Camp. Biochem. Physiol. Vol. IOIC, No. 3, pp. 561-569, 1992 Printed in Great Britain

03064492j92 $5.00 + 0.00 0 1992 Pergamon Press Ltd

THE AUTONOMIC INNERVATION OF THE LARGE INTESTINE OF THE TOAD (BUFO MARfNUS)

SUSAN MURPHY* and G. CAMPBELL

Department of Zoology, University of Melbourne, Parkville, Victoria 3052, Australia (Tel.: 03 344-6244; Fax: 03 344-7909)

(Received 9 July 199 1)

Abstract-l. A study was made of the pelvic and the splanchnic nerve supplies to the toad large intestine. 2. Stimulation of pelvic nerve fibres in the 9th and 10th spinal nerves caused a series of contractions

of the circular muscle, only the first of which was abolished by hyoscine. The entire response was blocked by d-tubocurarine. The response was not affected by capsaicin treatment.

3. Stimulation of the splanchnic nerves caused a rapid contraption followed by a prolonged relaxation. The relaxation was abolished by bretylium. The contraction was selectively antagonised by prolonged exposure to capsaicin. Splanchnic nerve stimulation also caused a slow, prolonged excitation that was abolished by bretylium.

4. Application of adrenaline caused relaxation of circularly cut strips of large intestinal wall, whereas substance P, acetylcholine, %hydroxytryptamine, somatostatin and galanin caused contraction.

5. The results suggest that stimulation of the pelvic nerves releases acetylcholine and a non-cholinergic co-transmitter from peripheral postganglionic neurons. Both the inhibitory response to splanchnic nerve stimulation and the subsequent slow excitation appear to be mediated by adrenergic nerves. The rapid capsaicin-sensitive excitation is likely to be due to release of substance P from antidromically activated afferent nerve fibres in the splanchnic outflow.

In the vertebrate series, the amphibians first show an autonomic outflow from the posterior spinal cord that can be distinguished anatomically as parasympa- thetic (Langley and Orbeli, 1910; see Nilsson, 1983). Part of that outflow, through the 9th and 10th spinal nerves of anuran amphibians, provides an excitatory innervation to the large intestine (Langley and Orbeli, 1910; Boyd et al., 1964). In mammals, the postgangli- onic neurons in the sacral parasympathetic pathway to the gut lie in the myenteric plexus (Burnstock, 1969). In one anuran amphibian, the toad Bufo marinus, some at least of the postganglionic neurons have been identified lying on pelvic nerve branches outside and just within the gut wall (Anderson and Campbell, 1989): they have the rounded unipolar shape typical of amphibian postganglionic neurons (Taxi, 1976) and are unlike the generally multipolar myenteric neurons (Anderson and Campbell, 1989).

The most recent physiological study of the parasympathetic outflow to the amphibian large in- testine was that of Boyd er al. (1964). They found that brief periods of pelvic nerve stimulation caused intes- tinal contractions that were abolished by muscarinic antagonists, suggesting that the innervation is cholin- ergic. But at that time it was thought that all auto- nomic postganglionic neurons acted by releasing either acetylcholine or (in amphibians) adrenaline. There has since been a growing realisation that autonomic neurons can act by releasing one or more of a large range of transmitter substances, including a considerable number of neuropeptides (Furness

- *To whom correspondence should be addressed.

et al., 1987; Gibbins, 1989). In relation to the toad large intestine, it has been found that many of the postganglionic parasympathetic neurons contain im- munoreactivity for the peptides somatostatin and galanin, most commonly localized in the same neur- ons (Osborne et al., 1988), and for the amine 5-hy- droxytryptamine (5-HT: Anderson and Campbell, 1989). These and the earlier physiolo~cal obser- vations suggest that parasympathetic regulation of the large intestine might involve co-transmission by acetylcholine, 5-HT, somatostatin and galanin. For this reason, the effects of pelvic nerve stimulation on the toad large intestine were reinvestigated.

