Cell Tissue Res (1989) 255:601 609 Cell and T't ute Resealv �9 Springer-Verlag 1989

Innervation of the gastrointestinal canal of the toad Bufo marinus by neurons containing 5-hydroxytryptamine-like immunoreactivity Colin Anderson* and Graeme Campbell Department of Zoology, University of Melbourne, Australia

Summary. The gut of the toad, Bufo marinus, was examined for evidence of enteric neurons containing 5-hydroxytrypta- mine-like immunoreactivity. Such neurons were absent from the stomach. They were present in the small intestine, with processes confined to the myenteric plexus. Immunore- active nerve cell bodies lay on branches of the pelvic nerves supplying the large intestine; fibres were found in the sub- mucosa of the posterior large intestine and in the muscularis externa of the anterior large intestine. It is concluded, on morphological grounds, that the neurons in the small intes- tine are interneurons, whereas those in the large intestine are postganglionic parasympathetic motoneurons.

Keywords: Gut Immunohistochemistry Neurons Sero- tonin - Bufo marinus (Anura)

We have previously described the distribution of 5-hydroxy- tryptamine (5-HT) in neurons in the gut of the toad Bufo marinus, as shown by formaldehyde-induced fluorescence histochemistry (Anderson and Campbell 1984). No neuro- nal 5-HT is seen in the stomach. 5-HT is found in both nerve cell bodies and fibres in the small intestine whereas only fibres are visualized in the anterior large intestine. However, it seems that the formaldehyde technique does not reveal the low levels of 5-HT that are readily demon- strable by immunohistochemistry. For example, a number of studies of mammalian enteric neurons, using the formal- dehyde technique (Baumgarten et al. 1970; Costa and Fur- ness 1971; Ahlman etal. 1973; Ahlman and Enerbfick 1974; Dubois and Jacobowitz 1974; Furness and Costa 1974, 1978; Diab et al. 1976), have failed to show the 5-HT content that has subsequently been demonstrated using im- munohistochemistry (Costa et al. 1982; Kurian et al. 1983; Griffith and Burnstock 1983; Dahlstrom and Ahlman 1983; Legay et al. 1984). Similarly, 5-HT-containing enteric neu- rons in some teleost species have been visualized by immu- nohistochemistry (Anderson and Campbell 1988) but not by formaldehyde histochemistry, even after loading with 5-HT (Anderson 1983).

Given that neurons containing low levels of 5-HT may have been missed in our earlier formaldehyde histochemical

* 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

study, we have examined the gastrointestinal tract of Bufo marinus again, using 5-HT immunohistochemistry. In par- ticular, we have set out to determine whether there are any 5-HT-containing neurons in the stomach and to locate the nerve cell bodies that give rise to the 5-HT-containing fibres found in the large intestine.

Materials and methods

Toads, Bufo marinus, captured in North Queensland, were held at 25~ in boxes containing expanded mica soaked in artificial pond water (composition in mM: NaC1, 67.6; KC1, 0.8; CaClz, 0.1; NaHCO3, 2.7; CuSO4, 0.007) under a light-dark cycle consistent with the site of capture (latitude 25~ light period varied between 13 h 40 min and 10 h 20 rain annually). The toads were held for up to 6 weeks and were not fed. Tissues from a total of 15 animals were examined. Tissue from a single animal was often divided and processed for different treatments. Each treatment was performed on tissues from at least three animals.

Toads were anaesthetized by immersion in 0.5% tricaine methanesulphonate (Rural Chemical Industries, Glenorie, N.S.W.) dissolved in tap water. Dissected tissues were incu- bated, where necessary, in Mackenzie's physiological solu- tion (composition in mM: NaC1, 116.2; KC1, 3.2; CaC12, 1.3; MgSO4, 1.4; NaHCO3, 20.0; NaH2PO4, 3.1; glucose, 16.7) bubbled with 5% CO2:95% O z and maintained at 25 ~ C. No attempt was made to study dietary, seasonal or circadian variation, but tissues were taken from freshly collected or stored animals, throughout the year and in the morning or the early afternoon, with no obvious differ- ences in histochemical results.

