Brain Research, 578 (1992) 115-121 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
115
BRES 17629
Time-related decreases in/z and 6 opioid receptors in the superficial dorsal horn of the rat spinal cord following a large unilateral dorsal rhizotomy D. Besse, M.C. Lombard and J.M. Besson Unitg de Recherche de Physiopharmacologie du Syst~me Nerveux (INSERM, U. 161) and Laboratoire de Physiopharmacologie de la Douleur, Ecole Pratique des Hautes Etudes, Paris (France)
(Accepted 26 November 1991) Key words: Quantitative autoradiography; Receptor down-regulation; Deafferentiation; Degeneration; Thin primary afferent fiber
The aim of the present study was to measure the time-related modifications of p and 6 opioid binding sites in the superficial layers of the dorsal horn of the rat spinal cord after a C4-T2 unilateral dorsal rhizotomy. Using specific ligands, namely [3H]DAMGO for/~ sites and [3H]DTLET for 6 sites, and a quantitative autoradiographic analysis, we have observed: (a) a decrease in binding on the ipsilateral side to the lesion as early as the first day postrhizotomy, the maximal loss being attained at 8 days postlesion, (b) after 8 days postlesion, the residual binding remains stable over the period of analysis (90 days), (c) the loss of ~ receptors (71-74%) is significantly more pronounced than the loss of 6 receptors (57-62%) and (d) affinities of postsynaptic/~ and 6 receptors are similar to those of the total receptor population in the superficial layers of the dorsal horn. Comparison of these results with the degeneration of primary afferent fibers reported in literature favors the localization of the majority of/~ and 6 opioid binding sites on fine diameter primary afferent fibers. INTRODUCTION The decrease in opioid receptors following dorsal rhizotomy was initially described by L a M o t t e et al. 22 in the superficial dorsal horn of the spinal cord of rhesus monkey. M o r e recently, using a u t o r a d i o g r a p h y of selective opioid ligands and large dorsal rhizotomies, several studies 2'15'a5 have r e p o r t e d quantitative data on the decrease of/~, 6 and/or r opioid receptors in the superficial layers (laminae I - I I ) of the rat dorsal horn. I n d e e d , at the level of the C 7 segment, we have shown that, 8 days after a unilateral C 4 - T 2 dorsal rhizotomy, the presynaptic c o m p o n e n t s of k~ and 6 opioid receptors were 76 and 61%, respectively. Several lines of evidence suggest that the presynaptic c o m p o n e n t of opioid receptors can be associated with fine d i a m e t e r p r i m a r y afferent fibers. (i) Thin A 6 and C p r i m a r y afferent fibers essentially project in laminae I and 111'14'18'22'23'30'32'34'40-42 which contain the highest levels of opioid binding sites. (ii) A massive loss of opioid binding sites in laminae I - I I is observed after neonatal t r e a t m e n t with capsaicin 8'11'28. (iii) In contrast, large Aft and A 6 hair follicle fiber arborizations are in laminae III, IV and V (see refs. in ref. 10) in which the density of opioid binding sites is very low. Therefore, we have m e a s u r e d by a u t o r a d i o g r a p h y the
modifications of kt and 6 opioid receptors related to the postrhizotomy delay (1-90 days) and we have c o m p a r e d our results with previous qualitative anatomical studies dealing with d e g e n e r a t i o n of primary afferent fibers in the superficial layers of the dorsal horn. MATERIALS AND METHODS Surgery Experiments were performed on 31 Sprague-Dawley albino rats, weighing 225-250 g. The surgical procedure was performed using ketamine (100 mg/kg i.p.) as general and xylocaine (2%) as local anesthetics. A dorsal hemilaminectomy of the C3-T 2 vertebrae was made on the right side and the C4-T2 dorsal roots were sectioned intradurally proximal to the ganglia, through small individual openings of the dura mater, under a dissection microscope. Care was taken to cause no damage to the blood vessels running along the dorsal roots. The wound was washed with saline and the muscles and skin sutured, layer by layer. Delays postrhizotomy were: 1, 2, 4, 8, 14, 30 and 90 days. Lesions were performed so that the animals were all the same age on the day of sacrifice. Three animals were used for each group. Three additional intact rats were included as controls. Saturation experiments were performed using 6 intact rats and 8 C4-T 2 rhizotomized rats (8 days postoperative). At the end of the survival time, rats were sacrificed by decapitation. Cervicothoracic C4-T2 spinal cord was removed rapidly, frozen in isopentane at -40°C and preserved at -80°C. Autoradiographic procedures Levorphanol (tartrate; SIGMA), [3H]DAMGO (Tyr*-D-Ala-GlyNMe-Phe-Gly-ol) (2.1 TBq/mmol; C.E.A. Saclay, France) and
Correspondence: D. Besse, Physiopharmacologie du Syst6me Nerveux (INSERM U161), 2 rue d'Al6sia, 75014 Paris, France.