Boyd et al. (1964) also found that stimulation of the sympathetic supply to the toad large intestine caused an inhibition, apparently mediated by adren- ergic nerve fibres, and occasionally, an initial cholin- ergic contraction. This result also seemed worth reinvestigation, for two reasons. First, many adrener- gic postganglionic neurons in Bufo (Morris et al., 1986), as in mammals (see Potter, 1988), have been found to contain neuro~ptide Y; the action of neuropeptide Y on amphibian intestinal muscle is not known. Second, studies in mammals have shown that the sympathetic pathways to the gut contain a popu- lation of axons of primary aflerent neurons that contain co-localized substance P and calcitonin gene- related peptide (CGRP: Molander et al., 1987; Ekblad et al., 1988), that are sensitive to the toxic actions of capsaicin, and that, when antidromically activated, have motor effects on the intestine (see Maggi and Meli, 1988). Similar fibres innervate the amphibian small intestine (Osborne and Campbell, 1986), but their presence in the large intestine has not been sought.

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562 SUSAN MURPHY and G. CAMPBELL

MATERIALS AND METHODS

Toads (Bufo mar&s) of either sex and weighing 80-l 50 g were captured in central Queensland. They were held at 25°C on expanded mica soaked with pond water, for up to two months without feeding.

Animals were anaesthetised in 0.5% tricaine methane- sulfonate (MS 222: Rural Chemical Industries. N.S.W.. Australia) ‘and opened ventrally. In some experiments; the splanchnic nerves that run from the third to the fifth, and occasionally the sixth, sympathetic chain ganglia to the prevertebral coeiiaco-mesenteric ganglion were tied off and detached from the right and left sympathetic chains. The large intestine, with the splanchnic nerves, the prevertebral ganglion and the posterior branch of the anterior mesenteric artery, was removed from the animal. In experiments where the pelvic nerve supply was investigated, the ninth and tenth spinal nerves were cut bilaterally at their point of emergence from the vertebral column and tied together. The spinal nerves were cut distal to the origins of &he pelvic nerves, the fascia through which the pelvic nerves reach the large intestine was freed from the body wall and the intestine was removed from the body cavity. In four of these experiments, the vertebral column was opened and the ventral roots of the ninth and tenth nerves were dissected free from the spinal cord and tied together. In these preparations the large intestine was removed along with a portion of dorsal body wall and posterior vertebral column, as it was difficult to free the tenth nerve roots from the urostyle without damage.

Some animals were subjected to splanchnic nerve section 6 weeks before use. Under MS-222 anaesthesia, a 1 cm longitudinal incision was made in the lateral body wall, immediately lateral to the tips of the lateral vertebral processes, commencing just posterior to the parotoid gland. Each splanchnic nerve in turn was iocated in the retroperi- toneal space and a segment of nerve was removed to prevent reconnection of the cut stumps. The area was dusted with antibiotic powder (Cicatrin; Wellcome) before reclosure.

Innervated preparations were pinned out on Sylgard in a flat water-jacketed, 50 ml organ bath filled with Mackenzie’s solution (composition in mM: NaCI, 11.5; KCI, 3.2; NaHCO,, 20; NaH,PO,, 3.1: MgSO,, 1.4; CaCi,, 1.3: D-ghICOSe, 16.7) at 25”C, bubbled with 95% 0,: 5% CO,. A cotton thread was passed through the double thickness of gut wall about 1 cm away from the pins and tied to a Grass FT03C force-displacement transducer. Activity of the an- terior (colonic) and posterior (rectal) muscle was recorded in the longitudinal or circular direction and was displayed on a Grass Model 79 polygraph.

Nerves were passed through bipolar platinum ring elec- trodes shielded in plastic. In splanchnic denervated and control animals, the anterior mesenteric artery was passed through a ring electrode for electrical stimulation. A Grass S9 stimulator was used to deliver I-msec pulses at 15 V, a voltage that gave submaximal responses that were reproducible over many hours.

In some experiments, the large intestine was removed from the animal and opened along the mesenteric edge. Strips of intestinal wall, about 3 x 10 mm, were cut in the circular direction. The strip was tied at one end to a tissue holder, immersed in a 3 ml plastic organ bath containing Mackenzie’s solution, and tied to a force transducer as described above.

Innervated preparations were mounted at an initial ten- sion of I g and strips were mounted at OS g. All prep- arations were given a single wash and were allowed to equilibrate for at least 30 min prior to nerve stimulation or application of agonists. Electrical stimulation and drug applications were made on a 15-min cycle. Agonists were applied to strips for I min and were then washed off.

