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Evidence for presynaptic 5-hydroxytryptamine3 recognition sites on vagal afferent terminals in the brainstem of the ferret
0306-4522/!90 $3.00+ 0.00
Neuroscience Vol. 38, No. 3, pp.667-673, 1990 Printed in GreatBritain
PergamonPressplc 0 1990 IBRO
EVIDENCE FOR PRESYNAPTIC 5-HYDROXYTRYPTAMINE, RECOGNITION SITES ON VAGAL AFFERENT TERMINALS IN THE BRAINSTEM OF THE FERRET R. A. LEsLm,*t D. J. M. REYNOLDS,$ P. L. R. ANDREWS,$D. G. GRAHAMB-SMITH,*.~ C. J. DAVIS~ and J. M. HARVEY$ *Oxford University-Beecham Centre for Applied Neuropsychobiology and SMRC Unit and University Department of Clinical Pharmacology, Radcliffe Infirmary, Woodstock Road, Oxford OX2 6HE, U.K. §Department of Physiology, St George’s Hospital Medical School, Cranmer Terrace,
London SW17 ORE, U.K. Abatrac-Antagonists acting at the 5hydroxytryptaminer receptor are potent anti-emetic agents in cases of cytotoxic- and radiation-induced vomiting, and binding sites for these compounds have been described in brainstem areas known to be involved in mediation of nausea and vomiting. We have used autoradiography to examine the distribution of one of these antagonists, [3Hlgranisetron in the caudal brainstem of the ferret, a commonly used animal model for physiological investigations of emesis. The highest density of binding sites was found to be in the dorsomedial region of the nucleus of the solitary tract, the principal terminus for gastric vagal afferent fibres. Lower levels of binding were observed in the area postrema and the dorsal motor nucleus of the vagus. Following unilateral nodose ganglion excision, displaceable binding of [3H]granisetronin the nucleus of the solitary tract was attenuated on the ipsilateral side by 65%. Bilateral subdiaphragmatic vagotomy abolished binding of [31Qranisetron in the entire dorsal vagal complex. These results provide strong circumstantial evidence that 5hydroxytryptaminer receptors are located on vagal afferent terminals in the ferret brainstem.
A number of drugs that act as 5_hydroxyttyptamine, (S-HTs) receptor antagonists are effective agents in treating vomiting caused by cancer chemotherapy or radiotherapy. *’ Furthermore, it has been shown that S-HT, recognition sites occur on membranes isolated from various regions of the central nervous system.6,‘*~20*28 A number of autoradiographic studies have indicated that a high density of binding sites for 5-HT, specific radioligands occurs in the dorsal vagal complex of the medulla oblongata,‘9*3’,35~M a region that is known to mediate the emetic reflex.** Waeber et al.” suggested, on the basis of the distribution of S-HT, receptor binding sites in the mouse hindbrain, that they may be located presynaptically on sensory nerve terminals. 5-HT, receptors have been well characterized in the peripheral nervous system and in particular in the vagus nerve.‘6,32*33 A recent reporti has shown that [‘H](3a-tropanyl)-1-H-indole-3-carboxylic acid ester (ICS 205-930), a 5-HT, receptor ligand, labels sites in the vagus nerve and the superior cervical ganglion. Studies have demonstrated that abdominal vagal
tTo whom correspondence should be addressed. Abbreuiutions: BRL 43694, granisetron; GR 38032F, ondansetron; HEPES, N-[2-hydroxyethyllpiperaxine-N’-2[ethanesulphonic acid]; S-HT,, 5hydroxytryptaminer; ICS 205-930, (3a-tropanyl)-1-H-indole-3-carboxylic acid ester. 667
afferent stimulation in the ferret can induce retching and vomitir@ and abdominal vagotomy has been shown to reduce. or abolish the emetic response to a variety of emetic stimuli including radiation and cytotoxic drugs. 3~14In view of the close association between 5-HT, receptors, vagal afferents and emesis, it seems possible that 5-HT, receptors in the dorsal vagal complex (which comprises the area postrema, the dorsal motor nucleus of the vagus and the nucleus of the solitary tract) might be associated with the vagal afferent fibres that terminate here.23 Studies to date have shown, by means of unilateral cervical nodosectomy in the rat, that binding of the 5-HT, receptor ligand [3H]granisetron (BRL 43694) is reduced by up to 75% in the nucleus of the solitary tract thus suggesting that these binding sites are located on vagal atTerent terminals.M The rat is an animal without a vomiting reflex, and it is therefore impossible to assess the anti-emetic effects of 5-HT, receptor antagonists using this animal model. In the present study we have investigated binding of [‘I-IjBRL 43694 in the dorsal vagal complex of the ferret, an animal in which the emetic reflex and the effects of several 5-HT, receptor antagonists have been well characterized4 In addition we have extended previous studies by investigating the effect of chronic abdominal vagotomy as well as nodosectomy in an attempt to gain an insight into the association of 5-HT, receptor binding sites in the dorsal vagal
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complex with specific vagus nerve.
peripheral
EXPERIMENTAL
projections
LESLIEet
of the
PROCEDURES
Adult ferrets (Mustelu putorius furo) (1.0-l 5 kg) were used in this study. Under anaesthesia (ketamine, lOmg/kg s.c., halothane and N,O/O,) animals underwent bilateral abdominal vagotomy (n = 4), bilateral abdominal greater splanchnic nerve transection (n = 2), control laparotomy (n = 3), unilateral cervical nodose ganglion excision (n = 3) or sham nodosectomy (n = 3). Incisions were closed in layers with sutures and treated with antibiotic (polybactrin and cicatrin). Postoperative recovery was uneventful in all cases. The completeness of nerve excision was confirmed in all nine animals by post mortemdissection or by histological examination of the excised ganglion. Ferrets were allowed to recover for 1I-18 days when they were reanaesthetized with an overdose of sodium pentobarbitone and perfused with 0.1 M phosphate buffer. Serial frozen sections were cut (12 pm in thickness) throughout the rostrocaudal extent of the nucleus of the solitary tract and mounted on gelatinized slides. Sections were incubated with 10 nM [-‘H]BRL 43694 (specific activity 21.2 Ci/mmol) in 50mM HEPES buffer, pH 7.4 at room temperature for 45 min. This concentration of the radioligand, as used in a previous study,)O was chosen to ensure that the binding sites would be exposed to a saturating concentration of the ligand (& = 1.21 nM).” Adjacent-sections were similarly incubated with the addition of unlabelled ondansetron (GR 38032F) (100 uM) or metoclopramide (100 PM) to define non-&& binding. All sections were washed twice for 10 s each in ice-cold 50 mM HEPES, dipped rapidly in ice-cold distilled water, air-dried and exposed to Hyperfilm along with tritium standards. The sections were subsequently stained with Cresyl Violet to allow accurate localization of binding.
al.