116 [3H]DTLET (Tyr*-o-Thr-Gly-Phe-Leu-Thr) (2.15 TBq/mmol; C.E.A. Saclay, France) were used in this study. Entire C4-T2 extents were cut (frontal sections 15 pm thick) on a cryostat (-20°C), thaw-mounted onto gelatinized slides and kept at -80°C up to incubation. Two sets of slides each bearing adjacent sections were made: one for [3H]DAMGO, the other for [3H]DTLET. Sections were brought to room temperature and incubated in 350 ml of 50 mM Tris-HCl buffer (pH 7.4) at 25°C for 60 min with either 3 nM [3H]DAMGO to label p sites or 3 nM [3H]DTLET to label 6 sites. The non-specific binding was determined using some spinal cord sections under the same conditions of incubation, in the presence of 1 pM levorphanol. At the end of the incubation, spinal cord sections were washed twice in 350 ml of ice-cold 50 mM TrisHC1 buffer (pH 7.4) for 10 min each, rinsed in ice-cold bidistillated water for 2 s and air-dried. For saturation experiments, spinal cord sections from C6 to C8 were successivelythaw-mounted onto gelatinized slides so that, for a given ligand, adjacent sections corresponded to different concentrations. Four sets of 8 slides were made corresponding to: total and non-specific [3H]DAMGO binding and total and non-specific [3H]DTLET binding. For both ligands, about 10 sections per rat were used for each of the 8 concentrations from 0.5 to 24 nM. Slides were kept at -80°C up to incubation. For incubation, slides at room temperature were put in incubation chambers. Spinal cord sections were incubated for 60 rain in 600 pl of 50 mM Tris-HCl buffer (pH 7.4) containing the tritiated ligand with (non-specific binding) or without (total binding) 1/~M levorphanol. At the end of the incubation, sections were washed twice in 350 ml of ice-cold 50 mM Tris-HCl buffer (pH 7.4) for 10 min each, rinsed in ice-cold bidistillated water for 2 s and air-dried. Labelled sections were then juxtaposed against tritium-sensitive films (Hyperfilm-3H, Amersham France, Les Ulis) and exposed for 15 weeks at 4°C. Autoradiograms were developed in Kodak D19 for 2-3 rain at 20°C, fixed for 5 min, washed for 20 min under running water arid dried. Autoradiograms were analysed by densitometry using a Biocom 200 analyser system (Biocom, France). According to Nissl poststaining sections, we measured binding in the area corresponding to laminae I and II of Rexed's classification as defined for the rat spinal cord26'38. Grey levels measured on autoradiograms were converted into binding site concentration values by reference to tritium standards (Amersham). Specific binding was calculated by subtracing, from total binding, the amount of binding measured from sections incubated for non-specific labeling. Except for the saturation study, measurements of receptor concentrations were performed every 160 pm, from C4 to T2 segments, on the intact side and on the rhizotomized side. This procedure gave a large number of values (7-10 in each spinal segment) and allowed detection of any differences in binding in successive spinal segments to be observed. For an initial analysis, results were expressed as specific binding concentrations (fmol/mg of tissue) on each side. In a second part of the study, results were expressed as ratios of binding measured in the rhizotomized side compared to the intact side. These ratios were compared between the different experimental situations. The 'time of half-decrease' (qr2) was defined as the time of 50% of the maximal decrease in binding on the rhizotomized side compared to the intact side. In saturation experiments, binding parameters at equilibrium (dissociation constant = Kd and specific binding capacity = Bmax) were determinated from the linear regression analysis of Scatchard. For each rat, each point relative to a given concentration corresponded to the mean of binding values obtained from about l0 spinal cord sections. Determination of successive spinal segments was made using an atlas constructed from Nissl-stained spinal cord sections. The statistical analysis was performed using either a Student t-test or a variance analysis (ANOVA), followed by the LSD multiple range test (Least Significant Difference).
