue from each animal was incubated in drug-free solution and solution containing only pargyline.

The effect of prolonged treatment with 5,7-DHT was examined. Two toads treated with 6-OHDA were injected with 5,7-DHT, given as two doses of 100 mg/kg each, 4 and 2 days prior to processing for fluorescence histochemistry.

In some toads (n= 12) the small intestine was extrinsically denervated. Toads were anaesthetised with MS 222. A lateral incision was made posterior to the axilla. The coeliac ganglion located at the common junction of the systemic arches, mesenteric artery and dorsal aorta, was removed along with as many coeliac nerve branches as were accessible. The body cavity was dusted with Cicatrin antibi- otic powder (Wellcome, Australia) and the incision closed with tissue glue (Ethicon bucrylat, Ethicon, Australia). Ani- mals were left for 10 days to allow degeneration of the severed axons. Pieces of intestine from surgically denervated animals were processed for fluorescence histochemistry after the following drug treatments: All animals received an injection of 100 mg/kg pargyline. After 15 min, nine animals received a further injection of the following drugs at the indicated doses and were left for 1 h before processing for fluorescence histochemistry: 5-HT (10mg/kg, n=3), 5-HTP (10 mg/kg, n= 1), tryptophan (10 mg/kg, n= 1), L- dopa (10mg/kg, n = l ; 100mg/kg, n= l ) , noradrenaline (1 mg/kg, n=2). The remaining three toads injected with pargyline received no further treatment and were left 75 min before processing for fluorescence histochemistry.

Serpasil was obtained from Ciba-Geigy (Sydney, Aus- tralia). All other drugs were obtained from Sigma (St. Louis, USA).

Results

Fluorescence histochemistry

Initially, the stomach, small intestine and large intestine were processed without prior treatment. All three areas contained only nerves showing the blue-green fluorescence typical of catecholamines. In the stomach and small intestine the adrenergic nerves were confined to the myenteric plexus and circular muscle layer and to perivascular nerve plexuses in all layers of the gut. In the large intestine the longitudinal muscle contained numerous longitudinally running adrenergic nerve bundles. Additional longitudinally running nerve trunks in the myenteric plexus gave rise to smaller adrenergic nerve bundles that entered the circular muscle layer. The large intestine also contained adrenergic nerves in perivascular plexuses. All adrenergic nerves in the alimentary tract of the toad were eliminated by treatment with 6-OHDA.

After 6-OHDA treatment had eliminated the adrenergic fibres a second population of fluorescent fibres was revealed in the small intestine only. These nerves showed the fast- fading yellow fluorescence typical of 5-HT. The yellow-fluorescent axons in the small intestine were organised as large, longitudinally running nerve trunks in the myenteric plexus (Fig. 1). Smaller nerve trunks and single varicose fibres formed a network throughout the myenteric plexus. No yellow-fluorescent axons were seen in the longitudinal or circular muscle, submucosa or around blood vessels. Scat- tered amongst the nerve trunks in the myenteric plexus were yellow-fluorescent cell bodies (Fig. 2). These neurones were

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Fig. 1. Tangential section through myenteric plexus of small intestine. Large, yellow-fluorescent nerve trunks are seen running parallel to the longitudinal muscle direction. Note also single, varicose fibres, x 350. This and all subsequent figures are of formaldehyde-induced fluorescence in 6-OHDA-pretreated tissue loaded in vitro with 5-HT

Fig. 2. Yellow-fluorescent cell body in a tangential section through the myenteric plexus of small intestine. Arrow indicates orientation of longitudinal muscle. The short processes are aligned with the circular muscle layer, x 430

Fig. 3. Whole mount of large intestine showing large, longitudinally running nerve trunks in the longitudinal muscle layer. Arrow shows the orientation of the longitudinal muscle, x 80

Fig. 4. Oblique section through the longitudinal muscle of the large intestine showing both large nerve trunks and single fibres, x 130

Fig. 5. Whole mount of large intestine. Nerve bundles of various sizes in circular muscle layer. Arrow shows orientation of longitudinal muscle layer, x 135

Fig. 6. Oblique section through the circular muscle layer of the large intestine showing small and large yellow-fluorescent nerve trunks. x 135

mult ipolar with short, branching processes. The short processes were generally aligned with the smooth muscle cells of the circular muscle layer. A single long process was often seen leaving the cell body and entering a nearby yellow- fluorescent nerve t runk of the plexus. The stomach and

large intestine showed no yellow-fluorescent fibres or cell bodies.

In some experiments the small intestine of otherwise untreated toads was extrinsically denervated. This opera- tion resulted in the loss of 90-95% of the adrenergic fibres.