Pretreatments

To avoid the possibility that 5-HT might be localized to adrenergic nerves, especially after loading with 5-HT (see below), all tissues were treated in vivo or in vitro with 6- hydroxydopamine hydrochloride (6-OHDA, Sigma, USA). In 4 animals, 6-OHDA (50 mg kg- 1), dissolved in antioxi- dant solution (composition in mM: NaC1, 111.2; KC1, 2.0; ascorbic acid, 11.4), was injected into the iliac lymph sac and tissues were used four days later. Alternatively, dis- sected tissues were treated in vitro for 2 h with 6-OHDA dissolved in a small volume of antioxidant solution and added to Mackenzie's solution to give a final concentration of 500 ~tM (see Costa et al. 1982). Any other drugs used

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to pretreat the tissue were then added to the 6-OHDA solu- tion for a further 1 h.

In an effort to increase immunoreactivity, some tissues were incubated for 1 h in Mackenzie's solution containing either 25 p.M 5-HT creatinine sulphate (Sigma, USA) or 250 p.M 5,7-dihydroxytryptamine creatinine sulphate (5,7-DHT, Sigma, USA) as used by Howe et al. (1982). Some tissue from 6-OHDA treated animals was maintained in drug-free Mackenzie's solution at 25 ~ C for 6-8 h in an attempt to achieve an increase in the endogenous levels of 5-HT.

5-HT immunohistochemistry

Tissues for immunofluorescence were treated as described by Costa et al. (1982), as follows: sections of gut 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~ The fixed tissue was given 9 x 10 min washes in 80% ethyl alcohol and stored in phos- phate-buffered saline (PBS, composition in mM: NaC1, 145.4; Na2HPO4, 1.07; NaHzPO4, 2.5). Prior to incubation with antibodies, the pieces were sometimes split into two layers, consisting of the muscularis externa and the submu- cosa respectively. The mucosa was usually scraped offprior to splitting the tissue. Histological sections were used for immunohistochemical studies of the stomach. Tissue from the stomach was fixed and washed as above but, after the final wash in 80% alcohol, it was left overnight at 4~ in 30% sucrose in PBS. The tissue was then frozen in liquid nitrogen onto the chuck of a cryostat and ,sectioned at 20 ~tm.

For whole mounts, rat monoclonal anti-5-HT (Sera- Lab, England) diluted 1:200 in PBS was applied; tissues were incubated in a moist chamber for 18-24 h at room temperature. Following 3 rinses in PBS the tissue was incu- bated in fluorescein isothiocyanate (FITC)-labelled sheep anti-rat IgG (Wellcome Laboratories, Beckenham, En- gland) for 1 h at room temperature, rinsed in three changes of PBS and mounted in 50% glycerol:50% bicarbonate buffer, pH 8.6. Sections were mounted on subbed slides and incubated overnight in 1:500 anti-5-HT. Following a 15 min wash in PBS, the sections were incubated for 1 h in 1:40 FITC-labelled sheep anti-rat IgG, washed in PBS and mounted in 50% glycerol as above.

Binding of the primary antibody in the large intestine was also visualised in whole mounts using horseradish per- oxidase (HRP) and the avidin-biotin link method with dia- minobenzidine (DAB) as the chromogen (Vectastain ABC kit, Vecta Laboratories, USA). Following primary antibody incubation at a concentration of 1:1000, whole mounts were washed in TRIS-buffered saline (TBS, 0.1 M, pH 7.6) and incubated for I h in anti-rat biotinylated secondary antibody at 1 : 100 in TBS. Following a TBS wash, the tissue was further incubated for 1 h in avidin-biotinylated HRP complex diluted 1:200. After a wash in TBS, the tissue was presoaked for 10 min in a 0.05% DAB solution in TBS containing 0.01 M imidazole, following which HzOz was added to a final concentration of 0.01%. The whole mounts were then rinsed and air dried onto slides, cleared in graded alcohols and xylene and mounted in DPX.