RESULTS In intact rats, autoradiograms reveal [ 3 H ] D A M G O and [3H]DTLET specific binding mainly concentrated in the superficial layers of the dorsal horn (Fig. 1). At this level, /~ binding sites are p r e d o m i n a n t compared to 6 binding sites. For [ 3 H ] D A M G O as well as for [3H]DTLET, binding is similar in both right and left sides (see Table I for the C7 segment). The non-specific binding is constant all along the C4-T 2 extent and corresponds to about 10 and 20% of the total binding for [ 3 H ] D A M G O and [3H]DTLET, respectively. In rhizotomized rats, autoradiograms revealed a loss in ~ and 6 binding on the rhizotomized side compared to the intact side (Fig. 1). The decrease in specific binding becomes more and more p r o n o u n c e d until 8 days postlesion (P.L.). For longer delays (15-90 days), there is no further significant decrease in either ~ or 6 binding. Specific binding values for the central C7 segment are reported in Table I. As regards specific binding values, on the intact side of rhizotomized rats, /~-specific binding for when considering the nificant variation in p
there is a tendency of decrease in some particular delays. However, delays altogether, there is no sigas well as 6 specific binding on the
intact side ( A N O V A : F7.16 = 1.10 for [ 3 H ] D A M G O and F7a6 = 0,90 for [3H]DTLET). O n the rhizotomized side, a significant decrease in binding is observed when compared to values of intact rats ( A N O V A : F7.16 ~-- 19.71 for [ 3 H ] D A M G O and F7,16 = 11.26 for [3H]DTLET). In fact, for [ 3 H ] D A M G O as well as for [3H]DTLET, a comparison of binding values for each group with that of intact rats reveals significant decreases after 2 days P.L. Considering ratios, a significant effect is revealed as early as the first day. For both ligands, decreases in the ratio are related to the postlesion delay until 8 days. After this time, no further loss of binding is observed up to 90 days P.L. Fig. 2 illustrates the time course o f p and 3 binding ratios in the central Cv segment. For short survival delays (1-3 days), the time-courses are similar for both p and 6 opioid receptors, However, a noticeable difference between the decreases in p and 6 receptors appears in the 3 - 8 days interval. Thus, the decrease in 6 receptors reaches a minimal ratio of about 0.40 while the decrease in p receptors is more marked, the minimal ratio being about 0.28. Delays corresponding to the 50% decrease of both /~ and 6 sites (see Materials and Methods) are ttr2 = 2.5 days. From 8 to 90 days, the proportions of remaining p receptors (0.26-0.29) persist at lower levels than those of 6 receptors (0.390.43). The m e a n difference between the two types of receptors is 13% (P < 0.001, Student-t = 7.7, df = 22) in the C7 segment.
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118 TABLE I Specific binding values for 3 nM [3H]DAMGO and 3 nM [3H]DTLET in laminae l-II of the C7 segment according to various delays following a C4-T2 right dorsal rhizotomy Values are expressed as mean concentration (fmol/rng tissue) + S.E.M. (n = 3 rats). Ratios correspond to rhizotomized side/intact side binding values. Intact
[3H]DAMGO Left Right Ratio [3HIDTLET Left Right Ratio
Rhizotomized 1
2
4
8
15
30
90days PL.
35.7±4.9 35.5±5.0
35.5±3.0 31.9+2.2
33.4+5.6 24.5+3.9 a
37.9±2.4 17.5±1.4 b
27.4+4.1 7.9-+2.0 c
25.5+1.6 7.4±0.4¢
31.9±7.1 7.4+0.3 c
25.2+6.6 6.9±0-8c
1.00+0.02
0.90+0.03 a
0.74+0.01 c
0.46+0.01 c
0.28-+0.03c
0.29+0.02 ¢
0.26+0.05 c
0.28+0.04¢
11.0+1.9 11.0+1.9
14.2+0.7 13.1+0.7
9.9+1.3 6.6+0.9 b
10.0±1.1 5.5±0.6¢
8.5+0.3 3.2+0.2 ¢
12.2+2.3 4.8-+1.1 c
9.1±1.1 3.6+0.3 c
10.6+1.1 4.6-+0.6 c
1.00+0.05
0.89+0.02 b
0.68+0.01 c
0.55+0.04 ¢
0.39+0.04 c
0.39±0.02 c
0.40+0.02 ¢
0-43+0.01¢
ap < 0.05, bp < 0.01 and eP < 0.001 as compared to data obtained in intact rats, for left (intact) side, right (rhizotomized) side and ratios (ANOVA followed by LSD test).
In order to detect any variation along the rostrocaudal axis of the spinal cord of either [ 3 H ] D A M G O or [3H]DTLET specific binding, we performed a detailed analysis over the C4-T 2 segments (Fig. 3). Considering each survival time, a progressive decrease in binding is
1.00
-
-7-
DTLET DAMGO
though quite similar effects are found in C6 and C s. Indeed, statistical analysis of ratios corresponding to C 6, C 7 and Ca segments does not reveal significant variation at any survival delays. In order to check whether the observed decrease in binding is due to a loss of sites or to a change in their affinity, saturation experiments were performed for intact and rhizotomized rats at 8 days E L . Binding characteristics, obtained in C6-C 8 segments (Table II), demonstrate that: (i) there is no significant variation in either
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observed over all the 7 segments. As expected, the C 7 segment corresponds to the level of maximal effect, al-
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# or 6 receptor affinity after unilateral C4-T 2 rhizotomy, (ii) specific binding capacities on the intact side of rhizotomized rats are similar to those of intact rats, (iii) specific binding capacities on the rhizotomized side are 62 and 55% l o w e r ' t h a n on the intact side for/a and 6 binding sites, respectively.
