Evidence for cholinergic vagal afferents and vagal presynaptic M1 receptors in the ferret

Pergamon

o197-om6(94)oooso-s

Neurochem Int Vol 25, No 5, pp. 455-464, 1994 Copyright © 1994ElsevmrScienceLtd Pnnted m Great Bntmn All rights reserved 01974)186/94 $7 00+0 00

EVIDENCE FOR CHOLINERGIC VAGAL AFFERENTS A N D V A G A L P R E S Y N A P T I C M1 R E C E P T O R S I N T H E FERRET D. J. M.

REYNOLDS l*, P. R. LOWENSTEIN2, J. M. MOORMAN 3, D. G. GRAHAME-SMITHL3and R. A. LESLIE3

University Department of Clinical Pharmacology, Radcliffe Infirmary, Woodstock Road, Oxford OX2 6HE 2Department of Physiology, Umvermty of Wales College of Cardiff, CardlffCF1 1SS 3Oxford Umvermy-SmithKhne Beecham Centre for Applied Nenropsychobiology, University Department of Clinical Pharmacology, Radcliffe Infirmary, Woodstock Road, Oxford OX2 6HE, U.K (Recewed 16 March 1994, accepted 19 May 1994) Aima'act--The distribution of muscanmc receptor binding was examined in the ferret brainstem vagal nuclei using the non-selective hgand [3H]qumuclidlnyl benzflate and the relatively Mt receptor-selective ligand [3H]p~renzepme. The highest density of receptor sites are found in the subnucleus gelatmosus and lower levels in the other subnuclei of the nucleus of the tractus sohtarlus and m the area postrema and dorsal motor nucleus of the vagus nerve. Dense binding was also seen in the adjacent hypoglossal nucleus Following umlateral cervacal nodose ganghon excision binding m the submicleus gelatinosus was attenuated lpsdateral to the lesion compared with the contralateral side. In contrast, [3H]pirenzepme binding was only seen in the subnucleus gelatlnosus and in no other region at th~s level of the bramstem. This binding was reduced in the subnucleus as a whole by 52% lpsilateral to a cervical vagotomy In the more rostral parts of the subnucleus gelatinosus, binding was undetectable ipsdateral to the lesion but more caudally, appreciable levels of binding persisted. Th~s distribution parallels the known rostro-caudal variation in cross-over of vagal afferent fibres in the ferret dorsal vagal complex and indicates a presynaptic localization of [3H]ptrenzepme binding sites on vagal afferent terminals The distnbuUon of binding of the high afiimty chobne uptake site blocker, [3H]hemlcholimum-3, was also examined in the ferret bramstem using autoradiography. High densities of [3H]hemtchohnium-3 binding were seen m the hypoglossal nucleus, the subnucleus gelatinosus and in the area postrema, with lower levels in the dorsal motor nucleus of the vagus, the tngeminal nucleus and other submiclei of the nucleus of the tractus sohtanus. After unilateral vagal denervation, binding of [3H]hemmholimum-3 was attenuated in the lpsllateral subnucleus gelatmosus but not in other regions of the caudal medulla oblongata These data proxade evidence that a proportion of vagal afferent fibres are chohnerglc and that ptrenzepme binding sites, which are likely to be Mt receptors, are located presynaptlcally on vagal afferent terminals.

The nature of the transmitters and modulators employed in vagal afferent neurotransmission is still poorly understood. There is evidence that vagal afterents may contain substance P (Lundberg et al., 1978, Gamse et al., 1979; Gilbert et al., 1980; M a c L e a n and Lewis, 1984), 5-HT (Gaudin-Chazal et al., 1982 ; Nosjean et al., 1990), calcitonin gene-related peptide (~ and fl), galanin, neurokinin A, dynorphin (Sternim, *Author to whom all correspondence should be addressed Abbreviations : 5-HT, 5-hydroxytryptatmne ; VIP, vasoactwe intestinal protein; CCK, cholecystokmin; QNB, qumuchdmyl benzilate; NTS, nucleus of the tractus solttanus ; AP, area postrema. 455

