Neuroscwnce Vol 43, No 1, pp 197-209, 1991 Printed m Great Britain

0306-4522/91 $3 00 + 0 00 Pergamon Press plc :Q 1991 IBRO

HIGH RESOLUTION RADIOAUTOGRAPHIC LOCALIZATION OF [lZSI]FK-33-824-LABELLED MU OPIOID RECEPTORS IN THE SPINAL CORD OF NORMAL A N D D E A F F E R E N T E D RATS C CrOUARDI~RES,*t A . BEAUDET,]~ J . - M ZAJAC,t J. CROS~ a n d R. QUIRION*§

*Douglas Hospital Research Centre and Department of Psychiatry, McGfll University, 6875 Blvd LaSalle, Verdun, Quebec H4H 1R3, Canada J'Laboratoire de Pharmacologie et de Toxicologic Fondamentales, C N R S, 205 Route de Narbonne, 31400 Toulouse, France ~Laboratory of Neuroanatomy, Montreal Neurological Instatute, 3801 Universaty Street, Montreal, Quebec H3A 2B4, Canada Akstract--Recent data have shown that [t25I]o-Ala2, MePhe 4, Met(o)olS-enkephahn (FK-33-824) 1S a highly selective and speofic mu opaoid receptor hgand [Moyse et al (1986) Pepttdes 7, 351-355] This probe was used here to investigate the detailed radioautographlc distribution of mu sites at various levels of the spinal cord [~25I]FK-33-824 binding sites were localized by both tritium-sensitive film and liquid emulsion radloautography in the spinal cord of naive and deafferented rats In naive ammals, high densities of mu sates were apparent within laminae 171 at all levels of the dorsal horn, with higher levels of labelling seen in layer Ill as compared to Iio in the lumbar segment Laminae Ill-IV contained about half the quantaties of binding observed m superficial layers. Relatively high densities of sites were also seen over lamina VI m the upper cervacal cord and throughout Clarke's column Within the latter, [~25I]FK-33-824 binding clearly spared the large perlkarya of the spinocerebellar neurons In the ventral horn, [~25I]FK-33-824 binding was mainly concentrated in layer IX, at the level of cervical and lumbar enlargements Labelled sates were confined to the neuropll, mostly spanng the soma of motoneurons Significant decreases m [~25I]FK-33-824 binding In laminae I-II (55%) and Ill-IV (28%) were detected four days following cervical (C3427) or lumbar (LI L6) rhlzotomies. These decrements were most evident at seven days post-lesaon at C3427 levels (93 and 76% In laminae I-II and Ill-IV, respectively) and recovered slightly thereafter up to 28 days post-lesion In contrast, dorsal rhlzotomles did not influence mu labelling in either the ventral horn or Clarke's column These results confirm the association of mu opiold binding sites with dorsal primary afferent fibres and demonstrate the presence of mu sites in Clarke's column and lamina IX of the ventral horn These findings suggest that endogenous oploads in the spinal cord play a role in sensory motor antegratlon as well as an the modulation of primary nociceptwe inputs

Several studies have d e m o n s t r a t e d a direct effect o f opiolds o n spinal sensory a n d m o t o r processlng. 4'9'18'20'67'78'80It was also f o u n d t h a t spinally a d m i n lstered oplolds exert at least part o f their effects locally by acting t h r o u g h specific oplold receptors. Is 2o23,79,80 Mu, delta a n d k a p p a oplold binding sites have been d e m o n s t r a t e d in the spinal cord o f a variety o f m a m mals 2.3,823,26,29 32,47,58,59,68.72,81,82including h u m a n s . 16'22'33 All three types a p p e a r to be c o n c e n t r a t e d in the substantla g e l a t l n o s a 378,10,22,233247,58,59,68,7276,81,82 a region of the dorsal h o r n enriched with opioid peptides,4,5 13.15,27,35,43,46,6167,73a n d a k n o w n site o f terraina t l o n of noclceptive p r i m a r y a f f e r e n t s . 6'12'24'49'5156,70.74 Surgical section o f the sciatic nerve, 23 or of dorsal root afferent fibres 7 8,10,23,47,58,59,82 result in a m a r k e d decrease in all three types o f oploid receptor within the dorsal h o r n which has been t a k e n as evidence for a presynaptic localization o f the receptors. A