Preparations labelled with FITC were examined on a Leitz Dialux 20 fluorescence microscope fitted with a Ploe-

mopak 2.3 incident light system and filter block I2. Whole mounts that had been reacted with DAB were examined in bright-field on a Leitz Dialux 20. Photomicrographs were taken with a Leitz Orthomat camera using Kodak Tri-X or Ilford FP4 film, both rated at ISO 400.

The specificity of antibody staining was tested by stain- ing with primary antiserum preabsorbed with 0.5 M 5-HT for 6 h at room temperature. A high concentration of 5-HT is necessary for successful preabsorption (see Costa et al. 1982), presumably because the antibody is raised not against 5-HT but against 5-HT conjugated with bovine se- rum albumen (see Milstein et al. 1983).

Results

Incubation of tissue with 5-HT or 5,7-DHT increased the 5-HT-like immunoreactivity (5-HTLI) in gut neurons but did not cause an obvious increase in the numbers, or change in type, of 5-HTLI elements observed. Prolonged incuba- tion in drug-free solution also caused an obvious increase of 5-HTLI in neurons.

Stomach

Cryostat sections of the stomach showed no endogenous neuronal 5-HTLI, although enterochromaffin cells were abundant in the mucosal epithelium. A few 5-HTLI fibres were seen in tissue pretreated with 5-HT or 5,7-DHT. The 5-HTLI fibres were smooth in outline and ran singly; they were confned to the myenteric plexus. No 5-HTLI nerve cell bodies were seen.

Small intestine

In the small intestine, all neuronal 5-HTLI was confined to the myenteric plexus, where both 5-HTLI nerve cell bod- ies and axons were visible. No 5-HTLI nerve cell bodies or processes were seen in the circular muscle layer or in the submucosa. The nerve cell bodies were only faintly im- munoreactive. Pretreatment with 5-HT or 5,7-DHT in- creased the 5-HTLI markedly. However, it was apparent that 5-HTLI nerve cell bodies varied in intensity of immu- noreactivity, even in tissue exposed to indoleamines. No change in neuronal density was detected along the length of the small intestine. The nerve cell bodies were evenly distributed around the circumference of the intestine.

All 5-HTLI neurons displayed a similar morphology (Fig. 1 C-D). Typically, the nucleus of each neuron was ec- centrically located. The perinuclear region of the nerve cell body was, on average, 48 lam long by 30 lam wide. Between five and ten tapering processes arose from the soma; these were arranged on both sides of the cell body. The processes were always aligned with the circular smooth muscle cells and averaged 40 ~m in length, the longest process seen be- ing 61 ~tm. All processes arising from a cell body appeared to terminate within a short distance of the soma. No axon- like process could be distinguished.

The myenteric plexus of the small intestine contained a network of 5-HTLI fibres that were mainly arranged in longitudinally running bundles (Fig. 1 A). Fibres running in the bundles were not obviously varicose and their course within the bundle was often contorted. Between the longitu- dinally running nerve trunks was a loose meshwork of sin- gle, varicose 5-HTLI fibres (Fig. 1 B). The varicosities were generally small, uneven in size and spacing, and often

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Fig. 1. Whole mounts showing FlTC-labelled 5-HT-immunoreac- tivity (5-HTLI) in B. marinus small intestine, pretreated in vitro with 6-OHDA and, except in A, with 5-HT. Scale bar is 100 gm for A and 50 gm for BE. A 5-HTLI in B. marinus small intestine not pretreated with exogenous indoleamine, showing the general longitudinal orientation of the bundles of fibres in the myenteric plexus. The arrow indicates the alignment of the longitudinal mus- cle. x 115. B As above, but pretreated with exogenous indoleamine and at higher magnification. Single, varicose axons and small bun- dles of axons are visible between the large, longitudinal bundles of 5-HTLI fibres. The arrow indicates the orientation of the longi- tudinal muscle cells, x 230. C-E Nerve cell bodies in the myenteric plexus of the small intestine. In each micrograph the long arrow indicates the orientation of the smooth muscle cells of the longitudi- nal muscle. Note that, in each case, the nerve cell bodies are parallel to the orientation of the circular muscle cells. C A pair of 5-HTLI nerve cell bodies (arrowheads). x 230. D A single, weakly immuno- reactive 5-HTLI nerve cell body (arrowhead) surrounded by strongly immunoreactive varicosities, x 230. E A cluster of 5-HTLI varicosities outlining a possible non-immunoreactive nerve cell body. x 230