Densitometric analysis was performed using a computerbased image analysis system (Kontron IBAS 2) on a minimum of 12 sections per animal. In control animals and animals with bilateral lesions, no right-left differences were observed and so data from both sides of the medulla oblongata were combined. Values for non-specific binding were subtracted from total binding to give displaceable binding. Materials [‘H]BRL 43694 and metoclopramide HCl were a gift from Beecham Pharmaceuticals and GR 38032F from Glaxo Group Research. Tritium standards and Hyperfilm were purchased from Amersham International.
RESULTS
Anatomy of the ferret dorsal vagal complex The anatomical relationships of the dorsal vagal complex of the ferret brainstem are illustrated in Fig. 1. In the ferret, unlike the rat, the rostra1 area postrema is a bilateral structure represented by lateral ridges which form an incomplete roof over the most caudal part of the fourth ventricle. The anatomy of the nucleus of the solitary tract of the ferret is similar to that of the rat, lying ventrolateral to the area postrema and dorsal to the dorsal motor nucleus of the vagus nerve. Caudal to the obex the nucleus of the solitary tract traverses the midline dorsal to the central canal to form a commissural subnucleus.
Fig. 1. Medium power light micrograph of Cresyl Violet-stained transverse section through the ferret dorsal hindbrain at the level of the area postrema (AP). At this level (the obex), the two halves of the area postrema are almost fused in the midline to form the most rostra1 portion of the roof of the central canal (CC). Prominent landmarks also visible are the dorsal motor nucleus of the vagus (DMX) and the hypoglossal nucleus (XII). The bulk of the nucleus of the solitary tract (NTS) lies medial to the solitary tract (arrow), which is difficult to resolve with this histological stain. CB, cerebellum; scale bar = 0.5 mm.
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Ferret brainstem 5-HT, receptors Table 1. Table showing levels of specific binding of [3H]BRL 43694 in ferret brainstem frozen sections measured by quantitative autoradiography Ap
NTS
Condition Control animals (n = 6) Splanchnic lesions (n = 2) Unilateral cervical vagus nerve and nodose lesions (n = 3) Ipsilateral Contralateral Bilateral abdbminal vagus nerve lesions (n = 4)
DMX
133 k 12.8 125 f 4.8 (NS)
13.2 + 12.6 56.8 + 9.8 @IS)
15.5 f 3.5 4.3 f 4.6**
44.1 + 16.0*** 122.2 + 1.7(NS) 0***
43.4 f 8.1** 51.1+ 5.5 (NS) 0***
10.9 + 10.3 (NS) 10.8 &-11.8 (NS) 0***
Values are expressed in fmol [-‘H]BRL 43694 specifically bound per mg wet weight of tissue and are the means of data taken from a minimum of 12 sections per animal k S.D. The data for binding in the brainstems of the ferrets with unilateral cervical vagus lesions are
expressed for brain regions ipsilateral and contralateral to the lesion. In the control (sham-operated) animals and in the animals with bilateral lesions, the data from both sides of the brainstem have been combined. NTS, nucleus of the solitary tract; AP, area postrema; DMX, dorsal motor nucleus of the vagus nerve; VN, vagus nerve. Data were analysed with an unpaired r-test using Student’s t-distribution; in each casq values obtained from animals in the treatment -aromx . were compared with those from controls. **P < 0.01, ***p Q 0.001. Distribution of S-hydroxytryptamine, sites in control animals
receptor binding
High levels of specific binding of [3H]BRL 43694 were seen in the dorsal vagal complex of the ferret (Table 1) and represented approximately 75% of total binding (cf. Figs 2E and 3A). In control animals no differences in the distribution or amounts of binding were observed between the right and left sides of the medulla oblongata. The binding occurred in the nucleus of the solitary tract throughout the commissural subnucleus, extending for approximately 0.5 mm caudal to the obex (Fig. 2A, D). The heaviest density of binding was observed in the most dorsomedial region of the nucleus of the solitary tract and extended for a distance of approximately 0.5 mm rostra1 to the obex (Fig. 2B, C, E, F). The average density of binding in the nucleus of the solitary tract was 133 + 12.8 fmol/mg wet weight of tissue (mean k SD., n = 6). A distinctive ring-shaped region of heavy binding occurred in all cases in the dorsomedial region of the nucleus rostra1 to the area postrema (Fig. 2F). Binding occurred throughout the rostrocaudal extent of the area postrema (Fig. 2E). The binding in this nucleus had a relatively even distribution and averaged 73.2 + 12.6 fmol/mg wet weight of tissue. Much lower levels of displaceable binding were observed within the dorsal motor nucleus of the vagus nerve, averaging 15.6 f 3.5 fmol/mg wet weight of tissue. No displaceable binding was observed in other medullary regions at the level of the nucleus of the solitary tract, such as in the reticular formation, the hypoglossal nucleus or the inferior olivary nucleus. The effects of nerve lesions on 5_hydroxytryptamine, receptor Iigand binding
Binding of [‘H]BRL 43694 in the nuclei of the solitary tract and area postrema of the two ferrets
with bilateral splanchnic nerve lesions did not differ significantly from that seen in the control animals (Table 1). There was a difference between the levels of binding in the dorsal motor nuclei of the vagus nerve of these animals and those in controls which was statistically significant (Table 1). Unilateral cervical vagus nerve and nodose ganglion excision resulted in a significant reduction of displaceable binding of [3H]BRL 43694 in the nucleus of the solitary tract (Table 1 and Fig. 3B). The attenuation of displaceable binding on the ipsilateral side was approximately 67% compared with that in control animals (see Table 1). The asymmetry of binding following unilateral cervical vagal lesion was most marked in the dorsomedial region of the nucleus of the solitary tract rostra1 to the area postrema (Fig. 3B). Displaceable binding in the area postrema was also significantly reduced in the cervical vagus nervelesioned group on the ipsilateral side by 41%. The binding seen in the dorsal motor nucleus of the vagus was not significantly affected by cervical nerve lesion (Table 1). Following bilateral abdominal vagotomy displaceable binding of [‘H]BRL 43694 was abolished in all regions of the dorsal vagal complex (Fig. 3C).