Drugs used were: acetylcholine chloride (ACh), adrenaline bitartrate, capsaicin (8methyl-N-vanillyl-6- nonenamide), 5-hydroxytryptamine creatinine sulphate

complex, hyoscine hydrobromide, neostigmine bromide, synthetic porcine neuropeptide Y (NPY), propranolol hydrochloride, synthetic porcine somatostatin, tetrodo- toxin, ~-tubocurarine chloride (all from Sigma, St. Louis, MO); bretyli~ tosylate (Serva, Heidelberg, Germany), synthetic porcine galanin, synthetic human/porcine~rat vasoactive intestinal peptide (VIP; Peninsula Labs, Belmont, CA); methysergide hydrogen maleinate (Sandoz, Base], Switzerland); phentolamine mesylate (Regitine; Ciba- Geigy); synthetic human calcitonin gene-related peptide (CGRP; Auspep, South Melbourne, Victoria, Australia); synthetic rat CGRP and substance P (Bachem, Torrance, CA). Capsaicin was dissolved in dimethyl sulphoxide. All other drugs were made up in distilled water.

RESULTS

After a period of equilibration, most preparations of the intact large intestine showed strong spon- taneous activity on a constant basal tension. Gener- ally, the spontaneous contractions were of greater amplitude in the rectum, and this masked weak contractile responses to nerve stimulation in some preparations. Isolated strips of large intestine did not show marked spontaneous activity, but rather main- tained a constant basal tension of 0.5 g. Responses to applied agonists were of greater amplitude on strips taken from the rectum.

Initially, in four preparations, the responses of the longitudinal muscle to splanchnic and pelvic nerve stimulation were examined. Stimulation of the splanchnic nerves at 10 Hz for 10 or 60 set caused an inhibition of spontaneous activity that lasted for 2 min. Stimulation of the pelvic nerves at 10 Hz for 10 set had no effect, but a 60-set period of stimu- lation caused a series of contractions at a frequency greater than the spontaneous frequency. However, these were no greater in amplitude than spon- taneous contractions, and, for this reason subsequent expe~ments were carried out on circular muscles.

Splanchnic nerve preparations

Stimulation of the splanchnic nerves at 10Hz for 60 set usually caused a biphasic response consisting of a contraction followed by a prolonged relaxation and cessation of spontaneous activity of the circular muscle in both the colon and the rectum (Fig. 1, first panel). In four of fifteen preparations, only con- tractile responses were observed in the colon. In the rectum, small contractile responses to nerve stimu- lation were often hidden by surrounding spontaneous contractions of equal or greater amplitude.

Where present, the contraction began within 10-20 set of the onset of nerve stimulation. It had an amplitude of 0.53 zf: 0.11 g (mean t_ SEM; N = 9), and lasted about 20-30 set, after which the inhibitory phase of the response usually began, lasting about 2 min.

The amplitude of the contraction was unaffected by hyoscine (10m6 M, N = 2) or d-tubocurarine (10e4 M, N = 2). In three preparations, the size of the contrac- tion was increased by 17-236% following treatment with bretylium (lo-’ M). In one preparation initially showing only inhibitory responses, bretylium treat- ment revealed a contraction in response to nerve stimulation. Tetrodotoxin (3 x 10m6 M) also in- creased the size of the contraction by 40 and 46% in

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Fig. 1. Responses obtained sequentially from a single splanchnic nerve-colon preparation. The first panel shows the mixed response to splanchnic nerve stimulation. In the second panel, capsaicin (CAP, 5 x 10m5 M) causes an initial contraction followed by a slow inhibition of tone: note the reduction of the excitatory response to splanchnic stimulation, The third panel shows the response to stimulation after a 90 min exposure to capsaicin, washout and recovery of tone (WASH): note the absence of initial excitation. In the fourth panel, the surviving inhibitory response has been abolished by bretylium (BRET, 10m5 M).

two preparations. In another, it abolished a purely inhibitory response and revealed a contraction, as did propranolol ( 10e6 M) in one experiment.

Hyoscine had no effect on the relaxation (N = 2). Bretylium abolished the relaxation in four of five preparations, as did propranolol in three prep- arations tested. Treatment with d-tubocurarine abol- ished the relaxation in two of three experiments. Tetrodotoxin also abolished the relaxation in three of four preparations.