placed eccentrically on the axon. The long axis of the vari- cosity was frequently at right angles to the axon, giving the fibre the appearance of being covered in short, uneven side branches.

The 5-HTLI nerve cell bodies were usually located on the longitudinally running 5-HTLI nerve bundles, and lay between the nerve t runk and the circular muscle layer. Each nerve cell body was intimately associated with a variable number of 5-HTLI varicosities (Fig. 1 D). Axons reached the nerve cell body from the overlying nerve trunks and enmeshed both the cell body and its processes in a sparse cluster of varicosities. The varicosities a round 5-HTLI cell bodies were more intensely immunoreactive and often larger than other varicosities in the myenteric plexus. Simi- lar pericellular arrangements of axons were seen around neurons that apparently lacked 5-HTLI, even in tissue pre- treated with 5-HT or 5,7-DHT (Fig. 1 E).

In a few instances, small 5-HTLI nerve bundles were seen to leave the myenteric plexus and enter the longitudinal muscle layer where they ran for a short distance before re-entering the myenteric plexus. The small nerve bundles seldom contained varicose axons.

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605

Large intestine

Some features of the ana tomy of the large intestine in Bufo are unusual. The large intestine is sac-like and about 4 cm long in an animal weighing 100 g. I t comprises distinct ante- rior and poster ior regions that Boyd e ta l . (1964) have equated to the mammal ian colon and rectum, respectively. The large intestine ends and the cloaca begins at the level of the openings of the ureters through the dorsal wall and of the b ladder through the ventral wall.

The anter ior large intestine is often dilated with fluid and is 1 2 cm in diameter. In this region, both longitudinal and circular muscle layers are thick. At regular intervals along the length, circumferential arteries and veins connect to a longitudinal artery and vein on the dorsal surface of the intestine.

The poster ior large intestine is a much narrower struc- ture that lacks circumferential vessels. At the extreme ante- rior end, the two muscle layers are int imately connected. More posteriorly, the muscle layers separate; they are even- tually are l inked only by thin strands of connective tissue and b lood vessels crossing a lymphat ic space. The longitudi- nal muscle layer comprises distinct bundles of smooth mus- cle that are anchored poster iorly to the pelvic girdle. The circular muscle is poor ly developed anteriorly and becomes even thinner posteriorly, disappearing just before the cloaca. The circular muscle, which is usually constricted, is firmly at tached to a well developed submucosa and mu- cosa. Together, these layers form a tube, which often winds within the loosely a t tached longitudinal layer. This par t of the intestine receives the pelvic nerves that arise mainly from the ninth and tenth spinal nerves (Boyd et al. 1964). On reaching the gut wall, near the ureteral openings, the nerves send branches that follow the longitudinal muscle bundles anteriorly towards the anter ior large intestine; they also send branches poster iorly towards the cloaca.

The cloaca lies within the pelvic girdle. It is lined with melanocytes and is surrounded by a b road band of striated muscle that forms a sphincter.