§To whom all correspondence should be addressed Abbrevtanons FK-33-824, (D-Ala2, MePhe 4, Met(o)olS)-enkephahn, DAGO, (D-Ala 2, MePhe 4, Gly-olS)-enkephahn

c o m p a r a b l e decrease is observed after injection of the selective neurotoxin, capsaicln, 17'26'57 suggesting t h a t these presynaptic receptors are mainly associated with fibres o f small calibre o f the type carrying noclceptlve information In a d d i t i o n to their d o c u m e n t e d role in the m o d u latlon o f noclceptlon, 4'9'75'8° spinal oplolds have been s h o w n to suppress volume-evoked micturltlon reflex in h u m a n s 65 a n d animals 19 a n d to alter the excitability o f m o t o r n e u r o n s a n d ventral h o r n lnterneurons. 2° In keeping with these latter effects, spastlclty was shown to be modified in h u m a n s following lntrathecal infusion o f m o r p h i n e 21 T a k e n together, these findlngs suggest t h a t the effects of spinal oplolds m a y not be confined to the dorsal horn. A few biochemical 72 a n d r a d i o a u t o g r a p h l c 81 binding studies o n samples f r o m the rat spinal cord have indeed reported the presence o f specific m u oplold binding sites within the ventral horn. M o s t such studies, however, using either non-selective (such as e t o r p h l n e or diprenorphlne) 3'16'22'47'58'59'75'76or selective 78A0,17,28328182 trltlated mu, delta or k a p p a hgands, merely report the presence

197

C GOUARDI~RESet al

198

of high d e n s m e s o f all types o f oplold binding sites m the s u b s t a n t l a gelatlnosa of the dorsal horn. In order to confirm the existence, a n d further d o c u m e n t the distribution o f m u receptors within the ventral h o r n a n d intermediate zone of the spinal gray matter, we have investigated the detailed a n a t o m i c a l distribution o f m u o p i m d receptors at all m a j o r segmental levels of the rat spinal cord using highr e s o l u t m n r a d l o a u t o g r a p h y after labelling with (D-AIa-', M e P h e 4, Met(o)olS)-enkephahn ([128I]FK-33824), a highly selective, high-affimty p r o b e 28.36 385258 ' Moreover, unilateral dorsal rhlzotomles 48 were p e r f o r m e d m order to determine m each segment the p r o p o r t i o n of m u sites assocmted with p r i m a r y afferent fibres EXPERIMENTAL PROCEDURES

Matertals [~25I]Na, trmum micro-scales and Hyperfilms were obtamed from Amersham Canada (Oakville, Ontano). Naloxone was purchased from RBI (Wayland, MA) and FK-33-824 obtained from Sandoz (Switzerland; Dr D. Roemer) Anunals were purchased from Charles River Laboratories Canada (St-Constant, Quebec). All other chemicals were obtained from standard commercial sources

Methods lodmatton of the radwhgand. FK-33-824 was mchnated and purified as described elsewhere, 58 Binding properties of the monolodmated compound (specific actwlty of 400 Cl/ mmol) were routinely tested on rat brain striatal sections before the actual experiments. At a concentration of 1 nM the amount of nonspecific binding (m the presence of 10/a M naloxone) accounted for approxamately 2% of total binding m spinal cord sectmns Surgwal procedures~dorsal rhzzotomy Adult male Sprague-Dawley rats (300-350 g) were anaesthetized with Nembutal (50 mg/kg, Lp ) and subjected to a dorsal lammectomy of vertebrae C3427 or of T13-LI In both cases, a longatudinal mctsmn of the dura was made with care to avoid damaging the blood vessels running along the cord The corresponding dorsal roots (C3--C7 and L1-L6, respectwely) were sectioned on the nght side next to the gangim Dorsal roots were then resected on part of their trajectory to prevent possible regeneration Care was taken to leave the ventral roots undamaged. The overlying muscle and skin were then sutured back m place and the rats allowed to recover for one, four, seven, 14, 21 or 28 days. Effect of rhizotomaes on [~25I]FK-33-824 binding was analysed by quanhtatlve radmantography and significance assessed using pmred Student's t-test, Tissue preparaUon. Nmve (n = 3) or deafferented (n = 3 per survival penod) rats were decapitated and their whole spinal cords were rapidly removed on ice Cords from nmve anunals were divided into 15 segmental blocks m order to sample the cerxacal and lumbar enlargements, as well as the upper and lower thoracic and sacral segments (three blocks per segment) In lesioned rats, the deafferented segment (cervmal or lumbar), as well as regions rostral and caudal to it were &vided into five blocks each All blocks were frozen m 2-methyl butane ( - 4 5 ° C ) and kept at - 8 0 ° C until needed Coronal sectmns (20ttm thick) were cut on a cryostat at - 17°C, thaw-mounted on gelatin-coated glass shdes, desiccated at 4°C under vacuum for 2 h and then kept at - 8 0 ° C until use Receptor bmdmg assay Secuons were warmed up to room temperature and immediately incubated m 50 mM Tns-HCI buffer (pH 7 4) at 25°C for 60 man with [mI]FK-33-824 at a final concentratmn of 0 5 nM (about 2 x l0 s d.p m/ml)