1992), glutamate (Granata and Reiss, 1983), metenkephahn (Lundberg et al., 1978), VIP (Lundberg et al., 1978, Gdbert et al., 1980), somatostatin (Lundberg et al., 1978, Gilbert et al., 1980; M a c L e a n and Lewis, 1984), acetylcholine (Helke et al., 1983) and G a s t r i n - C C K (Lundberg et al., 1978). As well as containing a variety of neuroactive substances, the visceral afferent terminals m the dorsal vagal complex (i.e. in the nucleus of the tractus solitarius, the area postrema and the dorsal m o t o r nucleus of the vagus nerve) bear a number of presynaptic receptors (Table 1) The presence of acetylcholine in the dorsal vagal complex has been demonstrated by a number of

456

I) I M RI~OlD'C, CIa[

Fable 1 Presynaphc receptors located on xag,d allercnt termmal~ the dorsal vagal ~_omplex

Muszanm~.chohnerg~c l; and ? opto~d Ang~otensmU GABA. and GABAa Cholecystokmm Vasopre,s~m ~-HT, TRH Substance P

m

(Leshe t't td 1989) (Shefl~er~; a/ 1981, Leshe, t a/ 1989) tD~."et a/, 19861 (Pratt and Bower~, 19921 (Ladenhe~m et al 19881 (Phdhp~ el a[, 1990) (Pratt and Bo~eI~ 19R9 Le,,hct'l a/ 19901 (Manaker and Zu~.dn 19931 (Manaker and Zm.t.h~, t993}

presynapnc chohnergtc terminals (Kuhm ~ ; a / . 1973} and ~ts d J s t n b u t m n m the bl,un correlate- ~ell ~J(h that o f c h o h n e acetyltransferase ( K m m i , i , ; , / / . 1981)1 ( h o l l n e r g t c receptor binding sites m the dol,,d vagal complex o f the ferret were examined using the non-selectwe r a d m h g a n d [~HJQNB and Ihe mole selective hgand [3H]plrenzepme The racholabelled c h o h n e uptake blocker, [~H]henuchohnlum-~ I HC-3). was ubed to label high alhmt 3 chohne uplake s~tes (Pascual e/a,l. 1990. 1991 ).

EXPERIMENTAL PROCEDURES

authors (reviewed tn Leshe. 1985, Palkowts, 1985) A l t h o u g h some chohnergm cell bodies have been identified within the nucleus o f the tractus sohtarlus, at the margins o f the dorsal m o t o r nucleus o f the vagus ( A r m s t r o n g et a l , 1988). 1t has generally been assumed that the major source o f acetylchohne Jii this nucleus is from afferent fibres The ewdence that acetylcholine functions as a n e u r o t r a n s m l t t e r in vagal afferent neurones has been largely based on immunocytochemical identification o f choline acetyltransferase a n d / o r acetylchohnesterase as markers for chohnergic terminals (Gwyn and Wolstencroft, 1968, Palkovlts and Jacobowltz, 1974. Helke et a l , 1983, Ternaux et a l , 1989) and on functional studies involving the use o f m u s c a r m i c chohnergm antagonist drugs (Tsubomura et a l , 1988, Sharkey et a l , 1991) Other studies have identified c h o h n e acetyltransferase m the nodose g a n g h o n and used th~s as an mdmatton that afferent vagal fibres may be chohnergIc (Palouzier et a l , 1987) A high concentration o f muscarmlc receptor recognition sites has been identified m the brainstem by a u t o r a d m g r a p h y using the nonspecific muscarinlc ligand [3H]quinuclidinyl benzflate (QNB) (Leshe et a l , 1989) In particular muscarlmc receptor sites are concentrated m the subnucleus gelattnosus of the nucleus o f the tractus sohtarIu~, the principal s~te o | termination o f vagal sensory fibres emanating from the proximal gut (Leslie et a l , 1982). Following urnlateral nodose ganglion excIsmn, Leshe et al 11989) observed a diminution o f [~H]QNB binding site density tpsflateral to the lesmn suggesting that some o f these receptor sites are located on vagal sensory termmals The present study was p e r f b r m e d in an attempt to gain more information a b o u t the possible subtype o f muscarmtc receptor that may be located on the vagal afferent fibres and terminals In addition the relatmnship between vagal afferent terminals and high affinity choline uptake sites was investigated. The high affintty chohne uptake system has been used ,t~ a m a r k e r for