Capsaicin (5 x 10e5 M; N = 6) caused a rapid contraction of 0.72 f 0.14 g, which lasted approxi- mately 50 set (Fig. 1, second panel). The spontaneous activity of the preparations decreased by 60-100% and the tone fell over the next 10-15 min. There was a concomitant fall in the amplitude of the nerve- mediated relaxation throughout the 90-min exposure to capsaicin (Fig. 1, second panel). In one prep- aration, capsaicin did not cause a contraction, but did cause the typical inhibition of activity. The initial contraction in response to nerve stimulation was never seen after addition of capsaicin to the prep- arations, nor did it return after washout of the drug and subsequent recovery of tone (Fig. 1, third panel). In three preparations, the inhibitory response to nerve stimulation was still visible, returned in full after washout, and was subsequently blocked by bretylium (Fig. 1, fourth panel). Application of the capsaicin vehicle, dimethylsulphoxide, had no effect (N = 2).

In three experiments, two adjacent strips of intesti- nal wall were removed and mounted as previously described. After an equilibration period, one strip of each pair was treated with tetrodotoxin for 30 min. Capsaicin was then added to both strips. In all cases, capsaicin caused an immediate contraction, and the strips without tetrodotoxin pretreatment showed a typical inhibition developing over the next 10-l 5 min. In two of the three experiments, each of the strips displayed strong spontaneous activity: in these, the fall of tone caused by capsaicin was about 50% smaller in the tetrodotoxin-treated than in the control strips. Following washout of capsaicin and recovery of tone, a second application of capsaicin caused inhibition in both control and tetrodotoxin-treated preparations.

Stimulation of nerves on mesenteric arterial branches distal to the coeliacomesenteric ganglion, had similar effects to splanchnic nerve stimulation. Following degenerative section of the splanchnic nerves, perivascular stimulation elicited only inhibi- tory responses in the rectum and colon (N = 7; Fig. 2). The appearance and duration of the relax- ation was similar to that seen in control preparations and it was also abolished by bretylium (N = 4). Application of capsaicin to denervated preparations caused an initial contraction that was no different from the response elicited in normal preparations, and only one of seven preparations failed to contract in response to the drug. In splanchnic denervated preparations, both the tone and the inhibitory response to perivascular stimulation were reduced after capsaicin treatment; however, the response to stimulation returned in full in three preparations after washout and recovery.

In seven of fifteen preparations taken from normal animals, and in four of seven splanchnic denervated preparations (Fig. 2), the first application of splanchnic or perivascular nerve stimulation to each preparation was followed, after the response described above, by an increase in the basal tension of up to 0.37g or by an increased amplitude of

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Fig. 2. Response of the colon to stimulation of the posterior mesenteric artery following degenerative section of the splanchnic nerves. Note the absence of an initial contraction and the prolonged increase in tone after the inhibitory

response.

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SUSAN MURPHY and G. CAMPBELL

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Fig. 3. Responses of the colon to stimulation of the 9th and 10th spinal nerves, connected to the colon by the pelvic nerves. Note the small initial contraction in the first panel which is abolished after treatment

with hyoscine (HYOS, 10m6 M), as seen in the second panel.

spontaneous contractions, or sometimes both. This effect began within 2-3 min after the onset of stimu- lation, i.e. after recovery from the relaxation, and it lasted for up to 20min. In each preparation, with a repetition of stimulus trains on a 15-min cycle, these after excitations appeared as a continuous elevation of tone or spontaneous activity. When the stimulus cycle was interrupted, the tone and activity gradually decreased to pre-stimulation levels. Furthermore, on application of bretylium or tetrodotoxin, drugs that abolished inhibitory responses to nerve stimulation, the activity also declined.

Pelvic nerve preparations

Stimulation of the 9th and 10th spinal nerves, connected to the large intestine by the pelvic nerves, at 10 Hz for 60 set caused a rapid contractile response in both colon and rectum (Fig. 3). As the responses in colon and rectum differed only in amplitude, all subsequent descriptions of the effects of stimulation refer only to the rectum, which displayed the stronger responses. There was no response to stimulation at frequencies lower than 5 Hz. The typical response was a series of three, or sometimes four, contractions which began within 10 set after the onset of stimu- lation (N = 15). The response lasted 104 + 5 set and reached a peak tension of 3.68 f 0.57 g.