Ad&tlonal secnons were mcubated in the presence of 10 ,u M naloxone to assess nonspeclfic binding Competition experunents were carned out on sections from naxve rats whereby increasing concentrations (3.0 100nM) of unlabelled (oAla 2, MePhe 4, Gly-olS)-enkephahn (DAGO), a selectwe mu agomst, ~9'45 were added to the mcubatmn medmm At the end of the incubation, shdes were transferred sequentially through six rmses of 1 mm each in Tns-buffer at 4~-C, and rapidly &pped m cold dlstdled water to remove excess salts Sections were then either mr-dned or fixed by lmmersmn (30mm) m an me-cold 3 5% glutaraldehyde solution in 50mM phosphate buffer m preparatmn for film or hqmd emulsmn radmautographic processing, respectwel~¢ A m d n e d sectmns were then apposed to tntium-sens~tlve films and exposed for two weeks In X-ray cassettes at room temperature F~lms were developed 64 and quantitated by computertzed unage analys~s (Eyecom 11, Spatial Data Systems, Melbourne, FL)63 For anatomical accuracy, the &fferent Rexed laminar subdw~slons66 were dehneated on the corresponding N~ssl-stamed sectmns based on diagrams by Paxinos and Watson, 6° and Molander et al ~4 Densltometnc measurements were performed within each layer and data were converted into equivalents of nCl bound per mg of tissue using standards 63 Glutaraldehyde-fixed sectmns were dehydrated m graded ethanols, defatted m xylene and rehydrated m an reverse series of ethanols as described. ~7 Sections were then coated by &ppmg m Kodak NTB-2 emulsmn diluted 1 1 with &stilled water and exposed for four to six weeks m hghttxght boxes at 4~C Exposed sectxons were developed m freshly prepared D-19 (4ram at 17~C), fixed m Kodak Ektaflo, stained with Cresyl Vmlet and cover-shpped with Permount RESULTS

Characteristws o f [125I]FK-33-824 bmdmg Previous kineUc a n d c o m p e t i t m n stu&es have s h o w n t h a t ['25I]FK-33-824 is a selectwe m u o p i m d receptor agomst which binds with a n a p p a r e n t dissociation c o n s t a n t (KD) of approximately 1 n M to rat brain sectmns. 36 38,55 Figure 1 confirms t h a t [~"5I]FK33-824 selectwely labels m u sites since D A G O , a selectwe m u agonlst, 39'45 competes with high affimty a n d in a c o n c e n t r a t i o n - d e p e n d e n t m a n n e r for all specific [12sI]FK-33-824 labelled sites. A s s u m i n g t h a t the K o value o f the binding of this ligand to sections o f rat spinal cord is similar to t h a t f o u n d in the brain, approximately 33% of available m u sites m a y be expected to be labelled at the c o n c e n t r a t m n (0.5 n M ) used m the present study

Radioautographw dtstributton binding in rat spmal cord

of

[125I]FK-33-824

The overall d i s t n b u t l o n of [t25I]FK-33-824 binding in various segments of the rat spinal cord, as detected in film a n d h q m d emulsion processed ra&oautographs, IS illustrated in Figs 2 a n d 3-5, respectively. As prevmusly reported for o t h e r opiold h g a n d s (see Introductmn), [125I]FK-33-824.1abelled m u o p m l d binding rues were m o s t heavdy c o n c e n t r a t e d m the superficial laminae of the dorsal horn, t h r o u g h o u t the length of the spinal cord (Figs 2, 3, Table 1). In addition, a n d m c o n t r a s t to m o s t previous reports, low to m o d e r a t e densities o f [~Sl]FK-33-824 binding were detected in laminae I I I - V I o f the dorsal horn, as welt as within

Mu oplold receptors m spinal cord

0 nM

5nM

199

~:~

-

"

7

10 nM

~,,v,~~...~,:~,

100 nM

"~:~

",-',z~:,""

Fig 1 Ra&oautographlc &strlbutmn of ['2SI]FK-33-824 binding sites m sections of the rat cerwcal spinal cord incubated m presence of increasing concentratmns of the selective mu op=old agomst, D A G O Note the progresmve &sappearance of the labelhng m both dorsal and ventral horns with the increasing concentratnons of D A G O A value corresponding to nonspeofic labelhng is observed in presence of 100 nM D A G O

the m t e r m e d l a t e gray m a t t e r a n d the v e n t r a l h o r n ( T 1 - L 2 , Figs 2, 3, T a b l e 1). This labelling w a s m o s t p r o n o u n c e d o v e r C l a r k e ' s c o l u m n ( T 1 - L 2 ; Figs 2, 3) as well as over l a m i n a I X at the level o f cervical a n d l u m b a r e n l a r g e m e n t s (Figs 2, 3; T a b l e 1). A t h i g h e r m a g n i f i c a t i o n o f liquid e m u l s i o n -

processed material, [12SI]FK-33-824 b i n d i n g within the dorsal h o r n was f o u n d to be particularly c o n c e n t r a t e d in l a m i n a I a n d in the t u n e r p o r t i o n o f l a m i n a II ( I h ) This was m o s t e w d e n t at l u m b a r levels, as illustrated in Fig. 4a,a'. M o s t o f the label was d e t e c t e d over the neuropil, a l t h o u g h c o n c e n t r a t i o n s o f g r a m s were also