Adult ferrets (MuatelaputoHu~ lure) (0 7 I 2 kg) of either sex were used m this study Under anaesthesia (ketamme, 10 mg/kg l m followed by halothane and N20) animals underwent unilateral cervical vagotomy and nodose ganglion removal (n = 5t or sham operatmn (,i = 2/ lnctsmns were closed m layers with sutures and a topmal antlbmttc apphed (polybactrm and ctcatrm) Postoperative recovery was une'~entful m all cases The completeness of the nerve transections and ganglion excisions was confirmed by post mortern dissection and histological examination of the exmsed ganghon Ferrets were allowed to recover for 14 days when they were re-anaesthetized w~th an overdose of ~odtum pentobarbUal and perfused transcardially with tee cold 0 ! M phosphate buffer Brains were removed and frozen m isopentane at - 4 0 C and transferred to a - 7 0 C freezer lor no more than 2 weeks Serial frozen sectmns from the brains of all ammals were then cut (12/~m) throughout the rostrocaudal extent of the dorsal vagal complex and mounted on gelatlntzed slides Autotadtographu receptor hmdmq u.~tml [ 'H/QA'B Shde mounted sections were incubated for 70 mm in 5 0 nM [~H]QNB (specific activity 42 Cl;mmoL New England Nuclear) in phosphate buffered sahne, pH 7 4, at room temperature They were then washed for 10 mm in phosphate buffer at 4' C, followed by a rinse m ice cold distilled water AdJacent shde-mounted secuons were stmdarly incubated but ~lth the addmon of 100 jiM atropine to identify nonspecific binding tutot adios# apht~ receptor hmdmq u ~mq [ ~H ]ptl eJl.=~Tmw Sections were incubated in 4 6 nM [3H]ptrenzepme (specific activity 744 Ct/mmol, New England Nuclear) lbr 1 h at 23 'C m 50 mM Trts buffer containing 7 mM KCI, 37 mM NaCI at pH 7 4 Slides were then washed tbr 5 mm m lee cold buffer, followed by a 10 s wash in distilled water AdJacent slides were incubated m the same way but m the presence of [0 itM atropine to identify non-specific binding A utoradtographtc bmdmg using [ 'H ]hemu hohnnon-3 Alternate sections of ferret bramstem from the ,,dine amreals used tbr pH]ptrenzepme binding were incubated m 10 nM [~H]HC-3 (specific actJvtty, 147 5 Cl/mmol, New England Nuclear), for 30 mm at 23 C m 50 mM glycylglycme buffer, pH 7 8, contamlng 200 mM NaCI (Vtckro) et al, 1985) Sections were then rmsed in me cold incubation buffer for a total of 2 mm and then dipped m lee cold distilled water for 10 ~ Non-,pectfic binding ~as determined as that present

Mt receptor binding m bramstem vagal nuclei m adjacent sections incubated m the same way but with the addmon of 10 /~M unlabelled hemicholinmm-3 m the incubation medium.

Image analysisand quantification of binding Excess t~did was removed from the washed sections and all sectaons wer~ then air dried either in a stream of cool (room temI~ature) air or by gentle warming (40°C) on a hotplate. After drying slides were exposed to tritlam sensitive film (Hyperfilm, Ametsham) along with brain paste standards for 4 weeks to enable sub~luent quantification. Films were then developed and sections were fixed in 4% paraformaldehyde and stained with cresyl violet for histological examination. DensRometric analysis was performed using a computerbased ~ analys~s system (MCID, Imaging Research, St Catharines, Canada). A minimum of 12 sections per animal was used for analysis. In unlesioned control animals no leftright dLffcfences in binding were detected and so data from both sides of the medulla oblongata were combined. Values for non-specific binding were subtracted from total binding to determine specific (displaceabie) binding. RESULTS

[3H] QNB binding in the ferret caudal medulla oblongata Binding of [3H]QNB was detected bilaterally, throughout the dorsal vagal complex and hypoglossal nucleus o f the 2 control (sham nodosectomy) ferrets. Heavy binding was seen in the subnucleus gelatinosus and was o f similar intensity to that seen in the hypoTable 2. [3H]QNBbinding m the ferret caudal medulla oblongata Condmon Control (n = 2) Vagotomy (n ffi 5) Ipsdatcral ~a¢1¢ Contralatcral side

bITS

AP

Hy~)oglossaln

9994-82

4334-65

11704-114

5934-61" 947 4-44

5194-71 485 + 21

1081 4-101 989 4-91

Data are in fnmi/mg proton and are expressed as the mean + S D NTS, nucleusof the tractus sofitarius, AP, area postrema. Values were compared in lesioned animals between the sides ipsilateral and contrahtteral to the lesion Significancewas determined usang a S t u d e n t ' s t-test * P < 0.05 Table 3 [~Hlplrenzcpine binding m the ferret caudal medulla oblongata Condmon Control (n = 2) Vagotomy (n = 5) Ipsdateral side Contralateral szde