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DISCUSSION
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Earlier investigations2'8A5'22'29'45 have shown a reduc0.00
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post-lesion delay (days) Fig. 2. Diagram showing the time-related decrease of 3 nM [3H]DAMGO and 3 nM [3H]DTLET specific binding on the rhizotomized side compared to the intact side. Measurements were performed at the C7 segment level, in laminae I-II of the dorsal horn, after a unilateral C4-T2 dorsal rhizotomy. Note that the maximal decrease for both [3H]DAMGO and [3H]DTLET binding is obtained after 8 days and that the half maximum effect titz is observed after 2.5 days. A significant difference between ratios of [3H]DAMGO and [3H]DTLET binding is observed from 4 to 90 days EL.
tion of opioid receptors in the dorsal horn after dorsal rhizotomy. The aim of the present study was to analyse the time-course of the decreases in # and 6 binding sites after a C4-T2 dorsal rhizotomy. From the present study several points arise, some of them being comparable to those obtained with [125I]FK-33-824 U-selective ligand) after a less extensive lesion in the rat 15. These points are: (a) both # and 6 receptor decreases begin as early as the first day postrhizotomy, (b) up to 3 days, the curves of /z and 6 receptor decreases related to the survival time are superimposable, (c) both/~ and 6 receptor losses are
119
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Fig. 3. Curves showing the rhizotomized vs. intact side ratios measured every 160/tm along the C4-T 2 spinal segments, following 1, 2, 4 and 8 days after a unilateral C4-T 2 dorsal rhizotomy. For [3H]DAMGO as well as for [3H]DTLET, each curve represents the mean values for 3
rats.
maximal at 8 days and remain stable at least over the analysed period (90 days), (d) 50% decreases in binding are obtained at tl/2 = 2.5 days for both [ 3 H ] D A M G O and [3H]DTLET, (e) the loss of/~ binding sites (71-74%) is significantly more pronounced than the loss of 6 binding sites (57-62%) and (f) affinities of postsynaptic/~ and receptors were found to be similar to those of the to-
TABLE II Binding characteristics at equilibrium for [3H]DAMGO and [3H]DTLET in laminae 1-I1 of intact rats and rats with a unilateral (right) C4--T2 dorsal rhizotomy
Data were measured from sections obtained from C6-C8 spinal segments which correspond to the center of the lesion in C4-T 2 rhizotomized rats. They were obtained by the linear regression analysis of Scatchard. Degree of correlations are comprised inbetween r = -0.948 and r = -0.999. Postlesion delay is 8 days. Results are expressed as mean + S.E.M. (n = 6 for intact and n = 8 for rhizotomized rats).
f H]DAMGO (0.5-24 nM)
[3H]DTLET (0.5-24 nM)
K~ (nM) Bma,
g~ (ng)
(fmol/mg) Intact Rhizotomized Left Right
B.~
(fmol/mg)
1.3+0.2
67.4+4.1
2.3+0.3
40.6+3.5
1.4+0.2 1.6+0.2
64.8+2.0 24.5+2.0 c
2.0+0.2 1.7+0.4
40.7+1.0 18.2+2.3 c
c p < 0.001 compared to values from intact rats.
tal receptor population in the superficial layers of the dorsal horn. So, it can be assumed that pre- and postsynaptic opioid binding sites have similar affinities for their own ligands. Although performed only for one P.L. delay (8 days), we think that this latter result can be extended to other P.L. delays since, at 8 days P.L., the maximal binding decrease is reached. If the decrease in binding was due to a modification of receptor affinity, it would be certainly detected at 8 days P.L. So, we can reasonably assume that the decreases in/~ and 6 binding observed in laminae I - I I are due to real losses of/~ and 6 binding sites rather than differences in receptor affinities. Two possible mechanisms can explain these decreases in ~ and 6 binding sites on the lesioned side as compared to the intact side: degeneration of neuronal elements associated with these binding sites or receptor regulation not directly linked to morphological changes. Unfortunately, our approach has not a sufficient accuracy to detect a modification of opioid receptor density on neuronal membranes. In the literature, there is no evidence for the existence of an alteration in opioid receptor regulation following dorsal rhizotomy. The only means to assess this phenomenon would be to visualize by electron microscopy the exact localization of opioid binding sites on neuronal membranes and to quantify their density. Although we cannot exclude a possible regulation of