Anterior large intestine. The dis tr ibut ion of 5-HTLI in the anterior large intestine was very different from that in the

Fig. 2. Whole mounts showing 5-HTLI in B. marinus large intes- tine, pretreated in vitro with 6-OHDA and 5-HT. A-B FITC-la- belled 5-HTLI in the anterior large intestine. A Axons forming large bundles in the longitudinal muscle (arrowheads) and myen- teric plexus. Scale bar= 100 ~tm; x 120. B Varicose axons and bun- dles of axons running in the circular muscle layer. Scale bar= 50 pm; x 270. C-F DAB-labelled 5-HTLI in posterior large intes- tine. C A group of immunoreactive nerve cell bodies on a branch of the pelvic nerves in the wall of the large intestine. The nerve trunk contains many 5-HTLI nerve fibres. Scale bar=501am; x 120. D 5-HTLI nerve cell bodies on a branch of the pelvic nerve

in the posterior large intestine. Each neuron has a single, smooth process, which in one case (arrow) appears to branch immediately after arising from the nerve cell body. Scale bar= 10 ~tm; x 800. E Whole mount of the longitudinal muscle layer only, showing many large bundles of 5-HTLI fibres aligned with the bundles of longitudinal muscle cells. The stars indicate ligaments that linked the longitudinal muscle layer to the circular muscle layer. Scale bar = 200 ~tm; x 50. F Single, varicose axons accompany the large bundles of 5-HTLI axons running in the bundles of longitudinal smooth muscle. Scale bar= 50 ~tm; x 200

small intestine. 5-HTLI fibres were found in the longitudi- nal and circular muscle layers, and in the region of the myenteric plexus. Throughout the thickness of the longitu- dinal muscle layer, there were large bundles of non-varicose 5-HTLI fibres arranged longitudinal ly (Fig. 2A). Bundles of fibres divided and rejoined to give a braided appearance to the plexus. Occasional small nerve bundles and individ- ual varicose axons were found between the large nerve trunks. Nerve trunks lying at the boundary between the longitudinal and circular muscle layers formed par t of the myenteric plexus and gave rise to small nerve t runks that entered the circular muscle layer and penetrated as far as the submucosa. Nerve trunks in the myenteric plexus ap- peared to be continuous with immunoreact ive nerve t runks in the myenteric plexus of the small intestine. There were very large numbers of single varicose axons in the circular muscle layer; these axons ran parallel to the circular smooth muscle cells (Fig. 2B). Their varicosities were narrow, smooth swellings evenly spaced along the axon, and were much more intensely immunoreact ive than the intervaricose regions. No 5-HTLI nerve cell bodies were seen and no perineuronal plexuses of 5-HTLI fibres were present.

Posterior large intestine. 5-HTLI fibres were present in pos- terior large intestine. It was also possible, in contrast to the anter ior large intestine, to locate 5-HTLI nerve cell bod- ies. The nerve cell bodies were mainly restricted to the pelvic nerves at the point near the ureters where the nerves entered the longitudinal muscle layer. The cell bodies, est imated to be several hundred, were closely applied to the surfaces of the nerve trunks and formed large ganglia of up to a hundred nerve cell bodies (Fig. 2 C). Nerve cell bodies were also found associated with longitudinal ly directed branches of the pelvic nerves within the longitudinal muscle layer, up to 1 cm anteriorly from the junct ion of the pelvic nerves with the large intestine. However, the number of nerve cell bodies decreased with distance from the main ganglia. A few nerve cell bodies were also found lying on the pelvic nerves just outside the large intestine. The immunoreact ivi ty of all 5-HTLI nerve cell bodies could be increased by expo- sure to exogenous 5-HT or 5 ,7-DHT for 1 h or by incubat- ing the large intestine in toad physiological saline without drug treatment for 6-12 h.

The 5-HT nerve cell bodies were always closely applied to the surface of a nerve trunk. Typically, the somata were round or fusiform with an eccentrically located nucleus (Fig. 2D). In many cases, it was impossible to discern any processes arising from the cell body amongst the mass of immunoreact ive fibres making up the nerve trunk. How- ever, in a number of cases, a single process arose from the soma and entered the underlying nerve bundle. In one instance, the process was seen to divide and extend two branches in opposi te directions along the nerve trunk (Fig. 2D). It was also apparen t that the end of the nerve cell body opposi te the nucleus was often associated with non-immunoreact ive structures (perhaps satellite cell pro- cesses or large axon varicosities) that appeared to indent the smooth outline of the neuron.