NTS

AP

Hypoglossaln

242 + 32

--

--

--

--

--

--

77 4-21" 161 _ 4 4

Data are m fmol/mg protein and are expressed as the mean4-S D NTS, nucleusof the tractus sohtanus, AP, area postrema Values were compared m les~oned ammals between the stdes lpsdateral and contralateral to the les~on Stgmfieancewas determined using a Student's t-test *P < 0 05

457

glossal nucleus (Fig. 1 and Table 2). Lighter binding was seen in the area postrema and in all the other subnuclei of the nucleus o f the t r ~ t u s solitarius as well as in the dorsal m o t o r nucleus o f the vagus (Fig. 1 and Table 2). Binding in the inferior olive was not displaceable by unlabelled atropine (Fig. 1). In sections taken from 2 ferrets which had previously undergone a unilateral nodosectomy, [3HI Q N B binding was markedly attenuated in the nucleus of the tractus solitarius ipsilateral to the lesion compared with the contralateral side (Fig. 1 and Table 2). In these animals, binding o f [3H]QNB appeared unaffected in other regions o f the caudal medulla oblongata.

[3H]pirenzepine binding in the ferret caudal medulla oblongata In contrast to [3H]QNB, binding o f [3H]pirenzepine was largely restricted to the dorsal vagal complex of the ferret medulla oblongata. In two sham-operated control ferrets, heaviest binding o f [3H]pirenzepine occurred bilaterally in the subnucleus gelatinosus (Fig. 2 and Table 3) with almost undetectable levels in the area postrema and other subdivisions of the nucleus of the tractus solitarius. There was no detectable binding in the dorsal m o t o r nucleus o f the vagus nerve or in the hypoglossal nucleus, or in any other structures of the caudal brainstem examined at this level. In five animals which had previously undergone a unilateral lesion of the nodose ganglion and cervical vagus nerve, [3H]pirenzepine binding was attenuated ipsilateral to the lesion by 52% compared with the contralateral side. C o m p a r e d with the two control animals, the binding in the subnucleus gelatinosus contralateral to the lesion may also have been reduced, although the numbers are too small to make an adequate comparison (see Table 3). The reduction m [3H]pirenzepine binding on the side ipsilateral to the lesion was not uniform throughout the rostro-caudal extent o f the subnucleus gelatinosus. In the most rostral portions o f the subnucleus, binding was undetectable on the lesioned side, whereas in the more caudal areas appreciable levels o f binding remained.

[3H]HC-3 binding in the ferret caudal medulla oblongata There was dense binding of [3H]HC-3 in the caudal medulla oblongata of the two sham-operated ferrets, in particular m the subnucleus gelatinosus, in the area postrema and m the hypoglossal nucleus. Lower levels of displaceable binding were observed in the other regions of the nucleus of the tractus solitarius, the

45~,

D I M Rl',i-,!OLI)<,c/~d

Fig 1 Negative zmages of,iutor,tdlogldms of [*H]QNB binding in the ferret caudal blaJnst.em (sk, i, 1,~l~ll binding of [*HJQNB in a sham-operated control ,lnnnal Note the den<,e binding in the h~ poglossal lltl~_lt:tl~ (XII) with lower levels n~ the nudetis of the tractus bohtanus (NTS) and the area po.strena,t (AP) fhc blndlng in the inferior ohvaiy nucleus (asterisk) was not displaced by excess unlabelled atropine Scdlc bar = I mm (B) is an autoradiogram generated oxe~ ,~ snnllar section from a ferret which h,id undergt~ne left-s~ded unilateral nodose ganglion exc,sion 2 v~eeks cdrher The drrou indicates the reduc{lon in binding In the NTS lpsllateral to the les~on