120 opioid binding sites not directly linked to morphologic changes, the explanation of our results by the degeneration of primary afferent fibers is supported by various pieces of experimental data: (a) several studies show that presynaptic opioid receptors are associated with fine diameter primary afferent fibers, (b) the time courses of/~ and 6 receptor decreases are comparable to the time course of primary afferent fiber degeneration and (c) from 8 to 90 days P.L., there is no modification of/t and 6 binding sites in the central segments totally deafferented by the large C4-T 2 dorsal rhizotomy. With regard to the loss of presynaptic opioid receptors, several pieces of evidence favor their location on primary afferent fibers 9'17'44 and specially on fine diameter fibers 11'28. Since opioid receptors are membranebound glycoproteins, the intensity of the loss in opioid binding can be considered as a clue of the degree of disorganization of membrane structures occurring when fibers degenerate. In this respect, results obtained with both [3H]DAMGO and [3H]DTLET are in good agreement with most of the studies describing degeneration ~6' 21,25 and ultrastructural alterations 5,6,16,t9,2°,25.27.31-33 of dorsal root afferents following dorsal rhizotomies. Indeed, according to these various anatomical studies, fine diameter primary afferent fibers (C and A6 fibers) projecting in laminae I and II appear to degenerate as early as the first day postrhizotomy 5'6'16, the degeneration process being maximal at 2-3 days P.L. 6'16'20'27'32. The delay corresponding to the total disappearance of degenerative primary afferent terminals in laminae I and II is more controversial. Differences can be attributed to species and to the nature (myelinated or unmyelinated) of afferent fibers2°'32. However, with only a few exceptions 6'21'32, all fine diameter primary afferent fibers have degenerated after 1 week 5'16'2°. Interestingly, the timecourse in the decrease in p and 6 binding sites in laminae I and II is similar to that observed for degeneration of fine diameter primary afferent fibers at this level. These findings, which are relevant to the hypothesis of presynaptic opioid receptors located on fine diameter primary afferent fibers, suggest that 50% of fine diameter primary afferent fibers have degenerated in the central segment at 2.5 days after a large dorsal rhizotomy and that virtually all of them have disappeared after 8
REFERENCES 1 Arvidsson, J. and Pfaller, K., Central projections of C4--C s dorsal root ganglia in the rat studied by anterograde transport of WGA-HRP, J. Comp. Neurol., 292 (1990) 349-362. 2 Besse, D.. Lombard, M.C., Zajac, J.M., Roques, B.P. and Besson, J.M., Pre- and postsynaptic distribution of/~, 6 and ~¢opioid receptors in the superficial layers of the cervical dorsal horn of the rat spinal cord, Brain Res., 521 (1990) 15-22.
days. In addition, since various anatomical 1"21"4° (but see refs. 7 and 43) and electrophysiologica124'41'42 studies have reported that the rostrocaudal projection of cutaneous fine diameter primary afferent fibers seems to extend massively over less than 3 segments beyond the segment of entry, in our conditions, the C 7 segment can be considered as almost totally deprived of these fibers. Thus, 8 days after the C4-T 2 dorsal rhizotomy, the remaining binding sites in the ipsilateral side of the C 7 segment could be the postsynaptic opioid binding sites. Interestingly, in the central C7 segment, a stable amount of/~ and 6 opioid receptors is measured on the rhizotomized side and on the intact side from 8 days to 90 days postlesion. This is in agreement with a previous study describing no change in rhizotomized side/intact side ratios between 15 days and 4 months P.L. 45. Nevertheless, the apparent invariability in the number of postsynaptic opioid receptors, could reflect either a genuine receptor stability or an equilibrium between various effects (i.e. transsynaptic degeneration, receptor up-regulation). Although the location of opioid receptors in the spinal cord is not yet known at the ultrastructural level, enkephalinergic terminals have been observed on the somata and proximal dendrites of spinothalamic neurons in superficial laminae 35'36. From this observation, it is likely that enkephalin released at the level of the synaptic site may bind to opioid receptors on somata and proximal dendrites of postsynaptic spinothalamic neurons. Interestingly, when transsynaptic degeneration is described 4'12' 13.19.39 but see refs. 3 and 37, it mainly consists of a reduction in dendritic length and complexity of branching 4. Thus, it is probable that opioid binding sites are not notably affected by transsynaptic degeneration. Moreover, when described, transsynaptic degeneration becomes evident after a certain delay (10-60 days) 4"12'13'19' 39. Such observations further support our assumption that the loss of opioid binding sites occurring before 8 days P.L. is essentially due to degeneration of fine diameter primary afferent fibers.
Acknowledgements. We thank Dr D.A. Dickenson for English correction and E. Dehausse and E Morain for drawings and photography.
3 Brown, A.G., Brown, P.B. and Fyffe, R.E.W., Effects of dorsal root section on spinocervical tract neurones in the cat, J. Physiol., 337 (1983) 589-608. 4 Brown, P.B.. Busch, G.R. and Whittington, J., Anatomical changes in cat dorsal horn cells after transection of a single dorsal root, Exp. Neurol., 64 (1979) 453-468. 5 Chung, K., McNeill. D.L., Hulsebosch, C.E. and Coggeshall, R.E., Changes in dorsal horn synapticdisc numbers followingunilateral dorsal rhizotomy, J. Comp. Neurol., 283 (1989) 568-577.