Hyoscine abolished the initial contraction in the response (N = 8; Fig. 3). Following hyoscine treat-

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ment, the response to nerve stimulation began about 45 set after the onset of stimulation and reached a peak tension of 3.91 + 0.60 g. Two preparations showed what appeared to be a small inhibition prior to the beginning of the hyoscine-resistant contrac- tions. In one preparation, stimulation at 10 or 30 Hz for only IOsec elicited a single brief contraction of 1.05 and 2.05 g respectively (Fig. 4). Both responses were abolished by hyoscine, suggesting that this contraction, and therefore the early component of the response to 60 set periods of stimufation, is mediated by release of acetylcholine. Hyoscine caused a fail in tone in three preparations and in these the peak response was also reduced.

The acetylcholinesterase inhibitor neostigmine (3 x 10-7M-10-S M; N = 4) did not increase re- sponses to either lo- or 60-set periods of stimulation. With the higher concentrations tested, the amplitude of the spontaneous contractions increased to such an extent that the contractile response to short periods of stimulation and the initial contraction in response to 60 set stimulation could no Ionger be distinguished.

The entire response to pelvic nerve stimulation was abolished by d-tubocurarine (N = 4). In two of four preparations, tetrodotoxin abolished the entire response. In a third, the response in the rectum was abolished, but the colonic response was appar- ently unaffected. In the remaining preparation, the

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Fig. 4. Responses of the colon to brief (IO-set) periods of pelvic nerve stimulation with 10 or 30 Hz (left panels). After treatment with hyoscine {HYOS, tom6 M) the responses are abolished (right panels).

ANS of Bufo large intestine 565

amplitude of the contractile response was reduced to the large intestine that was apparently identical to 20% of the control (although in this case the colonic the response elicited by stimulation of the peripheral response was abolished). The response was not nerves (N = 4). In the one preparation tested, affected by bretylium (N = 3) or by the 5-HT receptor hyoscine abolished the initial contraction of the antagonist, methysergide ( 10e6 M; N = 3). response, and tetrodotoxin blocked the remainder.

Responses to applied agonists The effect of capsaicin on the response to pelvic

nerve stimulation was tested in four experiments. In one of these, capsaicin had no effect, but in the remaining three, it caused a rapid contraction of 1.80 f 0.59 g, lasting 46 +_ 24 sec. A subsequent fade of spontaneous activity in the continued presence of capsaicin was seen in all four preparations as described above. During this period of inhibition, the amplitude of the contractile response to nerve stimu- lation was reduced by 70-95%. After washout and recovery from capsaicin treatment, the response to stimulation recovered in full.

All agonists were applied to circular strips of large intestine for 60 sec. Values are given for the threshold concentration and the highest concentration tested.

Acetylcholine (10-7-10-4 M; N = 3) caused an increase in basal tension of 0.1-0.5 g and increased both the amplitude and frequency of the spontaneous contractions superimposed on the increased tone. The effect of acetylcholine was abolished by hyoscine (N = 2).

Stimulation of the ventral roots of the 9th and 10th spinal nerves caused a contractile response in

Adrenaline (10-6-10-4 M; N = 3) caused a pro- longed relaxation starting within 30 set of application and lasting 2-5 min after washout (Fig. 5a). On

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Fig. 5. Effects of 60-set applications of putative transmitter substances on circular muscle strips of the colon. Responses are shown to: (a) adrenaline (AD, 10m4 M); (b) S-hydroxytryptamine (SHT, 10m4 M); (c) substance P (SP, lo-‘M); (d) somatostatin (SOM, IO-‘M); (e) galanin (GAL, IO-‘M);

(f) neuropeptide Y (NPY, 10m6 M).

566 SUSAN MURPHY and G. CAMPBELL

recovery from this inhibition, two preparations showed an increase of from 0.15 to 0.55 g in the amplitude of spontaneous contractions, lasting for up to 15 min. One of these strips also showed a 0.15 g increase in the basal tension. The inhibitory response to applied adrenaline was abolished by propranolol (N = 2), after which adrenaline caused an increase in the amplitude of spontaneous contractions starting about 3Osec after application. This response was sustained for up to 13 min after washout and was abolished by phentolamine (10m6 M; N = 2).

Application of 5-hydroxytryptamine (lo-‘- 10m4 M; N = 3) caused a 0.3-0.5 g increase in the amplitude of spontaneous contractions (Fig. 5b). The response was abolished by methysergide (N = 2).