DISCUSSION
Binding of the 5-HT3 receptor antagonist [‘H]BRL 43694 to the medulla oblongata of the ferret brain is highly localized to the dorsal vagal complex. In particular, binding is heaviest in the dorsomedial portion of the nucleus of the solitary tract with substantially less binding in the area postrema and even less in the dorsal motor nucleus of the vagus nerve. Many studies have shown that the majority of vagal afferent fibres project to the nucleus of the solitary tract in the medulla oblongata (e.g. the cat,” monkeyI and rat”). Studies in these species have all shown that the dorsomedial region of the nucleus of the solitary tract rostra1 to the obex (i.e. the
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et al.
Fig. 2. (A-C) Low power micrographs of transverse sections through the ferret medulla oblongata at the level OE (A) the commissural subnucleus of the solitary tract (NTS), (B) the mid-area postrema (AP), and (C) the rostra1 nucleus of the solitary tract (NTS), beyond the rostra1 tip of the area postrema (D-F).
Autoradiograms generated on sections taken from levels similar to those illustrated on the left of the figure. The sections used to generate the autor~o~s were previously incubated in [‘HjBRL 43694 and D-F represent total binding of the @and. In D, note the heavy binding in the commissural subnucleus of the solitary tract nucleus @ITS). Binding visible in the inferior olivary nuclei (arrows) is not displaceable. The different levels of binding over the AP and NTS are easily ~~in~is~able in E. The section used to generate this figure is slightly oblique, so that the left side indicates the caudal margin of the ring-shaped region of heavy binding referred to in the text and seen more clearly in F. F illustrates the heavy binding in the NTS in a brainstem region rostra1 to the AP. At this level a distinctive ring- or V-shaped pattern of heavy labelling is always noticeable. Scale bar = 2 mm.
subnucleus gelatinosus”) receives the largest number of vagai a&rent fibres relaying visceral information from the stomach.i’*‘2~17~24 The distribution of [%IJBRL 43694 binding sites in the ferret brainstem, then, is consistent with the possibility that they are located on gastric vagal afferent fibre terminals. Unilateral cervical vagal nerve lesion has been shown in the rat to result in a reduction of rH]BRL 43694 binding sites, particularly noticeable on the ipsilateral side.= We have now demonstrated the
same effect in the ferret. It has been demonstrated that the principal site of termination of vagal nerve afferent fibres in the ferret is in the ipsilateral nucbus of the solitary tract** but some crossover of fibres does occur, particularly in the more caudal regions of the nucleus.29 This distribution matches the pattern of ligand binding found in the present study following unilateral vagus nerve transection, in that the greatest attenuation of binding of PH]BRL 43694 occurred in the ipsilateral nucfeus of the solitary tract,
Ferret brainstem SHT, receptors
671
Fig. 3. (A) Autoradiogram generated over a section similar to that in Fig. 2B. The section had been incubated in [3H]BRL 43694 with the addition of unlabelled GR 38032F, so that this figure represents non-specific binding of the ligand. (B) Illustration of an autoradiogram generated over a section from an animal that had undergone a left nodosectomy. Note the marked reduction in binding over the dorsal vagal complex on the side ipsilateral to the lesion. (C) Autoradiogram produced from a section taken from an animal that had received a bilateral subdiaphragmatic vagotomy. The section illustrated here was taken at the level of the commissural subnucleus of the solitary tract. Note the absence of binding in the dorsal vagal complex. Scale bar = 2 mm.
particularly in its more rostra1 regions. The attenuation of binding that we observed in the nucleus of the solitary tract on the contralateral side in cervical
vagus nerve-lesioned animals did not reach statistical significance despite the fact that there is a small amount of crossover of the vagal atferent fibres at the level of the brainstem as discussed above. The reason for the observed attenuation of ligand binding in the dorsal motor nucleus of the vagus nerve after splanchnic nerve lesions is unclear. We are unaware of any evidence for a direct projection of splanchnic fibres to the dorsal motor nucleus of the
vagus, thus it seems unlikely that this difference in binding is due to SHT, receptor ligand binding sites on splanchnic fibres. There was a large variability in ligand binding values observed in this region (cf. Table l), a common feature of the quantitative autoradiographic radioligand binding technique when working at the limits of detection in very small anatomical regions. Cervical vagus nerve lesion is not selective for afferents originating in any single viscus, whereas subdiaphragmatic vagotomy selectively removes abdominal visceral afferents which appear to be very
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R. A. LESLIEet al.