Table 1 Quantitative laminar &stnbutnon of [t25I]FK-33-824 mu oplotd blndmg sntes at various segmental levels of the rat spinal cord Segment specific [t251]FK-33-824 binding (nCn/mg tissue) Spinal area I-II III IV V VI VII Clarke's column VIII-IX

C1 n =15

C5 n=20

T6 n =20

TI 1 n =12

L2 n = 18

L4 n =19

SI n=8

187+26 110_+36 49+25* -108-t-35

208+28 108_+29 36__+11 -83+4.0

202+33 82+19 33_+12 3 3_ 12 33_+12

206+22 91_+11 30-t-13 8 8 _+ 2 6 30-t-13

201+21 87-t-14 32+09 12 2 _+ 2 5 6l+15

212+27 111+32 28_+10

225+11 70_+23 41__+17

71_+26

112+16

Twenty-mlcrometre-thlck sections from each speofied segment of rat spinal cord were incubated with 0 5 nM [~25I]FK_33_ 824 and then autoradlograms from trmum-sensltlve films were analysed by computerized densltometry as described in the text Nonspeofic binding m the presence of 10#M naloxone was subtracted from all readings The averaged optncal densnty values ( m e a n + S D ) from (n) m&vldual determinations for each spmal segment (three to five consecutwe sections per shde, one to two shdes per segment) were converted to relatwe binding rote density values wtthm an area by reference to standards The lamination of the spinal cord was defined accordmg to Paxmos and Watson ~ or Molander et a154 C, cervical, T, thoracic, L, lumbar, S, sacral *The apparent density of [~25I]FK-33-824 sites is important m lamina V I m that spinal segment 12 8 + 4 3 nCl/mg tnssue The density of binding sites in lamina X is the same as observed m laminae V-VII m all segments At T6, areas V IX and Clarke's column are taken as one region

J

Fig, 2 Pseudocolour reconstruction of film radloautograms fllustratmg the distribution of [125I]FK-33-824-1abelled mu oplold bmdang sites m representative segments of the rat spinal cord Gray levels are contrast-enhanced and colour-coded from red (higher denstttes) to yellow, green and blue (blank values) Anatomical drawings on the right are based on camera-luclda drawings of corresponding Nlssl-stamed sections Nonspeclfic labelhng remaining m presence o f 10/aM naloxone is shown at the bottom right Labelhng m superficial lammae of the dorsal horn is evident at all segmental levels. Labelhng in the ventral horn is most pronounced at cervical, lumbar and sacral levels C, cervacal; L, lumbar, S, sacral, T, thoracic Numbers refer to segmental levels 200

Mu oplold receptors m spinal cord

201

Fig 3 Dark-field photomicrographs of sections from the rat spinal cord labelled with [125I]FK-33-824 and radloautographed using conventional dipping techniques High densities of sites are apparent m the superficial laminae of the dorsal horn (I, II) at all segmental levels Labelhng is also present m Clarke's column (T11) and in lamina IX of the ventral horn at the level of cervical and sacral enlargments C, cervical, L, lumbar, S, sacral, T, thoraoc

visible over certain neuronal perlkarya (Fig 5b) Discrete silver gram clusters were also apparent over cross-sectioned axons of incoming dorsal roots (Fig. 4c,c') Within Clarke's column, most of the label was similarly detected mainly over the neuropll in amidst the penkarya of spmocerebellar neurons In the ventral horn, [~25I]FK-33-824 binding was particularly dense in lamina IX, and mostly spared the perlkarya of large motoneurons (Figs 4b,b', 5a) At higher magnification, &screte silver grain alignments were apparent along the membrane of motoneuron perikarya and proximal processes (F~g 5)

Effectof dorsal rhtzotomy Unilateral dorsal rhlzotomles markedly affected [~25I]FK-33-824/mu oplold binding in the superficial laminae of the dorsal horn on the ipsllateral side, as shown m Fig 6 While no apparent decrease was seen one day post-lesion, marked decrements In specific [125I]FK-33-824 labelling were observed at four and seven days post-lesion, without significant recovery at least up to 21 days post-lesion (Fig 6) Histological examination of sections also supported the apparent absence of recovery from lesions (not shown) A quantitative analysis of the effects of dorsal rhizotomles