121 6 Coimbra, A., Sodre-Borges, B.P. and Magalhaes, M.M., The substantia gelatinosa Rolandi of the rat. Fine structure, cytochemistry (acid phosphatase) and changes after dorsal root section, J. Neurocytol., 3 (1974) 199-217. 7 Culbersom J.L. and Brown, P.B., Projections of hindlimb dorsal roots to lumbosacral spinal cord of cat, J. Neurophysiol., 51 (1984) 516-528. 8 Daval, G., Verge, D., Basbaum, A.I., Bourgoin, S. and Hamon, M., Autoradiographic evidence of serotonin 1 binding sites on primary afferent fibres in the dorsal horn of the rat spinal cord, Neurosci. Lett., 83 (1987) 71-76. 9 Fields, H.L., Emson, P.C., Leigh, B.K., Gilbert, R.ET. and Iversen, L.L., Multiple opiate receptor sites on primary afferent fibers, Nature, 284 (1980) 351-353. 10 Fitzgerald, M., The course and termination of primary afferent fibres. In P.D. Wall and R. Melzack (Eds.), Textbook of Pain, Churchill Livingstone, Edinburgh, 1989, pp. 46-62. 11 Gamse, R., Holzer, P. and Lembeck, F., Indirect evidence for presynaptic location of opiate receptors on chemosensitive primary sensory neurones, Naunyn-Schmiederberg's Arch. Pharmacol., 308 (1979) 281-285. 12 Gobel, S., An electron microscopic analysis of the transsynaptic effects of peripheral nerve injury subsequent to tooth pulp extirpations on neurons in laminae I and II of the medullary dorsal horn, J. Neurosci., 4 (1984) 2281-2290. 13 Gobek S. and Binck, J.M., Degenerative changes in primary trigeminal axons and in neurons in nucleus caudalis following tooth pulp extirpations in the cat, Brain Res., 132 (1977) 347-354. 14 Gobel, S., Falls, W.M. and Humphrey, E., Morphology and synaptic connections of ultrafine primary axons in lamina I of the spinal dorsal horn: candidates for the terminal axonal arbors of primary neurons with unmyelinated (C) axons, J. Neurosci., 1 (1981) 1163-1179. 15 Gouardrres, C., Beaudet, A., Zajac, J.-M., Cros, J. and Quirion, R., High resolution radioautographic localization of [125I]FK-33-824-1abelled mu opioid receptors in the spinal cord of normal and deafferented rats, Neuroscience, 43 (1991) 197209. 16 Heimer, L. and Wall, P.D., The dorsal root distribution to the substantia gelatinosa of the rat with a note on the distribution in the cat, Exp. Brain Res., 6 (1968) 89-99. 17 Hiller, J.M., Simon, E.J., Crain, S.M. and Peterson, E.R., Opiate receptors in cultures of fetal mouse dorsal root ganglia (DRG) and spinal cord: predominence in DRG neurites, Brain Res., 145 (1978) 396-400. 18 Jancso, G. and Kiraly, E., Distribution of chemosensitive primary sensory afferents in the central nervous system of the rat, J. Comp. Neurol., 190 (1980) 781-792. 19 Kapadia, S.E. and LaMotte, C.C., Deafferentation-induced alterations in rat dorsal horn: I. Comparison of peripheral nerve injury vs. rhizotomy effects on presynaptic, postsynaptic, and glial processes, J. Comp. Neurol., 266 (1987) 183-197. 20 Knyihar-Csillik, E., Csillik, B. and Rakic, P., Ultrastructure of normal and degenerating glomerular terminals of dorsal root axons in the substantia gelatinosa of the rhesus monkey, J. Comp. Neurol., 210 (1982) 357-375. 21 LaMotte, C., Distribution of the tract of Lissauer and the dorsal root fibers in the primate spinal cord, J. Comp. Neurol., 172 (1977) 529-562. 22 LaMotte, C., Pert, C.B. and Snyder, S.H., Opiate receptor binding in primate spinal cord: distribution and changes after dorsal root section, Brain Res., 112 (1976) 407-412. 23 Light, A.R. and Perl, E.R., Reexamination of the dorsal root projection to the spinal dorsal horn including observations on the differential termination of coarse and fine fibers, J. Comp. Neurol., 186 (1979) 117-132. 24 McMahon, S.B. and Wall, P,D., The distribution and central termination of single cutaneous and muscle unmyelinated fibres in rat spinal cord, Brain Res., 359 (1985) 39-48. 25 Maxwell, D.J., Fyffe, R.E.W. and Brown, A.G., Fine structure