largely gastric in origin. 24It is known that lesions of distal processes of pseudounipolar primary sensory neurons, including those of the vagus nerve, result in transganglionic degeneration of their central projections. 9.26It is therefore probable that the subdiaphragmatic vagal lesions reported in the present study resulted in a loss of abdominal and, in particular, gastric afferent projection neurons of the vagus nerve which terminate largely in the subnucleus gelatinosus of the nucleus of the solitary tract. The observation that bilateral subd~aphragmatic vagal lesion results in apparent total loss of binding of t3H]BRL 43694 in the dorsal vagal complex of the medulla strengthens our hypothesis that 5-HT, receptor binding sites are located presynaptically on abdominal, and probably gastric vagal afferent terminals. Interestingly, a recent pub~~tioni3 has provided evidence that MIT, receptor binding sites in the periphery are located on sensory afferent fibre terminals in the dorsal horn of the spinal cord. In that study, capsaicin was used to cause degeneration of somatic afferent Iibres in neonatal rats. This work and the current study, then, suggest that 5-HT, receptor binding sites may be located on both general and visceral somatic afferents. A number of other studies employing similar methods to those reported here have shown that vagal nerve lesions result in a loss of binding sites for some neurotransmitters and neuromodulators. For example, in the cat” muscarinic cholinergic binding sites are reduced in the nucleus of the solitary tract following unilateral cervical vagotomy. Similarly, binding sites for angiotensin II,’ cholecystokinin” as well as delta opioid (but not mu opioid) ligands25 are reduced following such surgery. Other receptor binding sites are unaffected by such a lesion, such as alpha noradrenegic sitesz5 It has been demonstrated in the case of muscarinic sites, that the loss of binding is probably due to a decrease in B,,, rather than
an affinity change following the lesion.25 These reports suggest that the loss of S-HT, receptor binding sites reported here and elsewhere are not simply a non-specific effect of surgery or cranial nerve lesion. The function of the 5-HT, receptor binding sites in the dorsal vagal complex is not known but their location on vagal afferents terminating in the subnucleus gelatinosus of the nucleus of the solitary tract is suggestive of a role in the modulation of afferent information conveyed in gastric or other gastrointestinal afferents. El~trophysiological studies in a variety of species have demonstrated that these vagai afferents convey information relating to the activity of the muscular wall of the gut and the chemical nature of the luminal contents.‘.” This afferent information is used for the genesis of non-painful visceral sensations, the extrinsic reflex control of gut function and for the activation of the vomiting reflex and hence the 5-HT, receptor binding sites in the subnucleus gelatinosus could be involved in the modulation of any of these parameters. While it is clear that the dorsal vagal complex may be a site for the anti-emetic e&&s of 5-HT, receptor antagonists, it does not follow that this is the only site or that it is the most important site. Others have argued that the site of anti-emetic action may be on M-IT, receptors located on the peripheral terminals of vagal afferents which, it is hypothesized, are activated by 5-HT released by emetic stimuli such as radiation or cytotoxic drugs.4 Wherever the site(s) of the anti-emetic action of 5-HT, receptor antagonists, this study has demonstrated that these agents probably play an important presynaptic modulatory role in the processing of abdominal visceral information. Acknowledgements-We are gratefui to Dr P. Blower of Beeeham Pharmaceuticals for the gift o~[~H]BRL 43694 and metoclopramide HCl and to Dr B. M. Bain of Glaxo Group Research for providing GR 38032F.
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D. (1988) Speculations on the structure/function relationship for vagal and splanchnic afferent endings supplying the gastrointestinal tract. J. uuton. nerv. Syst. 22, 175-180. II. Gwyn D. G. and Leslie R. A. (1979) A projection of vagus nerve to the area subpostrema in the cat. Brain Res. 161, 335-341.
12. Gwyn D. G., Leslie R. A. and Hopkins D. A. (1985) Observations on the afferent and efferent organization of the vagus nerve and the innervation of the stomach in the squirrel monkey. J. camp. Neural. 239, 163-175. 13. Hamon M., Gallisot M. C., Menard F., Gozlan H., Bourgoin S. and Verge D. (1989) 5-HT, receptor binding sites are on capsaicin-sensitive fibres in the rat spinal cord. Eur. J. Phurmac. 164, 315-322. 14. Hawthorn J., Ostler K. J. and Andrews P. L. R. (1988) The role of the abdominal visceral innervation and S-hydroxytryptamine M-receptors in vomiting induced by the cytotoxic drugs cyclophosphamide and cisplatin in the ferret. J. exp. Physiol. 73, 7-21. 15. Hoyer D., Waeber C., Karpf A., Neijt H. and Palacios J. M. (1989) [‘H]ICS 205-930 labels 5-HT, recognition sites in membranes of cat and rabbit vagus nerve and superior cervical ganglion. Nuunyn-Schmiedeberg’s Arch. Pharmac. 340, 396-402.