202

C GOLARD~RESet al

Fig 4 Light microscopic ra&oautograph~c distribution of[t~'~I]FK-labelted mu op~o]d binding sites m the dorsal horn (a, a'), ventral horn (b, b' ) and dorsal root (c, c' ) of the rat spinal cord a c Bright-field, a' c dark-field In a, a', the label ~s seen to pervade the entire substantla gelatlnosa, marginal layer and dorsat root entry zone Note that the inner layer of the gelatlnosa (Ih) is more intensely reactive than the outer zone (Ilo) In b, b', moderate labelhng densities are detected m the lateral aspect of the ventral ho)n (lamina IX) at the level of cervical and lumbar enlargements Note that the penkarya of motoneuron~ are virtually devoid of labelling (arrows) In c, ~', clusters of silver grains are apparent over the cross-sectioned dorsal root fibres close to their entry in the spinal cord These labelled binding sites presumably correspond to receptors m transit from the dorsal root ganghon at cervical a n d l u m b a r levels is summartzed in Tables 2 a n d 3 A t the cervical level (C5), [L~SI]FK-33-824 binding was virtually abolished from the superficial laminae o f the dorsal h o r n following rhlzotomies. In fact, less t h a n 7 % o f the original labelling was still present in these l a m i n a e seven days post-lesion There was an a p p a r e n t , a l t h o u g h no significant, recovery by day 28 with values reacbang less t h a n 18% of controls Binding m laminae III a n d IV was also affected by the

rhtzotormes, although to a lesser extent than m superfictal laminae. It also showed a more r o b u s t recovery by the f o u r t h week (Table 2). Labelling m the ventral h o r n was not affected by dorsal rbazotomies (Table 2) Likewise, outside o f lesioned areas (C 1/2 a n d C8/T1), [t25I]FK-33-824 binding was not altered, even m superficial dorsal h o r n laminae (not shown). Similar results were observed following dorsal rhlzotomles at the l u m b a r level (Table 3) [~zSI]FK-33-824 binding losses were restricted to laminae I - I V a n d

Mu opioid receptors in spinal cord

Fig. 5. Cellular localization of ['251]FK-labelledmu opioid binding sites in rat spinal cord. (a) Ventral horn. Silver grains are detected throughout layer IX where they appear to be confined to the neuropil surrounding motoneurons. Some may be found aligned along longitudinally sectioned motoneuronal processes (arrowheads). (b) Dorsal horn. Labelling in the substantia gelatinosa is apparent over both perikarya and neuropil. Note that some of the nerve cell bodies (arrows) are more heavily labelled than others.

became evident by day 4 post-lesion with no apparent significant modifications thereafter at least up to 28 days post-lesion (Table 3). However, the proportion of [Iz5I]FK-33-824associated with primary afferent fibres is apparently smaller at this level since maximal decreases in binding reached only 70% by day 7 (as opposed to 90% at cervical level; Table 2) of control values in superficial laminae (Table 3). Proportionally similar results were also obtained in laminae 111-IV (Tables 2, 3). In contrast, labelling in Clarke's column, at L1-L2 level, was not affected by dorsal root sections (not shown). DISCUSSION

This study confirms the presence of relatively high densities of mu opioid binding sites in the superficial layers of the dorsal horn and in the ventral horn, and provides the first demonstration of the existence of these sites in Clarke's column. Through the use of a high-resolution radioautographic t e c h n i q ~ e , ~the ~ - present ~ ~ . ~ ~results also revealed that within the ventral horn, mu opioid

binding is confined to lamina IX, where it pervades the neuropil surrounding motoneuron perikarya. These findings provide an anatomical substrate for a regulatory role of opioid peptides in motor function. Nature of ["'I]FK-33-824 binding

That [1251]FK-33-824selectively labels mu opioid sites in the rat spinal cord is supported in the present study by the loss of radioautographic labelling observed in all laminae when using the selective mu agonist, DAGO, as a competing agent.39*45 These results are in keeping with those of earlier studies carried out in other regions of the CNS which have shown that at the concentration used (0.5-1 nM), this radioligand was unlikely to significantly label other classes of opioid recept~rs.~*>~~." The generally broader radioautographic distribution of mu sites reported in the present as compared to earlier reports (see Introduction) is probably due to the higher specific activity, proteolytic stability and specific over total binding ratio (>go-95%) of our ligand28,36-38~52~55 as well as to the absence of quenching since it is i~dinated.~] These results illustrate the

204

C (JOLARDERES CI tl/

ld

4d

7d

21 d Fig 6 Effects of right dorsal rhlzotomy on the distribution of ['251]FK-33-824 binding in the rat cerwcai spinal cord at C5 level (middle of the deafferented area) one, four, seven and 21 days, post-lesion Note that by day 4, the densmes of labelhng are already decreased m laminae I and II of the dorsal horn By the seventh day, labelling in these superficml laminae has practically disappeared

i m p o r t a n c e o f using high-affinity, selective lodinated p r o b e s to d o c u m e n t the distribution of n e u r o t r a n s mltter receptors in those areas that c o n t a i n only low densities o f sites,