Substance P (lO-g-lO-6 M; N = 6) caused a single, rather weak contraction of up to 0.5 g that began very rapidly after application and was terminated by washout (Fig. 5~). Somatostatin (lO-s-lO-6 M; N = 6; Fig. 5d) and galanin (10-7-10-6 M; N = 6; Fig. 5e) each caused an increase in tone of up to 0.5 g, and an increase in the amplitude of the superimposed phasic contractions. The responses to somatostatin and galanin usually continued for some minutes after washout. Hyoscine and tetrodotoxin did not affect the responses to any of the peptides. When any of these drugs was reapplied on a 15-min cycle, there was no sign of developing desensitization.

Neuropeptide Y (10-7-10-6 M; N = 3) had no effect on application but, starting 2-3 min after washout, the basal tension gradually increased by 0.1-0.3 g, and the frequency of spontaneous con- tractions increased (Fig. 5f). Both effects were maintained for up to 30min.

Neither calcitonin gene-related peptide (IO-“- 10m6 M; N = 3) nor vasoactive intestinal peptide (10-7-10-6 M; N = 4) had any effect on strips of large intestine.

DISCUSSION

Pelvic nerves

Stimulation of the autonomic outflow in spinal nerves 9 and 10 of the toad caused contractions of the large intestine. In confirmation of Boyd et al. (1964) the response to brief (IO-30-set) periods of stimu- lation was abolished by a muscarinic antagonist and can be ascribed to a release of acetylcholine. How- ever, when stimulation was continued for 60 set or more at frequencies greater than about 5 Hz, the cholinergic contraction was a preface to a slowly developing contraction that was apparently not cholinergic, in that it was not prevented by hyoscine. Both responses could be elicited by stimulation of the ventral roots of the spinal nerves, central to the points of entry of sympathetic fibres (see Langley and Orbeli, 1911) and of dorsal root afferents that might have antidromic actions on the gut. Furthermore, the response to peripheral nerve stimulation was pre- vented by the nicotinic antagonist d-tubocurarine. Together, these results suggest that both the cholin- ergic and the non-cholinergic responses were medi- ated by parasympathetic pathways with peripheral ganglionic synapses.

There is considerable evidence that the sacral parasympathetic outflow in mammals also has non-

cholinergic excitatory effects on the large intestine. Atropine-resistant contractions can be evoked by pelvic nerve stimulation in vivo in the cat (Fiilgraff et al., 1964; Rostad, 1973; Fasth et al., 1980; Andersson et d., 1983) and rabbit (Langley and Anderson, 1895). In vitro, however, there seems to be little evidence for non-cholinergic responses in the rabbit (Lee, 1960), cat (Fiilgraff and Schmidt, 1964) or guinea-pig (Lee, 1960; Rand and Ridehalgh, 1965). This discrepancy cannot be accounted for simply by the use of low frequencies or short trains of stimu- lation. There seems little doubt, however, that both the cholinergic and non-cholinergic contractions of the mammalian large intestine are mediated by parasympathetic nerves since, in all cases tested but one (see Fiilgraff et al., 1964), the responses were abolished by nicotinic antagonists (Lee, 1960; Rand and Ridehalgh, 1965; Fasth et al., 1980; Andersson et al., 1983).

The nature of the non-cholinergic transmitter in the toad large intestine is not yet apparent. Further progress would be aided by an anatomical identification of the postganglionic neurons. Some of the postganglionic cells may be multipolar enteric neurons lying in the myenteric plexus, where they would not be readily distinguishable from the sensory and interneurons that presumably lie there. How- ever, it is also possible that the entire population of postganglionic neurons is represented by the rounded unipolar cell bodies that lie on pelvic nerve branches outside and immediately inside the rectal wall (Osborne et al., 1988; Anderson and Campbell, 1989). These neurons resemble other amphibian para- sympathetic postganglionic neurons; for instance, those in the heart (McMahan and Kuffler, 1971) and the urinary bladder (Bazanova et al., 1965). Nearly all of the unipolar neurons on the toad pelvic nerves contain somatostatin-like immunoreactivity, mostly with co-localized galanin-like immunoreactivity (Osborne et al., 1988). The cholinergic parasym- pathetic postganglionic neurons in the toad heart also contain co-localized galanin and somatostatin (Campbell et al., 1982; Morris et al., 1989), suggesting that the pelvic neurons may, by analogy, also be cholinergic. Finally, some of the pelvic neurons con- taining somatostatin with or without galanin also contain 5-hydroxytryptamine (Osborne et al., 1988).