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The denervated preparations showed yellow-fluorescent neurones and axons, similar in numbers and distribution to those seen after 6-OHDA treatment.

5-HT applied either in vitro or in vivo increased the intensity of the yellow fluorescence seen in both axons and cell bodies of the small intestine after 6-OHDA treatment. Exposure to exogenous 5-HT did not alter the observed distribution of yellow-fluorescent axons and cell bodies in the small intestine.

Exposure of the 6-OHDA-treated large intestine to exogenous 5-HT revealed axons that had not previously been visible in untreated tissue. Yellow-fluorescent axons were found in the longitudinal muscle, organised as large longitudinally running nerve trunks (Figs. 3, 4). In the myenteric plexus, longitudinally running yellow-fluorescent nerve trunks gave rise to smaller nerve trunks that entered the circular muscle (Figs. 5, 6). The small nerve trunks visible in the circular muscle did not penetrate into the submucosa. No yellow-fluorescent nerve trunks were visible around blood vessels in the large intestine. In contrast to the small intestine, no fluorescent cell bodies were seen in the the large intestine.

The stomach did not show any yellow-fluorescent axons or cell bodies even after exposure to exogenous 5-HT. No 5-HT containing mast cells were seen with fluorescence histochemistry in any part of the gut.

The effect of reserpine on the yellow fluorescence in the small intestine was examined. A single 20 mg/kg dose of reserpine given 2 days prior to processing was sufficient to eliminate any yellow-fluorescent cell bodies or axons in the small intestine. When reserpine treatment was followed by treatment in vitro with exogenous 5-HT, yellow fluorescence could be restored to the myenteric plexus of the small intestine. This treatment also resulted in the appearance of yellow fluorescence in axons in the large intestine. In both small and large intestine the distribution of the yellow- fluorescent axons and cell bodies was as described above.

5-HTP and tryptophan were also used in an attempt to restore fluorescence after reserpine treatment. Trypto- phan did not restore yellow fluorescence to the small intestine following reserpine treatment. This result held for both in vivo and in vitro exposure to tryptophan for periods of up to 3 h. Even after inhibition of monoamine oxidase with pargyline, tryptophan did not restore yellow fluorescence. In contrast, treatment of reserpinised tissue with 5-HTP in vitro resulted in restoration of yellow fluorescence. The distribution of yellow fluorescence resulting from exposure to 5-HTP was similar in all respects to the native yellow fluorescence. Comparable results were obtained with the reserpinised large intestine: incubation with tryptophan in vitro did not produce yellow-fluorescent nerves; incubation with 5-HTP in vitro produced yellow fluorescence with a distribution similar to that seen after 5-HT loading. In the reserpinised stomach, neither tryptophan nor 5-HTP treatment revealed yellow-fluorescent axons.

Treatment with the neurotoxin 5,7-DHT did not result in an obvious degeneration of the yellow-fluorescent axons. It was apparent, however, that the 5,7-DHT was being taken up into the yellow-fluorescent neurones as the colour of the fluorophore produced after 5,7-DHT was noticeably different from that in control tissue. The fluorophore in the treated tissue was a distinctive pale yellow colour. This effect was most obvious when the endogenous stores of

5-HT had been depleted with reserpine and the gut was subsequently exposed to 5,7-DHT for 1 h before processing for FIF. The pale yellow fluorophore was probably due to the fluorescence of 5,7-DHT itself.

Following 6-OHDA treatment, no catecholamine-containing axons were seen in the stomach, small intestine or large intestine. Exposure to L-dopa, dopamine or noradrenaline after 6-OHDA treatment did not produce any fluorescent axons in the gut. Treatment with L-dopa did cause some cells in the myenteric plexus, most probably neurones, to show a blue-green fluorescence. The number of fluorescent cells revealed was dependent on the dose of L-dopa given: in the small intestine, the number of cells revealed was far in excess of the number of yellow-fluorescent cell bodies.

In thin-layer chromatograms of mucosa-free small or large intestine from untreated animals, compounds that co- chromatographed with 5-HT and tryptophan standards could be detected. This was true for all three solvent systems used. The colour reaction with o-phthaldialdehyde of the 5-HT-like and the tryptophan-like compounds was also similar to 5-HT and tryptophan standards, respectively. No 5-HTP-like compound could be detected.

Discussion

Formaldehyde-induced fluorescence histochemistry showed that the intestine, but not the stomach, of Bufo rnarinus contains apparently 5-HT-containing (tryptaminergic) neurones. No 5-HT-containing mast cells were seen. It can then be concluded that the 5-HT that was found in chromato- graphs of extracts of small or large intestine with the mucosa removed, i.e. without enterochromaffin cells, repre- sents neuronal stores of 5-HT.