of normal and degenerating primary afferent boutons associated with characterized spinocervical tract neurons in the cat, Neuroscience, 12 (1984) 151-163. 26 Molander, C., Xu, Q., Rivero-Melian, C. and Grant, G., Cytoarchitectonic organization of the spinal cord in the rat: II. The cervical and upper thoracic cord, J. Comp. Neurol., 289 (1989) 375-385. 27 Murray, M. and Goldberger, M.E., Replacement of synaptic terminals in lamina II and Clarke's Nucleus after unilateral lumbosacral dorsal rhizotomy in adult cats, J. Neurosci., 6 (1986) 3205-3217. 28 Nagy. J.I., Vincent, S.R., Staines, W.M.A., Fibiger, H.C., Reisine, T.D. and Yamamura, H.I., Neurotoxic action of capsaicin on spinal substance P neurons, Brain Res., 186 (1980) 435444. 29 Ninkovic, M., Hunt, S.E and Kelly, J.S., Effect of dorsal rhizotomy on the autoradiographic distribution of opiate and neurotensin-like immunoreactivity within the rat spinal cord, Brain Res., 230 (1981) 111-119. 30 Proshansky, E. and Egger, M.D., Dendritic spread of dorsal horn neurons in cats, Exp. Brain Res., 28 (1977) 153-166. 31 Ralston, H.J., Dorsal root projections to dorsal horn neurons in the cat spinal cord, J. Comp. Neurol., 132 (1968) 303-330. 32 Ralston, H.J. and Ralston, D.D., The distribution of dorsal root axons in laminae I, II and III of the macaque spinal cord: a quantitative electron microscope study, J. Comp. Neurol., 184 (1979) 643-684. 33 Ralston, H.J. and Ralston, D.D., The distribution of dorsal root axons to laminae IV, V and VI of the macaque spinal cord: a quantitative electron microscopic study, J. Comp. Neurol., 212 (1982) 435-448. 34 Rethelyi, M., Preterminal and terminal axon arborizations in the substantia gelatinosa of cat's spinal cord, J. Comp. Neurol., 172 (1977) 511-528. 35 Ruda, M.A., Opiates and pain pathways: demonstration of enkephalin synapses on dorsal horn projection neurons, Science, 215 (1982) 1523-1525. 36 Ruda. M.A., Coffield, J. and Dubner, R., Demonstration of postsynaptic opioid modulation of thalamic projection neurons by the combined techniques of retrograde horseradish peroxydase and enkephalin immunohistochemistry, J. Neurosci., 4 (1984) 2117-2132. 37 Sedevic, M.J., Capowski, J.J. and Mendell, L.M., Morphology of HRP-injected spinocervical tract neurons: effect of dorsal rhizotomy, J. Neurosci., 6 (1986) 661-672. 38 Steiner, T.J. and Turner, L.M., Cytoarchitecture of the rat spinal cord, J. Physiol., 222 (1972) 123-124. 39 Sugimoto, T. and Gobel, S., Dendritic changes in the spinal dorsal horn following transection of a peripheral nerve, Brain Res., 321 (1984) 199-208. 40 Sugiura, Y., Lee, C.L. and Perl, E.R., Central projections of identified, unmyelinated (C) fibers innervating mammalian skin, Science, 234 (1986) 358-361. 41 Traub, R.J. and Mendell, L.M., The spinal projection of individual identified A-6- and C-fibers, J. Neurophysiol., 59 (1988) 1-55. 42 Traub, R.J., Sedivec, M.J. and Mendell, L.M., The rostral projection of small diameter primary afferents in Lissauer's tract, Brain Res., 399 (1986) 185-189. 43 Traub, R.J., Solodkin, A. and Ruda, M.A., Calcitonin generelated peptide immunoreactivity in the cat lumbosacral spinal cord and the effects of multiple dorsal rhizotomies, J. Comp. Neurol., 287 (1989) 225-237. 44 Young III, W.S., Wamsley, J.K., Zarbin, M.A. and Kuhar, M.J., Opioid receptors undergo axonal flow, Science, 210 (1980) 76-78. 45 Zajac, J.M., Lombard, M.C., Peschanski, M., Besson, J.M. and Roques, B.P., Autoradiographic study of/~ and 6 opioid binding sites and neutral endopeptides 24-11 in rat after dorsal root rhizotomy, Brain Res., 477 (1989) 400-403.