[125I]FK-33-824/mu opwld sttes m the substantta gelatmosa As reported earher, 3,78,16,22.32,47,59,68,76,8182 m u oplold binding sites were f o u n d to be highly c o n c e n t r a t e d in superficial laminae (I, II) of the dorsal horn. The

s u b s t a n t l a gelatinosa was enriched with m u sites along its entire rostrocaudal extent, with no evidence of segmental e n r i c h m e n t The high-resolution radioa u t o g r a p h i c technique used in the present study allowed a l a m i n a r heterogeneity in the distribution o f m u oplold binding within the s u b s t a n t i a gelatmosa to be distinguished This was most evident at the level of the l u m b a r e n l a r g e m e n t where labelling densities were clearly higher in the inner (IIl), as c o m p a r e d to the outer p o r t i o n o f l a m i n a II (IIo). Previously, a

Table 2 Relative effects of dorsal rhlzotomy on [125I]FK-33-824 mu oploid specific binding in the cervical spinal cord of the rat Days after rhlzotomy Laminae

1

4

7

14

21

28

I-II Ill-IV VIII-IX

102.8 + 2 2 108.3___75 102.1__+141

48 1 _+ 3.9*** 69.6+3.9*** 1263+119

7 1 __+2 4*** 235_+79** 947+174

12 0 + 3 2** 313_+78"** 893+80

10.2 _.+0 8*** 402+100" 94.6+76

18 3 _+ 1 9*** 426_+54*** 938+58

Binding densities were analysed at the level of segment C5 (middle of the deafferented area) at various times post-lesion The results are expressed as percentage of the specific binding density of the control contralateral side' means + S.E.M of six determinations for each survival period However, a very recent study 7°a has suggested possible slight decreases of opioid receptors on the contralateral side following unilateral rhizotomies Thus, greater decreases than those reported here may be found if compared to data obtained from naive animals *P < 0.05, "*P < 0.01, ***P < 0.001 compared to control side Moreover, a two-way ANOVA of the above results from four to 28 days and within laminae 1-IV indicates that: (i) for each survival period, although significant, the decrease in laminae Ill-IV is less pronounced (P < 0 01) than in laminae I-II, (n) for each spinal area, the results are not statistically different between seven to 28 days post-lesion, although being significantly distinct (P < 0.001) from four day values

Mu oplold receptors in spinal cord

205

Table 3 Relative effects of dorsal rhlzotomy on [125I]FK-33-824 mu oplold specific binding in the lumbar spinal cord of the rat Days after rhlzotomy Laminae

1

4

7

I I1 III IV VIII IX

101 7 + 1 2 1036+34 1109+65

42.2+44"** 74.2+85* 1041+102

293+32"** 805+44*** 1081+52

14 385+29"* 590+71" 912+89

21

28

384+48"** 543+99* 888+126

386+43"** 618+19"** 968+67

Binding densities were analysed at the level of segment L4 (middle of the deafferented area) at various times post-lesion The results are expressed as percentage of the specific binding density of the control contralateral side means + S E M of six to 29 determinations for each survwal period However, a very recent study TM has suggested possible shght decreases of oplold receptors on the contralateral side following umlateral rhlzotomles Thus, greater decreases than those reported here may be found if compared to data obtained from nawe animals *P < 0 05, **P < 0 01, ***P < 0 001 compared to control side Moreover, a two-way ANOVA of the above results from four to 28 days and within laminae I IV indicates that (1) for each survwal period, although significant, the decrease m laminae III IV is less pronounced (P < 0 01) than m laminae I II, (11) for each spinal area, the results are not statistically &fferent from four to 28 days post-lesion similar distribution o f oplo~d binding sites h a d been reported in h u m a n , 22 but to o u r knowledge neither m rat~, ~8,32,588182 n o r m m o n k e y 47~5° spinal cord. In this context, it should be recalled that n e u r o n s located in II0 (stalked cells a n d some of the Islet cells) are either noclceptlve-speclfiC or multlreceptlve whereas those present in IIi are only a c t w a t e d by noxious stimuli. ~2 Moreover, within l a m i n a II, the finely m y e h n a t e d afferents, supposedly A3 fibres with high-threshold m e c b a n o r e c e p t o r s , a p p e a r to terminate preferentially w~thm II056 whereas the u n m y e h n a t e d C fibre afferents have been claimed to be the only primary afferent fibers to t e r m i n a t e in lamina II1, this g r o u p of C fibres originates largely, ff not exclusively, from the skin 5J 56 The intense [~25I]FK_33_824 labelling observed m sublayer 1h, together with the massive d r o p in binding observed after r h l z o t o m y (see b e l o w ) c o u l d therefore imply t h a t the majority of m u sites detected in layer II (at least in the l u m b a r region) are associated with i n c o m i n g C-fibres This i n t e r p r e t a t i o n also c o n f o r m s with o u r o b s e r v a t i o n t h a t within the s u b s t a n t i a gelatmosa, m u oplold binding sites are mainly assocmted w~th elements o f the neuropll, G w e n the p u r p o r t e d opposite role of substance P a n d oplold-related peptldes in the m o d u l a t i o n of pain transmission, 4°788° it is o f interest to c o m p a r e the distribution of these two classes of substances with that of opIold receptors in the superficial laminae of the dorsal h o r n Substance P-lmmunoreactwe fibres in rat dorsal h o r n are f o u n d to be mainly c o n c e n t r a t e d within the outer, as opposed to the tuner p o r t i o n of lamina II. 4~ This is in c o n t r a s t to the & s t r l b u t l o n of m u oplold binding sites which were s h o w n here to be m o r e heawly c o n c e n t r a t e d over layer Ill t h a n over layer Iio G i v e n the evidence for a p r e d o m i n a n t association of m u sites with i n c o m i n g C fibres, m a n y of which have been s h o w n to c o n t a i n substance p 4 2" 42 ~,46,61 a n d the d e m o n s t r a t i o n of oploxd binding sites over substance P - l m m u n o r e a c t l v e dorsal root ganglion cells, 59 this discrepancy is m o r e likely to reflect a lack o f association of m u receptors with substance P-containing fibres o f central r a t h e r t h a n peripheral origin Peptldes derived from all three k n o w n oplold pre-