Mj receptor binding in brainstem vagal nuclei

Fig. 2. Negative images of autoradiograms generated over ferret brainstem sections incubated with [3H]pirenzepine. (A) is the binding in a shaln-operated control animal and clearly shows the very localized nature of [3H]pirenzeplne binding w~thm the subnucleus gelatlnosus of the N'IS (arrow) and in no other brainsteln region at this level. (B) shows a similar secUon from a ferret wluch had undergone left sided nodose ganglion excision 2 weeks earlier. The reduction m [3H]pirenzelnne binding in the subnucleus gelatlnosus is clearly seen (arrow). Scale bar = I ram

459

460

1) I ~.1 R I ; ' , d l l l ) ' - , , ,

:/

I~lg 3 Negatl,,e images of a ~adlogr,lms of [~H]H(-3 binding in the lerret caudal bram~tem IA~ is t,~t,~ binding of [~H]HC-3 in a .qlam-operaled control amm,iI Note the dense binding m the hypoglo,,,~,tl nut.lcu, (XII). the nucleu~ of the tractu~ solltarlu~, (arlo~) and the area postrema (arrow headt. Ulth lowel le\ cl~ of binding in the mlerlor Ollvary nucleus (asterisk) Scale bar = 1 rain (B) is an ,lUtOladlo~zraln gcner,Hcd m e t a similar section from a ferret which had undergone left-sided unilateral nodose galagllon c,~cl>mn 2 v~eeks earher The arrov~ Indicates the reduct,on m binding m the NTq lpsll,lter.tl I,, the le~fon

Mj receptor binding in brainstem vagal nuclei dorsal motor nucleus of the vagus nerve and in the inferior olivary nucleus (Fig. 3). A low level of binding of [3H]HC-3 was also observed in the spinal trigeminal nucleus and throughout the reticular formation. In sections taken from five animals which had previously undergone unilateral nodosectomy and cervical vagotomy, binding of [3H]HC-3 was seen to be attenuated in the subnucleus gelatmosus on the side ipsilateral to the lesion (Table 4 and Fig. 3). Binding of [3H]HC-3 in other regions of the caudal medulla oblongata was unaffected by the vagal lesions. DISCUSSION

A number of early studies used the demonstration of the degradative enzyme acetylcholinesterase to infer the presence of cholinergic terminals within the subnucleus gelatinosus and the commissural subnucleus of the nucleus of the tractus solitarius (Gwyn and Wolstencroft, 1968; Palkowts and Jacobowitz, 1974). More recently, techniques using immunocytochemistry to identify the synthetic enzyme choline acetyltransferase have been used m an attempt to provide more reliable localization of cholinerglc neurones and nerve terminals. In this way Henderson and colleagues (1991) have defined the organization of the dorsal vagal complex in the ferret. These authors found little evidence of chohnergic primary afferent terminals in the subnucleus gelatinosus but extensive choline acetyltransferase-positive terminal axonal arborization in all of the other subnuclei of the vagal visceral nucleus and suggested that acetylcholine has an important role in secondary processing of visceral afferent impulses. Vagal afferent denervatlon (following nodose ganglion excision or subnodose cerwcal vagotomy) has been reported to result in decreased choline acetyltransferase activity in the nucleus of the tractus solitarius in the rat (Helke et al., 1983). This could be interpreted as evidence for vagal cholinergic afferents. However, the observation that a reduction in choline acetyltransferase activity Table 4. [3H]HC-3 binding m the ferret caudal medulla oblongata Condmon Control (n = 2) Vagotomy (n = 5) Ipsflateral side Contralateral sade

NTS

AP

Hypoglossal n

506±25

644_+110

452±53

305+45* 504 ± 86

413+94 470 ± 85

472+53 483 -+ 85

Values arc m fmol/mg protein and are expressed as the mean + S D NTS, nucleus of the tractus sohtarius, AP, area postrema Values were compared m lestoned animals between the sides lpsflateral and contralateral to the les~on Slgmficanee was deternuned using a Student's t-test *P < 0.05