In the small intestine, tryptaminergic cell bodies were confined to the myenteric plexus. The localisation suggests an interneuronal function, although no 5-HT-containing nerve fibres were seen to form obvious pericellular baskets on other neurones. It is also possible that the fibres in the plexus innervate the nearer layers of the longitudinal or circular muscle. In the large intestine, no fluorescent neuronal perikarya were found but nerve fibres were seen in the myenteric plexus and in both longitudinal and circular muscle layers. Again, an interneuronal function is possible but the distribution suggests more clearly an innervation of the musculature. The origin of the fibres in the large intestine is obscure. They might arise from intrinsic perikarya that store so little 5-HT that they cannot be visualised. They might also arise from neurones extrinsic to the large intestine, lying either in the small intestine or in the posterior autonomic outflow.

The tryptaminergic neurones could be seen only after removal of catecholamine fluorescence by 6-OHDA treatment or by extrinsic denervation. The tryptaminergic neurones are not artefacts of the adrenergic denervation proce- dures, because they can also be seen in untreated tissue with 5-HT immunohistochemistry (C. Anderson, unpub- lished observations). While the neurones in the small intestine were visible with routine histochemistry, axons in the large intestine could be seen only after loading the tissue with 5-HT. Previous histochemical studies in this laboratory (Read and Burnstock 1968a, b; Gillard and Read 1971) on B. rnarinus intestine failed to find a tryptaminergic innervation, presumably because the large intestine only was

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studied without adrenergic denervat ion and exposure to exogenous 5-HT. Wong et al. (1971) repor ted that Bufo melanostictus has yellow-fluorescent nerve fibres, presumed to contain 5-HT, a round submucous b lood vessels and in the core of villi. No such fibres were found in B. marinus. It is p robable that Wong et al. (1971) misidentified adrenergic fibres, for their histochemical technique, f ixation in an aqueous neutral formalin solution, is unlikely to be better than the " F A G L U " technique (Furness et al. 1977) which does not show 5-HT-containing axons (Blessing et al. 1978). The experiments of Wong et al. (1971) would need to be repeated using animals pre t reated with 6 - O H D A to eliminate the possibil i ty of misidentifying adrenergic nerve fibres.

This paper adds further support to the idea that t ryptaminergic enteric neurones may occur throughout the verte- bra te series (Goodr ich et al. 1980). The presence of such neurones has been shown by formaldehyde- induced fluorescence in cyclostomes (Honma 1970; Baumgarten e t a l . 1973 ; Goodr ich et al. 1980; Sakharov and Sal imova 1980), a chondros tean (Sal imova and Feher 1982) and teleosts (Watson 1979; Anderson 1983). Immunohis tochemis t ry has shown only a very sparse innervat ion by neurones containing a 5-HT-like mater ial of the gut of an e lasmobranch (Holmgren and Nilsson 1984), while the same technique shows numerous 5-HT-containing enteric neurones in mammals (Costa et al. 1982). No study has yet shown endogenous 5-HT in avian or repti l ian enteric neurones. I t is unlikely that the 5-HT-containing enteric neurones are a func- t ionally homogeneous group. F o r example, the teleostean neurones provide an extensive plexus of fibres to the circular muscle and submucosa (Watson 1979; Anderson 1983). The mammal ian neurones are, like those in the small intestine of Bufo, confined to the enteric plexuses.

The histochemical results presented here suggest that the enteric t ryptaminergic neurones in B. marinus cannot synthesise 5-HT from the dietary precursor t ryptophan, and are not damaged by the neurotoxin 5,7-dihydroxytrypto- phan. In these respects the neurones differ from both the well-known central t ryptaminergic neurones of mammals (Boadle-Biber 1982; Baumgarten et al. 1982) and, accord- ing to certain reports (see Gershon 1982), mammal ian enteric neurones. However, a recent immunohis tochemical study of 5-HT-containing neurones in the guinea-pig intestine (Costa e t a l . 1982) produced results comparable to those found for Bufo. Whatever the functional similarities between the t ryptaminergic neurones in Bufo and guinea- pig intestines, the neurones of Bufo did not take up catecholamines or their precursors. They therefore cannot be regarded as " amine hand l ing" neurones as described by Furness and Costa (1978).

Acknowledgement. This work was supported by the Australian Re- search Grants Scheme.

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Accepted June 18, 1984

evidence for 5-hydroxytryptamine in neurones in the gut of the toad, bufo marinus

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