16. Ireland S. J. and Tyers M. B. (1987) Pharmacological characterization of S-hydroxytryptamine-induced depolarization of the rat isolated vagus nerve. Br. J. Phurmuc. 90, 229-238. 17. Kalia M. and Mesulam M.-M. (1980) Brain stem projections of sensory and motor components of the vagus complex in the cat I. The cervical vagus and nodose ganglion. J. camp. Neural. 193, 435-465. 18. Kilpatrick G. J., Jones B. J. and Tyers M. B. (1987) Identification and distribution of 5-HT, receptors in rat brain using radioligand binding. Nature 330, 746-748. 19. Kilpatrick G. J., Jones B. J. and Tyers M. B. (1988) The distribution of specific binding of the 5-HT, receptor ligand [3H]GR 65630 in rat brain using quantitative autoradiography. Neurosci. Let?. 94, 156-160. 20. Kilpatrick G. J., Jones B. J. and Tyers M. B. (1989) Binding of the 5-HT, ligand, [3H]GR 65630, to rat area postrema, vagus nerve and the brains of several species. Eur. J. Pharmac. 159, 157-164. 21 Ladenheim E. E., Speth R. C. and Ritter R. C. (1988) Reduction of CCK-8 binding in the nucleus of the solitary tract in unilaterally nodosectomized rats. Brain Res. 474, 125-129. 22 Leslie R. A. (1985) Neuroactive substances in the dorsal vagal complex of the medulla oblongata: nucleus of the tractus solitarius, area postrema, and dorsal motor nucleus of the vagus. Neurochem. Znf. 7, 191-211. 23. Leslie R. A. and Gwyn D. G. (1984) Neuronal connections of the area postrema. Fedn Proc. 43, 2942-2943. 24. Leslie R. A., Gwyn D. G. and Hopkins D. A. (1982) The central distribution of the cervical vagus nerve and gastric afferent and efferent projections in the rat. Brain Res. Bull. 8, 37-43. 25. Leslie R. A., Murphy K. and Robertson H. A. (1989) Nodose ganglionectomy selectively reduces muscarinic cholinergic and delta opioid binding sites in the dorsal vagal complex of the cat. Neuroscience 32, 481-492. 26 Majumdar S., Mills E. and Smith P. G. (1983) Degenerative and regenerative changes in central projections of glossopharyngeal and vagal sensory neurons after peripheral axotomy in cats: a structural basis for central reorganization of arterial chemoreflex pathways. Neuroscience 10, 841-849. 27 Miner W. D. and Sanger G. J. (1986) Inhibition of cisplatin-induced vomiting by selective S-hydroxytryptamine m receptor antagonism. Br. J. Pharmac. 88, 497-499. 28. Nelson D. R. and Thomas D. R. (1989) [3H]-BRL 43694 (granisetron), a specific ligand for 5-HT, binding sites in rat brain cortical membranes. Biochem. Phnrmac. 38, 1693-1695. 29 Odekunle A. and Bower A. J. (1985) Brainstem connections of vagal afferent nerves in the ferret: an autoradiographic study. J. Anat. 140, 461-469. 30 Pratt G. D. and Bowery N. G. (1989) The 5-HT, receptor ligand [‘HI-BRL 43694 binds to presynaptic sites in the nucleus tractus solitarius of the rat. Neuropharmacology 28, 1367-1376. 31. Reynolds D. J. M., Leslie R. A., Grahame-Smith D. G. and Harvey J. M. (1989) Localization of 5-HT, receptor binding sites in human dorsal vagal complex. Eur. J. Pharmac. 174, 127-130. 32 Richardson B. P., Engel G., Donatsch P. and Stadler P. A. (1985) Identification of serotonin M-receptor subtypes and their specific blockade by a new class of drugs. Nufure 316, 126-131. 33. Round A. and Wallis D. I. (1987) Further studies on the blockade of 5-HT depolarizations of rabbit vagal afferent and sympathetic ganglion cells by MDL 72222 and other antagonists. Neuropharmacology 26, 39-48. 34. Taber E. (1961) The cytoarchitecture of the brainstem of the cat. I. Brainstem nuclei of the cat. J. camp. Neural. 116, 27-69. 35. Waeber C., Dixon K., Hoyer D. and Palacios J. M. (1988) Localization by autoradiography of neuronal 5-HT, receptors in the mouse CNS. Eur. J. Pharmac. 151, 351-352. 36. Waeber C., Hoyer D. and Palacios J. M. (1989) S-Hydroxytryptamine, receptors in the human brain: autoradiographic visualization using [‘HJICS 205-930. Neuroscience 31, 393-400. (Accepted 2 May 1990)
Neuroscience Vol. 38, No. 3, pp.667-673, 1990 Printed in GreatBritain
PergamonPressplc 0 1990 IBRO
EVIDENCE FOR PRESYNAPTIC 5-HYDROXYTRYPTAMINE, RECOGNITION SITES ON VAGAL AFFERENT TERMINALS IN THE BRAINSTEM OF THE FERRET R. A. LEsLm,*t D. J. M. REYNOLDS,$ P. L. R. ANDREWS,$D. G. GRAHAMB-SMITH,*.~ C. J. DAVIS~ and J. M. HARVEY$ *Oxford University-Beecham Centre for Applied Neuropsychobiology and SMRC Unit and University Department of Clinical Pharmacology, Radcliffe Infirmary, Woodstock Road, Oxford OX2 6HE, U.K. §Department of Physiology, St George’s Hospital Medical School, Cranmer Terrace,
London SW17 ORE, U.K. Abatrac-Antagonists acting at the 5hydroxytryptaminer receptor are potent anti-emetic agents in cases of cytotoxic- and radiation-induced vomiting, and binding sites for these compounds have been described in brainstem areas known to be involved in mediation of nausea and vomiting. We have used autoradiography to examine the distribution of one of these antagonists, [3Hlgranisetron in the caudal brainstem of the ferret, a commonly used animal model for physiological investigations of emesis. The highest density of binding sites was found to be in the dorsomedial region of the nucleus of the solitary tract, the principal terminus for gastric vagal afferent fibres. Lower levels of binding were observed in the area postrema and the dorsal motor nucleus of the vagus. Following unilateral nodose ganglion excision, displaceable binding of [3H]granisetronin the nucleus of the solitary tract was attenuated on the ipsilateral side by 65%. Bilateral subdiaphragmatic vagotomy abolished binding of [31Qranisetron in the entire dorsal vagal complex. These results provide strong circumstantial evidence that 5hydroxytryptaminer receptors are located on vagal afferent terminals in the ferret brainstem.