The immunoreact ive material was evenly distr ibuted throughout the nerve cell body but there were often differ- ences in intensity between neurons. Most nerve cell bodies were intensely stained after prolonged incubat ion in physio- logical solution or exposure to 5-HT or 5,7-DHT, al though even these treatments left some neurons only weakly immu-

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Fig. 3. Whole mounts of B. marinus posterior large intestine, pre- treated in vitro with 6-OHDA and 5-HT, showing DAB-labelled 5-HT-immunoreactivity. A Two 5-HTLI fibres (arrows) running

in the pelvic nerves immediately outside the large intestine. Scale bar = 50 ~tm; x 300. B Bundles of 5-HTLI fibres forming a submu- cosal plexus running along the submucosal side of the circular

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LM

CM

Post.

Ant.

SM

\ ) 'i

Fig. 4. Diagram summarising the innervation of the large intestine of B. marinus. In the anterior region (A), the longitudinal muscle layer (LM) contains longitudinally oriented nerve trunks contain- ing 5-HTLI fibres. The myenteric plexus containing 5-HTLI fibres but no 5-HTLI nerve cell bodies lies on the serosal surface of the circular muscle layer (CM). Numerous varicose 5-HTLI fibres can be seen within the circular muscle layer, which is thick in this region. There are no 5-HTLI fibres in the submucosa (SM) at this level, whereas the mucosa (M) contains many 5-HTLI enter- ochromaffin cells. In the posterior region (P), the pelvic nerves (PN) enter the longitudinal muscle. 5-HTLI nerve cell bodies lie on the pelvic nerves and on the associated nerve plexus in the longitudinal muscle. The longitudinal muscle is widely separated from the circular muscle layer, but is connected to it by thin liga- ments of connective tissue containing 5-HTLI nerve bundles. The circular muscle layer is much reduced in thickness and contains very few 5-HTLI fibres. The submucosa contains a plexus of 5-HTLI fibres; numerous non-neural 5-HTLl cells lie immediately under the mucosa. The mucosa does not contain 5-HTLI enteroch- romaffin cells

muscle layer in the most anterior part of the posterior large intes- tine. Note the innervation of the circular muscle layer by single varicose 5-HTLI axons at this level. Scale bar=50 p.m; x 300. C A connective tissue ligament joining the longitudinal muscle (top) to the circular muscle/submucosa (dissected away). Bundles of 5-HTLI fibres run down the ligaments into the submucosa. Scale bar=50 ~tm; x 230. D A single fibre from the submucosa of the posterior large intestine showing the thick calibre and smooth, non-varicose appearance typical of fibres in this plexus. Scale bar - 20 p.m ; x 650. E Whole mount of circular muscle and submucosa with the mucosa removed. Thick, non-varicose 5-HTLI fibres form an open plexus in the submucosa. Scale bar=50 ~tm; x 320. F A preparation of the submucosa, with the mucosa left intact, from the most distal part of the posterior large intestine. No enterochro- rnaffin cells are present, but strongly immunoreactive amoeboid cells (presumably mast cells) lie in the submucosa immediately be- neath the mucosa. Scale bar - 50 ~tm ; x 330. G A group of 5-HTLI mast cells lying under the mucosa in the mid part of the posterior large intestine. Scale ba r -20 ~tm; x 550. H The same field as in G, but focused on the mucosa to show 5-HTLI enterochromaffin cells. Scale bar=20 lam; x 550

noreactive. It was difficult to determine whether the weakest staining was specific or simply represented non-specific staining of non-immunoreact ive neurons. In tissue that had not been treated to increase immunoreactivity, only a few nerve cell bodies were strongly immunoreactive and the ma- jority of the cells were only weakly positive.