Each of the three substances co-localized in toad pelvic neurons was tested as a potential transmitter of the non-cholinergic contraction. The contractions evoked by exogenous 5-HT were abolished by methy- sergide, suggesting an action on 5-HT, or 5-HT2 receptors (Costa11 and Naylor, 1990). However, as the response to stimulation was unaffected by methy- sergide, it is not likely that 5-HT is involved in the response. Nor does 5-HT appear to be the transmitter mediating the atropine-resistant contractions in the cat colon (Fasth et al., 1980). Galanin and somato- statin caused slow sustained contractions that were not affected by muscarinic receptor blockade or by tetrodotoxin, indicating a direct action on the muscle. In other preparations, it has proved possible to use tachyphylaxis to repeated applications of somato- statin or galanin for pharmacological dissection of a neurogenic response: e.g. somatostatin tachyphylaxis was used to show a somatostatin component of the

ANS of Bufo large intestine 561

vagal cardioinhibition in the toad (Campbell et al., 1982); galanin tachyphylaxis has been used to show a galanin component of excitation of rat ileal muscle to stimulation of enteric neurons (Muramatsu and Yanaihara, 1988). However, there was no sign of tachyphylaxis to either peptide in the toad large intestine. It may be that intestinal smooth muscle is not easily desensitized in the same way that cardiac muscle is in this species (see Campbell et al., 1982), as Osborne and Campbell (1986) did not observe tachyphylaxis to repeated applications of substance P to toad ileal smooth muscle. In short, while the histochemical evidence may suggest a role for somatostatin or galanin in the non-cholinergic trans- mission from the pelvic nerves to the large intestine, no experimental evidence can yet be produced for it.

It is of course possible that the pelvic nerves release a non-cholinergic transmitter that is not somatostatin or galanin. Osborne et al. (1988) found a population of small unipolar nerve cell bodies containing VIP- like immunoreactivity in the pelvic nerve branches to the toad large intestine. VIP is a marker of some inhibitory neurons in mammalian gut (see Fahrenkrug, 1988) and may mediate inhibition of smooth muscle contractions in chicken rectum (Komori and Ohashi, 1990). The VIP cell bodies in the toad rectum may represent inhibitory neurons, and could account for the occasional appearance of weak inhibitory responses prior to the hyoscine- resistant contractions in the toad large intestine. However, synthetic mammalian VIP had no effect, at least in the exposures tested here, and it is possible that even if VIP was released from pelvic neurons in the toad it may have no effect on the muscle. In the cat, Andersson et al. (1983) found that although VIP is released by pelvic nerve stimulation, this peptide has no motor effect on colonic muscle, although Eklund et al. (1979) previously reported that VIP caused colonic contraction. As yet, the role, if any, of VIP in transmission to the toad large intestine is unknown.

One nerve element that might have been expected to pass through the pelvic nerves to the large intestine was, to some extent surprisingly, absent. Antidromic stimulation of a class of capsaicin-sensitive primary afferent neurons that pass through the sympathetic pathways to the gut causes contraction of the small intestine (Osborne and Campbell, 1986) and, as will be argued below, the large intestine. They are present in the sacral outflow to the amphibian urinary bladder, at least in the frog (Runu: Bowers and Kolton, 1987). Such fibres may also supply the bladder in Bufi, as nicotinic antagonists do not abolish excitatory responses to pelvic nerve stimu- lation (Burnstock et al., 1963). If such fibres were present in the sacral outflow to the large intestine, it would be expected firstly, that some part of the excitatory response to stimulation would remain after ganglion blockade. Secondly, if dorsal root afferent fibres are involved, ventral root stimulation should not exactly mimic the response evoked by peripheral nerve stimulation. The exact similarity of the two responses suggests that capsaicin-sensitive afferent fibres do not arise from the sacral spinal cord in the toad. The presence of pelvic visceral afferent neurons projecting through the sacral dorsal roots is well

established in mammals (see Bahns et al., 1987), but such fibres, if present in the toad, may not have antidromic motor effects on the hindgut.

Splanchnic nerves

Stimulation of the splanchnic nerve supply to the large intestine had three distinct effects; initial excitation, slow relaxation, and a very slow and prolonged excitation. The inhibitory response to splanchnic nerve stimulation obtained by Boyd et al. (1964) was apparently mediated by release of adrenaline from sympathetic nerves to act on /I- adrenoreceptors, as the relaxation was abolished by dichloroisoprenaline and bretylium. This finding was confirmed in the present study, as bretylium or propranolol abolished the inhibitory response to stimulation.