A number of drugs that act as 5_hydroxyttyptamine, (S-HTs) receptor antagonists are effective agents in treating vomiting caused by cancer chemotherapy or radiotherapy. *’ Furthermore, it has been shown that S-HT, recognition sites occur on membranes isolated from various regions of the central nervous system.6,‘*~20*28 A number of autoradiographic studies have indicated that a high density of binding sites for 5-HT, specific radioligands occurs in the dorsal vagal complex of the medulla oblongata,‘9*3’,35~M a region that is known to mediate the emetic reflex.** Waeber et al.” suggested, on the basis of the distribution of S-HT, receptor binding sites in the mouse hindbrain, that they may be located presynaptically on sensory nerve terminals. 5-HT, receptors have been well characterized in the peripheral nervous system and in particular in the vagus nerve.‘6,32*33 A recent reporti has shown that [‘H](3a-tropanyl)-1-H-indole-3-carboxylic acid ester (ICS 205-930), a 5-HT, receptor ligand, labels sites in the vagus nerve and the superior cervical ganglion. Studies have demonstrated that abdominal vagal
tTo whom correspondence should be addressed. Abbreuiutions: BRL 43694, granisetron; GR 38032F, ondansetron; HEPES, N-[2-hydroxyethyllpiperaxine-N’-2[ethanesulphonic acid]; S-HT,, 5hydroxytryptaminer; ICS 205-930, (3a-tropanyl)-1-H-indole-3-carboxylic acid ester. 667
afferent stimulation in the ferret can induce retching and vomitir@ and abdominal vagotomy has been shown to reduce. or abolish the emetic response to a variety of emetic stimuli including radiation and cytotoxic drugs. 3~14In view of the close association between 5-HT, receptors, vagal afferents and emesis, it seems possible that 5-HT, receptors in the dorsal vagal complex (which comprises the area postrema, the dorsal motor nucleus of the vagus and the nucleus of the solitary tract) might be associated with the vagal afferent fibres that terminate here.23 Studies to date have shown, by means of unilateral cervical nodosectomy in the rat, that binding of the 5-HT, receptor ligand [3H]granisetron (BRL 43694) is reduced by up to 75% in the nucleus of the solitary tract thus suggesting that these binding sites are located on vagal atTerent terminals.M The rat is an animal without a vomiting reflex, and it is therefore impossible to assess the anti-emetic effects of 5-HT, receptor antagonists using this animal model. In the present study we have investigated binding of [‘I-IjBRL 43694 in the dorsal vagal complex of the ferret, an animal in which the emetic reflex and the effects of several 5-HT, receptor antagonists have been well characterized4 In addition we have extended previous studies by investigating the effect of chronic abdominal vagotomy as well as nodosectomy in an attempt to gain an insight into the association of 5-HT, receptor binding sites in the dorsal vagal
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complex with specific vagus nerve.
peripheral
EXPERIMENTAL
projections
LESLIEet
of the
PROCEDURES
Adult ferrets (Mustelu putorius furo) (1.0-l 5 kg) were used in this study. Under anaesthesia (ketamine, lOmg/kg s.c., halothane and N,O/O,) animals underwent bilateral abdominal vagotomy (n = 4), bilateral abdominal greater splanchnic nerve transection (n = 2), control laparotomy (n = 3), unilateral cervical nodose ganglion excision (n = 3) or sham nodosectomy (n = 3). Incisions were closed in layers with sutures and treated with antibiotic (polybactrin and cicatrin). Postoperative recovery was uneventful in all cases. The completeness of nerve excision was confirmed in all nine animals by post mortemdissection or by histological examination of the excised ganglion. Ferrets were allowed to recover for 1I-18 days when they were reanaesthetized with an overdose of sodium pentobarbitone and perfused with 0.1 M phosphate buffer. Serial frozen sections were cut (12 pm in thickness) throughout the rostrocaudal extent of the nucleus of the solitary tract and mounted on gelatinized slides. Sections were incubated with 10 nM [-‘H]BRL 43694 (specific activity 21.2 Ci/mmol) in 50mM HEPES buffer, pH 7.4 at room temperature for 45 min. This concentration of the radioligand, as used in a previous study,)O was chosen to ensure that the binding sites would be exposed to a saturating concentration of the ligand (& = 1.21 nM).” Adjacent-sections were similarly incubated with the addition of unlabelled ondansetron (GR 38032F) (100 uM) or metoclopramide (100 PM) to define non-&& binding. All sections were washed twice for 10 s each in ice-cold 50 mM HEPES, dipped rapidly in ice-cold distilled water, air-dried and exposed to Hyperfilm along with tritium standards. The sections were subsequently stained with Cresyl Violet to allow accurate localization of binding.
al.
Densitometric analysis was performed using a computerbased image analysis system (Kontron IBAS 2) on a minimum of 12 sections per animal. In control animals and animals with bilateral lesions, no right-left differences were observed and so data from both sides of the medulla oblongata were combined. Values for non-specific binding were subtracted from total binding to give displaceable binding. Materials [‘H]BRL 43694 and metoclopramide HCl were a gift from Beecham Pharmaceuticals and GR 38032F from Glaxo Group Research. Tritium standards and Hyperfilm were purchased from Amersham International.
RESULTS
Anatomy of the ferret dorsal vagal complex The anatomical relationships of the dorsal vagal complex of the ferret brainstem are illustrated in Fig. 1. In the ferret, unlike the rat, the rostra1 area postrema is a bilateral structure represented by lateral ridges which form an incomplete roof over the most caudal part of the fourth ventricle. The anatomy of the nucleus of the solitary tract of the ferret is similar to that of the rat, lying ventrolateral to the area postrema and dorsal to the dorsal motor nucleus of the vagus nerve. Caudal to the obex the nucleus of the solitary tract traverses the midline dorsal to the central canal to form a commissural subnucleus.
Fig. 1. Medium power light micrograph of Cresyl Violet-stained transverse section through the ferret dorsal hindbrain at the level of the area postrema (AP). At this level (the obex), the two halves of the area postrema are almost fused in the midline to form the most rostra1 portion of the roof of the central canal (CC). Prominent landmarks also visible are the dorsal motor nucleus of the vagus (DMX) and the hypoglossal nucleus (XII). The bulk of the nucleus of the solitary tract (NTS) lies medial to the solitary tract (arrow), which is difficult to resolve with this histological stain. CB, cerebellum; scale bar = 0.5 mm.