cursors I are also highly c o n c e n t r a t e d in the substantaa gelatlnosa o f the spinal cord ~3~2735466173 There ~s general agreement t h a t e n k e p h a h n - a n d d y n o r p h l n related pept~des are m o r e a b u n d a n t t h a n POMC-/3endorphln-derlved ones ~4 ~ 27536~80 Moreover, i m p o r t a n t segmental differences in the c o n c e n t r a t i o n s of these various oplold peptldes have been rep o r t e d t0536183 In the present study, we did not o b t a i n clear evidence for the existence of a rostrocaudal s e g m e n t a t i o n of m u oplold receptors In the s u b s t a n t l a gelatlnosa This is in c o n t r a s t to o t h e r oplold receptor classes, especially the k a p p a 2 sub-type 20~2 These resuits could be taken to indicate that different oplold peptldes act as e n d o g e n o u s h g a n d s at the m u oplold receptor visualized here The precise nature of the e n d o g e n o u s hgand(s) which physiologically actlvate(s) m u oplold receptors remains to be firmly established, but pharmacological d a t a point t o / / - e n d o r p h l n and to some of the C-terminal extended forms o f the e n k e p h a h n s as prime candidates ~46,, ~7~, A large p r o p o r t i o n of enkephalln- a n d d y n o r p h m l m m u n o r e a c t w e n e u r o n s in the substantla gelatlnosa are part of an extensive network of p r o p n o s p l n a l a n d / o r ascending projection n e u r o n s t~ t~27~46,~6~ ~ Since a large p r o p o r t i o n of m u oplold binding sites disappears following rhlzotomy, it is unhkely that many, if any, of these c o r r e s p o n d to autoreceptors associated with these cells Dorsal rhtzotomy and mu receptors

In

agreement

with

the

results

of

prewous

studies, 78t°lTa7585~82 unilateral dorsal root sections

were f o u n d to ehclt a marked, lpsllateral loss of m u OplOld receptors in the dorsal h o r n of the spinal cord. G i v e n its relatively rapid onset, this loss is unlikely to result from transneuronal degeneration, which should theoretically not occur before three weeks post-les~on (see Ref. 82 and references therein) Thus, it seems that a large n u m b e r o f m u opioid receptors are associated with primary afferent fibres a n d / o r axon terminals, especially in laminae I a n d II However, a p r o p o r t i o n of m u sites also appears to be located postsynaptlcally since a small percentage of [~28I]FK-33-824 binding is still present in laminae I II, a n d more evidently in

206

( GOUARDI~RESet al

laminae Ill-IV, following dorsal rhizotomy at both cervical and lumbar levels In these two segments, the tame-course of the effects of the rhmotomy was slmdar although the reductions m mu sites were always more pronounced m the superficml (I-II) than m the deeper (III-IV) laminae Highly significant losses in [125I]FK-33-824 binding were already evident four days post-lesion, but maximal reductions (up to 82-93%) were observed after seven days. Later there was an apparent, partial recovery, although this was highly variable and not consistent from one lamina and/or segment to the next. No evidence of [125I]FK-33-824 receptor losses was seen at any time in deeper laminae (VIII-IX) or in Clarke's column, indicating that within these areas, mu opioid binding sites are unhkely to be associated to any large extent with primary afferent fibres Large-diameter myehnated primary afferent nerve fibres send projections mainly to laminae III and IV of the dorsal horn whilst, as already stated, the majonty of A6 and C fibres terminate within layers I and II) 2'24,34,43'56'69 Consequently, the results of the present dorsal rhizotomy experiments suggest that a major proportion of mu binding sites detected in the rat dorsal horn is associated with small dmmeter A3 and C fibre terminals, and a minor, but still sigmficant, proportion is present on large diameter fibres terminating m layers III-IV 2434 It could be argued that residual binding sites labelled in the dorsal horn following dorsal rhizotomy are associated with primary afferent fibres originating from adjacent segments that were not deafferented However, these fibres are generally relatavely short and do not project to more than three segments. 9'12'24'25'34'69 Thus, following large deafferentations (C3-C7, L1-L6) such as the ones performed here, it could be assumed that at the centre of the lesion (C5 or L4), inputs from adjacent segments are mimmal (but see Ref. 9). The remaining percentage of binding sites (10-40%) therefore probably truly reflects the labelling of postsynaptic mu opiold receptors. This interpretation 1S supported by the observation that numerous spmothalamic neurons receive direct enkephallnergm inputs m the superficial layers of the dorsal horn 67and by the recent demonstration of a direct postsynaptlc effect of morphine in the substantla gelatinosa of deafferented rats. l° [~25I]FK-33-824/mu and I X