Processes from 5-HTLI nerve cell bodies located in the posterior large intestine formed large nerve trunks and sin- gle varicose fibres that ran anteriorly on bundles of longitu- dinal muscle (Fig. 2E-F) . The longitudinal nerve trunks were continuous with the similar nerve trunks in the longi- tudinal muscle layer and myenteric plexus of the anterior large intestine. A second layer of 5-HTLI fibres was found forming a relatively sparse network of longitudinal fibres on the submucosal surface of the circular muscle layer (Fig. 3B). Individual varicose 5-HTLI axons were not found in the circular muscle layer of the extreme posterior large intestine, in contrast to the anterior large intestine. 5-HTLI fibres first appeared in the circular muscle near the point where the circular and longitudinal muscle layers separated. A few 5-HTLI fibres were also found in some of the branches of the pelvic nerves outside the gut (Fig. 3A).

A loose plexus of immunoreactive fibres was present in the submucosa (Fig. 3E-F) . This plexus was present throughout the posterior, but not the anterior, large intes- tine. The fibres making up this plexus were usually smooth and thick (Fig. 3 D). They did not form a close association with the epithelial cells of the mucosa nor did they envelop blood vessels. Some of the submucosal fibres appeared to arrive in the submucosa via small nerves running down the connective tissue ligaments connecting the two muscle layers (Fig. 3C). Alternatively, fibres may have arrived from the anterior in pelvic nerve branches running toward the cloaca on the surface of the circular muscle. The distribution of 5-HTLI nerve cell bodies and processes in the large intestine is summarised diagramatically in Fig. 4.

Preabsorption of the ant ibody with 5-HT prevented the appearance of any immunoreactive structures.

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Non-neuronal 5-HT-containing cells

Enterochromaffin cells containing 5-HTLI (Fig. 4G) were found in the mucosal epithelium throughout the alimentary tract except for the extreme posterior large intestine (Fig. 4E). The enterochromaffin cells were elongated with a centrally located nucleus. The ends of the cell were drawn out into processes that extended towards the lumenal and submucosal edges of the epithelium. In the posterior large intestine, a second type of 5-HTLI cell was present lying on the mucosal side of the submucosa (Fig. 4F, H). These were flattened ceils, oval or amoeboid in outline. The 5-HTLI in the cells was in distinct large granules packing the cytoplasm.

Discussion

Table 1 summarizes the distribution of 5-HTLI neuron so- mata and fibres in Bufo marinus alimentary canal. 5-HT immunohistochemistry showed all of the neural structures in the small and the anterior large intestines that had been described previously using formaldehyde-induced fluores- cence histochemistry (Anderson and Campbell 1984). The reported absence of 5-HT-containing neurons from the stomach has also been confirmed here; the few fibres that are seen in the stomach after loading with 5-HT or 5,7-DHT may be adrenergic axons. The observations were worth re- peating because it is now clear that stores of neuronal 5-HT are not always revealed by formaldehyde-induced fluores- cence: thus 5-HT is not found using this technique in neu- rons in the stomach of the teleost Salmo gairdneri (Ander- son 1983) although it has subsequently been demonstrated to occur there by immunohistochemistry (Holmgren et al. 1985).

In the previous study, monoamine histofluorescence did not reveal the axons in the anterior large intestine unless the tissue had been pretreated with 5-HT or 5-hydroxytryp- tophan (Anderson and Campbell 1984). Immunohisto- chemistry visualized the fibres without any prior treatment. It appears that the neurons in the large intestine do contain native 5-HT but at lower levels than neurons in the small intestine.

Morphology and possible functions of the 5-HT neurons

The distribution of nerve cell bodies and their processes provides evidence for the probable physiological roles of

Table 1.5-HT innervation of the gastrointestinal tract

Stomach Small somata/ intestine Fibres somata/

Fibres

Large intestine

Anterior Posterior somata/ somata/ Fibres Fibres

Longitudinal - / - - / - - / + + / + muscle

Myenteric - / + + / + - / + - / + plexus

Circular - / - - / - - / + - / - muscle

Submucosa - / - - / - - / - - / +

5-HT-containing neurons in the various parts of the alimen- tary canal. Overall, the 5-HT-containing neurons of the small and large intestine have so little in common, apart from the fact that they contain 5-HT, that it is difficult to believe that the two populations represent a single neuro- nal class.