461

occurred in the absence of light microscopic changes of afferent degeneration in rats with subnodose vagotomy led these authors to postulate that the enzyme is transynaptically regulated: hence cholinergic neurones are postsynaptic to vagal afferents in the nucleus of the tractus solitarius. In contrast, Armstrong and colleagues (1983) faded to identify any choline acetyltransferase immunoreactivity in the nucleus of the tractus solitarius or the area postrema. HC-3 is a potent and selective inhibitor of the high affinity choline uptake system (Guyenet et al., 1973) and the autora&ographic distribution of [3H]HC-3 binding has been proposed as a selective and quantifiable marker of cholinergic presynaptic terminals (Vickroy et al., 1985 ; Pascual et al., 1991). Regional mapping of [3H]HC-3 binding m the rat brain closely corresponds with the distribution of choline acetyltransferase and acetylcholinesterase (Beckenstein and Wooten, 1983, Rainbow et al., 1984). The binding of [3H]HC-3 in the brainstem of the two control ferrets was densest over the hypoglossal nucleus, the subnucleus gelatinosus, the dorsal motor nucleus of the vagus nerve and the area postrema. The two cramal nerve motor nuclei contain cholinerglc somata, but the nucleus of the tractus solitarius and area postrema contain few if any cholinergic cell bodies. This suggests that the choline uptake sites labelled in the nucleus of the tractus sohtarms and area postrema may be present on chohnergic terminals. Many studies have shown that the majority ofvagal afferent fibres project to the nucleus of the tractus solitarius in the cat (Gwyn and Leslie, 1979 ; Gwyn et al., 1979), the monkey (Gwyn et al., 1985) and the rat (Leslie et a l , 1982). In all of these species, the most dorsomedial regmn of the nucleus of the tractus solitarius (corresponding to the subnucleus gelatinosus) receives the largest number of vagal afferent fibres and these are primarily gastric in origin. The distribution of vagal terminals in the ferret has been less thoroughly investigated and details of viscerotop~c representation in dorsal vagal complex of this species are lacking. However, it has been demonstrated that the principal site of termination of vagai afferent fibres m the ferret is in the ipsilateral nucleus of the tractus solitarius (Odekunle and Bower, 1985; Fltzakerley and Lucier, 1988) but some crossover of fibres does occur, especially in the more caudal regions of the nucleus (Odekunle and Bower, 1985). The observation that [3H]HC-3 binding is significantly reduced in the ipsilateral nucleus of the tractus sohtarius compared with the contralateral side after unilateral cervical vagotomy in five ferrets is consistent with a presynaptic locaUon of high affinity

402

l) I M

R) ",",,:)z n~', ,~,~"a /

choline uptake sites on vagal afferent terminals, and thus suggests that a proportion of these afferent fibres are chohnerg~c There was no s~gmticant l eduction nt binding In the area postrema after vagal leslon,ng. which suggests that the majority of high affinity choline uptake sites in th~s nucleus are not present on vagal terminals All muscarmlc receptors appear to be labelled by [~H]QNB with little selectlvlt> between subtypes (Vanderheyden et a l , 1990) The muscarlnlC antagonist, p~renzeplne, is often described as an Mt receptor selective hgand (Vllar6 et a l . 1992). but it should be noted that its affinity for Mt receptors is only about 10-fold greater than its affimty for M~ receptors (Vanderheyden et a l , 1990) [~H]plrenzepme binding was performed at a concentration 4 6 nM which is at or near its Ka value at M~ receptor sites (Watson #I a . / , 1983) and the majority of binding sites identified with this hgand in this studv are likely to represent M~ receptors (see below) MuscarlnlC receptor binding in the mammalian bramstem has been reported by a number of investigators (Yamamura el" a l , 1983, Vvamsley ct a l , 1984, Pedlgo and Brlzzee, 1985, Spencer e¢ a l . 1985, Cortes and Palaclos, 1986, Mash and Potter. 1986, Hyde cl a l , 1988, Leslie el a / , 1989, Qulrlon el a / , 1989, Vllar6 el a l , 1992, Male2 and Se~bold, 1993) In those studms which have attempted to differentiate between M) and Me receptors, there has been general agreement that there are few' M~ receptor binding sites in the bralnstem and the maJority ofmuscarmlc receptors m this brain region are of the Me receptor subclass (Yamamura el a l , 1983, Wamsley ez a / , 1984, Spencer er a l , 1985. Mash and Potter, 1986, Qulrlon et al,1989,Vllaroetal,1992) Mash and Potter(198b) identified low densities of putative M, receptors (defined as [~H]QNB binding sites which could be displaced by unlabelled plrenzepme, but not by unlabelled carbachol) in the substantla gelatlnosa of the trlgemmal nucleus and in the inferior olive but not in the dorsal vagal complex At least 6 studies examining the binding of [3H]plrenzepine and [ ' H ] Q N B m malnmahan brain incorporated bramstem sections which Included the dorsal vagal complex and all of these concluded that the muscarlmc binding seen in the nucleus of the tractus sohtarlus was to Me receptor sites (Yamamura el a l , 1983, Wamsley el a l , 1984, Spencer et a l , 1985, Mash and Potter, 1986, Qmrlon el a l , 1989, Vllar6 el a l , 1992) The present results m the ferret also indicate dense binding of [3H]QNB m the subnucleus gelatlnosus of the nucleus of the tractus sohtarlus and in the hypoglossal nucleus, with lighter binding in the area