opiotd sites

m

lammae

VIII

A major finding of the present study is the detection of significant quantities of mu opioid binding sites in Clarke's column and in lamana IX of the ventral horn This relatively heavy labelling was in contrast to the virtual absence of mu binding in the intermediate cell column of the thoracic segment. The later result suggests that this receptor class is unlikely to be directly involved m the integration of sympathetic autonomic functions at the spinal level. 25[~25I]FK-33-824bindmg appeared somewhat more intense at the level of the

sacral mtermediolateral cell column indicating that endogenous oplolds may be involved m the regulation of parasympathetic tone. 2~ In this regard, it is of interest to note that opiates and opioid peptldes can modulate pelvic visceral organ function and micturltion reflex in animals ~ and humans. 6" The recent demonstration of the presence of dynorphm B-like lmmunoreactive materials m sacral primary afferents terminating in deep ventral laminae~ could also relate to some of these behavioural effects Labelhng m lamina IX was found by high-resolution radloautography to mostly spare the perlkarya of motoneurons, suggesting that mu opiold receptors within the ventral horn are not directly associated with these cells Admittedly, the presence of silver grain alignments along the perikarya and proximal dendrites of certain motoneurons could be taken as an indication of the presence of opiold receptors on the membrane, if not m the cytoplasm of these cells. However, evidence from studies carried out on dorsal root ganglia59 or elsewhere m the neuraxls3~38 suggests that neurons which express mu opiotd receptors should exhibit internal labelling under the present experlmental conditions Labelled sites detected in the immediate surroundings of lower motoneurons are therefore more likely to be located either on interneurons or on descending projection fibres. They are unlikely to be located on primary afferent fibres terminating m the ventral horn gwen the results of the present deafferentation expenments They could, however, be assocaated with coeruiospmal axons wtuch are known to tenmnate on motoneurons7~ and the cell bodies of which have been shown to express high levels of mu oplold receptors? s'4°'527576 This projection has been shown to be implicated m the control of locomotion7~ and is sensmve to opiates "~ In any event, the fact that mu oploid binding sites were particularly concentrated in the lateral segment of lamina IX, especially at the cervical level, suggests that opioids may be important m the regulation of limbs, as opposed to postural/trunk m o v e m e n t s ) 4"25'7) [~2SI]FK-33-824 binding in Clarke's column was, as m the ventral horn, mainly associated with the neuropd. For the reasons stated above, mu opiold receptors are therefore likely to be associated either with interneurons or with incoming afferent axons. Clarke's column is known to receive proprlospinal primary afferents from muscles. 14 These fibres are unlikely to carry mu opioid binding sites, however, given the absence of decrease of [125I]FK binding m tbas structure following dorsal rbazotomy. Nonetheless, the presence of relatively high concentrations of mu receptors throughout Clarke's column prowde further support for a role of endogenous opiolds in sensory motor integration. CONCLUSION It is clear that mu oploid receptors are more broadly distributed in the rat spinal cord than was

Mu oplold receptors in spinal cord originally described. In addition to the high densities o f sites located on primary afferents terminating m the substantia gelatinosa, m u sites are also f o u n d m relatively high c o n c e n t r a t i o n s in lamina IX o f the ventral h o r n as well as in Clarke's column. These findings suggest that m addition to the m o d u l a t i o n o f noclceptlve reformation, m u oplold receptors are associated with s e n s o r l m o t o r integration in various segments o f the spinal cord

207

Acknowledgements--This research was supported by grants from the Medical Research Council of Canada (MRC to A B and R Q ) and the CNRS, France (to J C ) C G was sponsored by a Visiting Scientist award from the MRC and the CNRS A B holds a MRC Soentlst award and R Q and "Chercheur-Boursler" award of the "Fonds de la recherche en sant6 du Qu6bec" The help and expert counselling of M Jean R~chard w~th the dorsal rh~zotomy ~s gratefully acknowledged The expert secretarial assistance of Mrs J Currie is acknowledged

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2()9

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