The splanchnic nerve fibres mediating inhibition are largely preganglionic in the splanchnic nerves, since the response to stimulation there was abol- ished by d-tubocurarine. Presumably, most of the ganglionic synapses in these inhibitory pathways are located in the coeliaco-mesenteric ganglion, since the response to perivascular stimulation sur- vived a degenerative section of the splanchnic nerves proximal to the ganglion.

It seems that adrenergic nerves also cause the long-term excitatory response to nerve stimulation, as it was abolished by bretylium. The slow excitation was similar to the excitatory response caused by applied adrenaline, apparently mediated through c(- adrenoreceptors. But the delayed, sustained contrac- tion evoked by nerve stimulation was suggestive of a peptidergic transmission. Since NPY is colocalized in adrenergic perivascular nerves in all eutherian mam- mals studied (see Potter, 1988, for review) and in the toad (Morris et al., 1986), the effects of NPY on the toad large intestine were tested. It, too, produced a slow excitatory response. So it remains possible that stimulation of adrenergic nerves to the gut causes release of an NPY-like peptide that acts on the muscle layer, as has also been postulated for the cat rectum (see Hellstrom et al., 1989).

The initial contraction reported here appears to be the same as the small, rapid excitatory response reported for this preparation after adrenergic neuron or ganglion blockade by Boyd et al. (1964). But whereas they found that the contraction was blocked by atropine, we found no such effect of hyoscine; it may be that atropine had acted non-specifically on a weak non-cholinergic response. Similar, but much larger, excitatory responses in the toad small intestine have been ascribed to a release of substance P or a similar peptide from antidromically-activated pri- mary afferent fibres (Osborne and Campbell, 1986), like those described as having motor effects on mam- malian gut (see Maggi and Meli, 1988; Mayer et al., 1990). This class of afferent fibre, commonly contain- ing substance P co-localised with CGRP, shows specific desensitization after prolonged exposure to capsaicin (Maggi and Meli, 1988). In the toad large intestine, the excitatory response to stimulation was indeed abolished following exposure to a high con- centration of capsaicin. It was also absent in prep- arations taken from animals subjected to splanchnic nerve section, which should have caused degeneration

568 SUSAN MURPHY and G. CAMPBELL

of primary afferent fibres. The similarity of the excitatory response to nerve stimulation to the con- tractions evoked by applied substance P and by capsaicin supports the idea that here, as in the toad small intestine, the responses are mediated by substance P released from afferent fibres.

CGRP causes transient contractions of the circular muscle of the guinea-pig ileum (Holzer et al., 1989) and relaxation of the longitudinal muscle (Barth6 et al., 1987), and has been suggested as the transmit- ter mediating relaxation in the isolated rat duodenum (Maggi et al., 1986). Desensitization to CGRP re- duces the inhibitory response evoked by capsaicin application to mammalian gut (Takaki et al., 1989; Mayer et al., 1990). However synthetic mammalian CGRP had no effect on the toad large intestine.

The presence of an apparently normal contractile response to capsaicin in preparations taken from splanchnic nerve-sectioned animals, in which there was no contractile response to perivascular stimu- lation, is anomalous. It is unlikely that some of the splanchnic nerve branches escaped sectioning, which suggests that there must be other routs by which capsaicin-sensitive afferent fibres supply the large intestine. As explained above, it seems that such fibres do not enter via the sacral spinal nerves.

The reason why capsaicin induces such a pro- nounced inhibition of tone is not clear. Capsaicin directly releases substance P and CGRP, and appar- ently, VIP (see Maggi et al., 1990) from primary afferent nerves which have motor effects on the mammalian intestine (Maggi et al., 1987; Maggi and Meli, 1988). The prolonged inhibition may be caused by CGRP acting on smooth muscle in rabbit and guinea-pig (Takaki et al., 1989; Mayer er al., 1990) or by VIP in human gut (see Maggi et al., 1990). However, as previously described, synthetic CGRP and VIP had no effect on toad intestinal smooth muscle. Capsaicin also causes inhibition of intestinal activity by direct stimulation of extrinsic sympathetic nerves or of non-adrenergic, non-cholinergic intra- mural nerves and has a direct depressant action on smooth muscle (Maggi et al., 1987). The inhibitory action of capsaicin on the toad large intestine did not show progressive desensitization, which might suggest that it was acting directly on the muscle.

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