postrelna and dorsal motor nucleus ol the x t i g r i s ller~,c Detailed assessment of the subnucleal organlzatmn ol [~H]QNB binding In the cat ( Leslie er a l . 1989, Male x and Seybold, 1993) reve,lls a ~er~ slnnlal distribution to that seen m the ferret with the highest level.-, ~)1 binding m the area correspond,ng to the subnucleus gelatlnosus In the cat ,rod lcrret there t', much less muscarmlc binding in the area postrelna. ~ Inch is in contrast with the study of Pedlgo and Brlzzee (I 985} where high levels were reported m honlogenates c,t bovine area postrema Alea postrema homogenates, however, are likely to be contaminated with undcllying subnucleus gelatlnosus and this appalent difference in,i} not be SO nnporldn[ Leslie and colleagues (1989) combined ['HIQNB binding with unilateral vagotom) and ldentllied a diminution of muscarmlc binding sites ~psllateral to the lesion This reduction m binding ~as explained b5 a reduction in the B,.... with no effect on A,). which is In keeping with a reduced number of ~cccptor sacs after leslonlng and suggests lhat muscarlmc receptors are present on ~agal afferent terminals m the nucleus of the tractus sohtarlus and m partlculal m the ~ubnucleus gelatlnosus Thl~ hndlng of a ~cducmm EI1 [~H]QNB binding sites ~as also seen u/ lhe terret The binding in more rostral areas of the nucleus w'a,, abolished lpmlateral to the ~agal lesion but nl the more caudal parts of the nucleus the effects ol Ihe lesion were less marked Th~s is consistent with the known rostro-caudal variation In the degree of cross-o~er ot xagal afferent fibres m the ferret (Odekunle and Bower, 1985), The more rostral vagal prolccuons ale mainly lpsllateral whereas apprecmble cross-o\ei of the more caudal proJections doe.~ occur In contrast to previous studies, high levels of ['HI plrenzeplne binding were seen in the subnucleu5 gelatlnosus but in no other bralnstem area {at the level of the dorsal vagal complex, J e from I mm caudal to the obex to 2 mm rostral to it) Furthermore. this binding was attenuated b3 unilateral vagotomy, again particularly In the more rostlal portions of the subnucleus No pH]plrenzeplne binding ~as seen in the hypoglossal nucleus or the mlerlor olive The binding of [~H]plrenzeplne in the dorsal vagal complex was very localized and this may explain whx In prexlous studies It had been overlooked These results provide extdence that muscarlnlC receptors are located presynaptlcally on vagal afferent terminals In the nucleus of the tractus sohtarlus The contrast between the distribution of binding of [3H]QNB and [~H]plrenzeplne in the bralnstem and the relative selective affinity of plrenzeplne for M~ receptors, supports the hypothesis that a proportnm

Mt receptor bindmg in brainstem vagal nuclei of these presynaptic muscarinic receptors are o f the Mt receptor subtype. Presynaptic M~ receptors have been described elsewhere in the brain where, for example, they mediate inhibition of excitatory synaptic transmission in the hippocampus (Sheridan and Sutor, 1990) and in the olfactory cortex (Williams and Constanti, 1988). In the periphery, excitatory presynaptic M~ receptors (like 5-HT3 receptors) facilitate noradrenaline etflux from cardiac sympathetic neurones (Habermeier-Muth et al., 1990). The majority of brainstem muscarinic receptors are not labelled by [3H]pirenzepme and this is consistent with the previous evidence that they are o f the M2 subtype. Brainstem [3H]pirenzepine binding sites appear to be located exclusively on vagal afferent terminals and this suggests that they play a role in the modification of visceral afferent firing and might be another site of action of anticholinergic drugs used in the treatment of emesis, gastrointestinal motility disorders and peptic ulceration.

REFERENCES

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