669
Ferret brainstem 5-HT, receptors Table 1. Table showing levels of specific binding of [3H]BRL 43694 in ferret brainstem frozen sections measured by quantitative autoradiography Ap
NTS
Condition Control animals (n = 6) Splanchnic lesions (n = 2) Unilateral cervical vagus nerve and nodose lesions (n = 3) Ipsilateral Contralateral Bilateral abdbminal vagus nerve lesions (n = 4)
DMX
133 k 12.8 125 f 4.8 (NS)
13.2 + 12.6 56.8 + 9.8 @IS)
15.5 f 3.5 4.3 f 4.6**
44.1 + 16.0*** 122.2 + 1.7(NS) 0***
43.4 f 8.1** 51.1+ 5.5 (NS) 0***
10.9 + 10.3 (NS) 10.8 &-11.8 (NS) 0***
Values are expressed in fmol [-‘H]BRL 43694 specifically bound per mg wet weight of tissue and are the means of data taken from a minimum of 12 sections per animal k S.D. The data for binding in the brainstems of the ferrets with unilateral cervical vagus lesions are
expressed for brain regions ipsilateral and contralateral to the lesion. In the control (sham-operated) animals and in the animals with bilateral lesions, the data from both sides of the brainstem have been combined. NTS, nucleus of the solitary tract; AP, area postrema; DMX, dorsal motor nucleus of the vagus nerve; VN, vagus nerve. Data were analysed with an unpaired r-test using Student’s t-distribution; in each casq values obtained from animals in the treatment -aromx . were compared with those from controls. **P < 0.01, ***p Q 0.001. Distribution of S-hydroxytryptamine, sites in control animals
receptor binding
High levels of specific binding of [3H]BRL 43694 were seen in the dorsal vagal complex of the ferret (Table 1) and represented approximately 75% of total binding (cf. Figs 2E and 3A). In control animals no differences in the distribution or amounts of binding were observed between the right and left sides of the medulla oblongata. The binding occurred in the nucleus of the solitary tract throughout the commissural subnucleus, extending for approximately 0.5 mm caudal to the obex (Fig. 2A, D). The heaviest density of binding was observed in the most dorsomedial region of the nucleus of the solitary tract and extended for a distance of approximately 0.5 mm rostra1 to the obex (Fig. 2B, C, E, F). The average density of binding in the nucleus of the solitary tract was 133 + 12.8 fmol/mg wet weight of tissue (mean k SD., n = 6). A distinctive ring-shaped region of heavy binding occurred in all cases in the dorsomedial region of the nucleus rostra1 to the area postrema (Fig. 2F). Binding occurred throughout the rostrocaudal extent of the area postrema (Fig. 2E). The binding in this nucleus had a relatively even distribution and averaged 73.2 + 12.6 fmol/mg wet weight of tissue. Much lower levels of displaceable binding were observed within the dorsal motor nucleus of the vagus nerve, averaging 15.6 f 3.5 fmol/mg wet weight of tissue. No displaceable binding was observed in other medullary regions at the level of the nucleus of the solitary tract, such as in the reticular formation, the hypoglossal nucleus or the inferior olivary nucleus. The effects of nerve lesions on 5_hydroxytryptamine, receptor Iigand binding
Binding of [‘H]BRL 43694 in the nuclei of the solitary tract and area postrema of the two ferrets
with bilateral splanchnic nerve lesions did not differ significantly from that seen in the control animals (Table 1). There was a difference between the levels of binding in the dorsal motor nuclei of the vagus nerve of these animals and those in controls which was statistically significant (Table 1). Unilateral cervical vagus nerve and nodose ganglion excision resulted in a significant reduction of displaceable binding of [3H]BRL 43694 in the nucleus of the solitary tract (Table 1 and Fig. 3B). The attenuation of displaceable binding on the ipsilateral side was approximately 67% compared with that in control animals (see Table 1). The asymmetry of binding following unilateral cervical vagal lesion was most marked in the dorsomedial region of the nucleus of the solitary tract rostra1 to the area postrema (Fig. 3B). Displaceable binding in the area postrema was also significantly reduced in the cervical vagus nervelesioned group on the ipsilateral side by 41%. The binding seen in the dorsal motor nucleus of the vagus was not significantly affected by cervical nerve lesion (Table 1). Following bilateral abdominal vagotomy displaceable binding of [‘H]BRL 43694 was abolished in all regions of the dorsal vagal complex (Fig. 3C).
DISCUSSION
Binding of the 5-HT3 receptor antagonist [‘H]BRL 43694 to the medulla oblongata of the ferret brain is highly localized to the dorsal vagal complex. In particular, binding is heaviest in the dorsomedial portion of the nucleus of the solitary tract with substantially less binding in the area postrema and even less in the dorsal motor nucleus of the vagus nerve. Many studies have shown that the majority of vagal afferent fibres project to the nucleus of the solitary tract in the medulla oblongata (e.g. the cat,” monkeyI and rat”). Studies in these species have all shown that the dorsomedial region of the nucleus of the solitary tract rostra1 to the obex (i.e. the
670
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tESLiE
et al.
Fig. 2. (A-C) Low power micrographs of transverse sections through the ferret medulla oblongata at the level OE (A) the commissural subnucleus of the solitary tract (NTS), (B) the mid-area postrema (AP), and (C) the rostra1 nucleus of the solitary tract (NTS), beyond the rostra1 tip of the area postrema (D-F).
Autoradiograms generated on sections taken from levels similar to those illustrated on the left of the figure. The sections used to generate the autor~o~s were previously incubated in [‘HjBRL 43694 and D-F represent total binding of the @and. In D, note the heavy binding in the commissural subnucleus of the solitary tract nucleus @ITS). Binding visible in the inferior olivary nuclei (arrows) is not displaceable. The different levels of binding over the AP and NTS are easily ~~in~is~able in E. The section used to generate this figure is slightly oblique, so that the left side indicates the caudal margin of the ring-shaped region of heavy binding referred to in the text and seen more clearly in F. F illustrates the heavy binding in the NTS in a brainstem region rostra1 to the AP. At this level a distinctive ring- or V-shaped pattern of heavy labelling is always noticeable. Scale bar = 2 mm.