Small intestine. The present observations support the pre- vious suggestion (Anderson and Campbell 1984) that the 5-HT-containing neurons function as interneurons, 5-HT terminals being detected around enteric neurons. It is also possible that the axons innervate the smooth muscle layers. The few small bundles of 5-HTLI axons seen running in the longitudinal muscle layer may innervate this muscle layer. In addition, the numerous 5-HTLI axons running throughout the myenteric plexus might innervate the longi- tudinal muscle layer, as do tertiary strands of the myenteric plexus in the guinea-pig ileum (Gabella 1972; Llewellyn- Smith et al. 1985). There is, however, no direct evidence for an innervation of the mammalian circular muscle layer by axons lying in the myenteric plexus.

5-HTLI axons in the small intestine probably arise, in the main part, from the intrinsic 5-HTLI nerve cell bodies in the myenteric plexus. Some of the fibres in the small intestine may also arise from the 5-HTLI cell bodies found in the posterior large intestine. Bundles of immunoreactive axons are seen to cross the border between the small and large intestines. It is unlikely that these fibres pass from the small to the large intestine; axons in the small intestine are normally visible with formaldehyde-induced fluores- cence without prior exposure to indoles but no such axons are found in the large intestine (Anderson and Campbell 1984).

Large intestine. It is probable that all of the 5-HTLI nerve fibres in the large intestine and cloaca arise from the 5-HT- containing neurons lying in ganglia on branches of the pel- vic nerves, where the nerves reach the posterior large intes- tine. The nerve cell bodies in the large intestine are strik- ingly different from those seen in the small intestine. Where- as the cell bodies in the small intestine have a multipolar shape typical of amphibian myenteric neurons (Gunn 1951 ; Wong et al. 1971), those in the posterior large intestine are unipolar and are reminiscent of neurons in amphibian para- sympathetic and sympathetic ganglia (Taxi 1976). Since the 5-HT-containing nerve cell bodies in the large intestine oc- cur along pelvic nerve branches, they are presumably para- sympathetic neurons; even though they lie in or near the gut wall, they have retained a morphology more typically found in discrete autonomic ganglia.

The parasympathetic 5-HTLI neurons in the large intes- tine also differ markedly from the enteric 5-HTLI neurons in the small intestine in the distribution and possible func- tion of their axons. In the anterior large intestine, 5-HT- containing axons run in large bundles in the myenteric plex- us and longitudinal muscle layer and in a very dense array of varicose axons in the circular muscle layer, whereas some varicose axons are also seen in the longitudinal muscle. In the posterior large intestine, the 5-HT-containing inner- vation of the muscle layers is sparse and the bulk of the axons lie in the submucosa, not obviously associated with a particular target tissue. No pericellular arrays of axons innervating nerve cell bodies can be found. Hence, there is no evidence that the 5-HT-containing axons in the large

intestine function as interneurons. Most probably, they are motor to the circular muscle layer and, to a lesser extent, the longitudinal muscle layer in the anterior large intestine. Their function in the posterior large intestine is obscure.

Stimulation of the parasympathetic pelvic nerves in B. marinus causes contraction of both muscle layers in the large intestine: it has been proposed that the postganglionic fibres involved are cholinergic because the response is abol- ished by atropine (Boyd et al. 1964). It is possible that the nerve cell bodies lying on the pelvic nerve branches in the large intestine are these same cholinergic postganglionic neurons. If so, some or all of them may contain both 5-HT and acetylcholine. Applied 5-HT causes contraction of the large intestine (Boyd et al. 1964); it thus would be worth re-examining this nervous transmission in case it should prove to be a co-transmission involving acetylcholine and 5-HT.

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Accepted October 17, 1988