subnucleus gelatinosus”) receives the largest number of vagai a&rent fibres relaying visceral information from the stomach.i’*‘2~17~24 The distribution of [%IJBRL 43694 binding sites in the ferret brainstem, then, is consistent with the possibility that they are located on gastric vagal afferent fibre terminals. Unilateral cervical vagal nerve lesion has been shown in the rat to result in a reduction of rH]BRL 43694 binding sites, particularly noticeable on the ipsilateral side.= We have now demonstrated the
same effect in the ferret. It has been demonstrated that the principal site of termination of vagal nerve afferent fibres in the ferret is in the ipsilateral nucbus of the solitary tract** but some crossover of fibres does occur, particularly in the more caudal regions of the nucleus.29 This distribution matches the pattern of ligand binding found in the present study following unilateral vagus nerve transection, in that the greatest attenuation of binding of PH]BRL 43694 occurred in the ipsilateral nucfeus of the solitary tract,
Ferret brainstem SHT, receptors
671
Fig. 3. (A) Autoradiogram generated over a section similar to that in Fig. 2B. The section had been incubated in [3H]BRL 43694 with the addition of unlabelled GR 38032F, so that this figure represents non-specific binding of the ligand. (B) Illustration of an autoradiogram generated over a section from an animal that had undergone a left nodosectomy. Note the marked reduction in binding over the dorsal vagal complex on the side ipsilateral to the lesion. (C) Autoradiogram produced from a section taken from an animal that had received a bilateral subdiaphragmatic vagotomy. The section illustrated here was taken at the level of the commissural subnucleus of the solitary tract. Note the absence of binding in the dorsal vagal complex. Scale bar = 2 mm.
particularly in its more rostra1 regions. The attenuation of binding that we observed in the nucleus of the solitary tract on the contralateral side in cervical
vagus nerve-lesioned animals did not reach statistical significance despite the fact that there is a small amount of crossover of the vagal atferent fibres at the level of the brainstem as discussed above. The reason for the observed attenuation of ligand binding in the dorsal motor nucleus of the vagus nerve after splanchnic nerve lesions is unclear. We are unaware of any evidence for a direct projection of splanchnic fibres to the dorsal motor nucleus of the
vagus, thus it seems unlikely that this difference in binding is due to SHT, receptor ligand binding sites on splanchnic fibres. There was a large variability in ligand binding values observed in this region (cf. Table l), a common feature of the quantitative autoradiographic radioligand binding technique when working at the limits of detection in very small anatomical regions. Cervical vagus nerve lesion is not selective for afferents originating in any single viscus, whereas subdiaphragmatic vagotomy selectively removes abdominal visceral afferents which appear to be very
672
R. A. LESLIEet al.
largely gastric in origin. 24It is known that lesions of distal processes of pseudounipolar primary sensory neurons, including those of the vagus nerve, result in transganglionic degeneration of their central projections. 9.26It is therefore probable that the subdiaphragmatic vagal lesions reported in the present study resulted in a loss of abdominal and, in particular, gastric afferent projection neurons of the vagus nerve which terminate largely in the subnucleus gelatinosus of the nucleus of the solitary tract. The observation that bilateral subd~aphragmatic vagal lesion results in apparent total loss of binding of t3H]BRL 43694 in the dorsal vagal complex of the medulla strengthens our hypothesis that 5-HT, receptor binding sites are located presynaptically on abdominal, and probably gastric vagal afferent terminals. Interestingly, a recent pub~~tioni3 has provided evidence that MIT, receptor binding sites in the periphery are located on sensory afferent fibre terminals in the dorsal horn of the spinal cord. In that study, capsaicin was used to cause degeneration of somatic afferent Iibres in neonatal rats. This work and the current study, then, suggest that 5-HT, receptor binding sites may be located on both general and visceral somatic afferents. A number of other studies employing similar methods to those reported here have shown that vagal nerve lesions result in a loss of binding sites for some neurotransmitters and neuromodulators. For example, in the cat” muscarinic cholinergic binding sites are reduced in the nucleus of the solitary tract following unilateral cervical vagotomy. Similarly, binding sites for angiotensin II,’ cholecystokinin” as well as delta opioid (but not mu opioid) ligands25 are reduced following such surgery. Other receptor binding sites are unaffected by such a lesion, such as alpha noradrenegic sitesz5 It has been demonstrated in the case of muscarinic sites, that the loss of binding is probably due to a decrease in B,,, rather than
an affinity change following the lesion.25 These reports suggest that the loss of S-HT, receptor binding sites reported here and elsewhere are not simply a non-specific effect of surgery or cranial nerve lesion. The function of the 5-HT, receptor binding sites in the dorsal vagal complex is not known but their location on vagal afferents terminating in the subnucleus gelatinosus of the nucleus of the solitary tract is suggestive of a role in the modulation of afferent information conveyed in gastric or other gastrointestinal afferents. El~trophysiological studies in a variety of species have demonstrated that these vagai afferents convey information relating to the activity of the muscular wall of the gut and the chemical nature of the luminal contents.‘.” This afferent information is used for the genesis of non-painful visceral sensations, the extrinsic reflex control of gut function and for the activation of the vomiting reflex and hence the 5-HT, receptor binding sites in the subnucleus gelatinosus could be involved in the modulation of any of these parameters. While it is clear that the dorsal vagal complex may be a site for the anti-emetic e&&s of 5-HT, receptor antagonists, it does not follow that this is the only site or that it is the most important site. Others have argued that the site of anti-emetic action may be on M-IT, receptors located on the peripheral terminals of vagal afferents which, it is hypothesized, are activated by 5-HT released by emetic stimuli such as radiation or cytotoxic drugs.4 Wherever the site(s) of the anti-emetic action of 5-HT, receptor antagonists, this study has demonstrated that these agents probably play an important presynaptic modulatory role in the processing of abdominal visceral information. Acknowledgements-We are gratefui to Dr P. Blower of Beeeham Pharmaceuticals for the gift o~[~H]BRL 43694 and metoclopramide HCl and to Dr B. M. Bain of Glaxo Group Research for providing GR 38032F.
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