115
BRES 17629
Time-related decreases in/z and 6 opioid receptors in the superficial dorsal horn of the rat spinal cord following a large unilateral dorsal rhizotomy D. Besse, M.C. Lombard and J.M. Besson Unitg de Recherche de Physiopharmacologie du Syst~me Nerveux (INSERM, U. 161) and Laboratoire de Physiopharmacologie de la Douleur, Ecole Pratique des Hautes Etudes, Paris (France)
(Accepted 26 November 1991) Key words: Quantitative autoradiography; Receptor down-regulation; Deafferentiation; Degeneration; Thin primary afferent fiber
The aim of the present study was to measure the time-related modifications of p and 6 opioid binding sites in the superficial layers of the dorsal horn of the rat spinal cord after a C4-T2 unilateral dorsal rhizotomy. Using specific ligands, namely [3H]DAMGO for/~ sites and [3H]DTLET for 6 sites, and a quantitative autoradiographic analysis, we have observed: (a) a decrease in binding on the ipsilateral side to the lesion as early as the first day postrhizotomy, the maximal loss being attained at 8 days postlesion, (b) after 8 days postlesion, the residual binding remains stable over the period of analysis (90 days), (c) the loss of ~ receptors (71-74%) is significantly more pronounced than the loss of 6 receptors (57-62%) and (d) affinities of postsynaptic/~ and 6 receptors are similar to those of the total receptor population in the superficial layers of the dorsal horn. Comparison of these results with the degeneration of primary afferent fibers reported in literature favors the localization of the majority of/~ and 6 opioid binding sites on fine diameter primary afferent fibers. INTRODUCTION The decrease in opioid receptors following dorsal rhizotomy was initially described by L a M o t t e et al. 22 in the superficial dorsal horn of the spinal cord of rhesus monkey. M o r e recently, using a u t o r a d i o g r a p h y of selective opioid ligands and large dorsal rhizotomies, several studies 2'15'a5 have r e p o r t e d quantitative data on the decrease of/~, 6 and/or r opioid receptors in the superficial layers (laminae I - I I ) of the rat dorsal horn. I n d e e d , at the level of the C 7 segment, we have shown that, 8 days after a unilateral C 4 - T 2 dorsal rhizotomy, the presynaptic c o m p o n e n t s of k~ and 6 opioid receptors were 76 and 61%, respectively. Several lines of evidence suggest that the presynaptic c o m p o n e n t of opioid receptors can be associated with fine d i a m e t e r p r i m a r y afferent fibers. (i) Thin A 6 and C p r i m a r y afferent fibers essentially project in laminae I and 111'14'18'22'23'30'32'34'40-42 which contain the highest levels of opioid binding sites. (ii) A massive loss of opioid binding sites in laminae I - I I is observed after neonatal t r e a t m e n t with capsaicin 8'11'28. (iii) In contrast, large Aft and A 6 hair follicle fiber arborizations are in laminae III, IV and V (see refs. in ref. 10) in which the density of opioid binding sites is very low. Therefore, we have m e a s u r e d by a u t o r a d i o g r a p h y the
modifications of kt and 6 opioid receptors related to the postrhizotomy delay (1-90 days) and we have c o m p a r e d our results with previous qualitative anatomical studies dealing with d e g e n e r a t i o n of primary afferent fibers in the superficial layers of the dorsal horn. MATERIALS AND METHODS Surgery Experiments were performed on 31 Sprague-Dawley albino rats, weighing 225-250 g. The surgical procedure was performed using ketamine (100 mg/kg i.p.) as general and xylocaine (2%) as local anesthetics. A dorsal hemilaminectomy of the C3-T 2 vertebrae was made on the right side and the C4-T2 dorsal roots were sectioned intradurally proximal to the ganglia, through small individual openings of the dura mater, under a dissection microscope. Care was taken to cause no damage to the blood vessels running along the dorsal roots. The wound was washed with saline and the muscles and skin sutured, layer by layer. Delays postrhizotomy were: 1, 2, 4, 8, 14, 30 and 90 days. Lesions were performed so that the animals were all the same age on the day of sacrifice. Three animals were used for each group. Three additional intact rats were included as controls. Saturation experiments were performed using 6 intact rats and 8 C4-T 2 rhizotomized rats (8 days postoperative). At the end of the survival time, rats were sacrificed by decapitation. Cervicothoracic C4-T2 spinal cord was removed rapidly, frozen in isopentane at -40°C and preserved at -80°C. Autoradiographic procedures Levorphanol (tartrate; SIGMA), [3H]DAMGO (Tyr*-D-Ala-GlyNMe-Phe-Gly-ol) (2.1 TBq/mmol; C.E.A. Saclay, France) and
Correspondence: D. Besse, Physiopharmacologie du Syst6me Nerveux (INSERM U161), 2 rue d'Al6sia, 75014 Paris, France.
116 [3H]DTLET (Tyr*-o-Thr-Gly-Phe-Leu-Thr) (2.15 TBq/mmol; C.E.A. Saclay, France) were used in this study. Entire C4-T2 extents were cut (frontal sections 15 pm thick) on a cryostat (-20°C), thaw-mounted onto gelatinized slides and kept at -80°C up to incubation. Two sets of slides each bearing adjacent sections were made: one for [3H]DAMGO, the other for [3H]DTLET. Sections were brought to room temperature and incubated in 350 ml of 50 mM Tris-HCl buffer (pH 7.4) at 25°C for 60 min with either 3 nM [3H]DAMGO to label p sites or 3 nM [3H]DTLET to label 6 sites. The non-specific binding was determined using some spinal cord sections under the same conditions of incubation, in the presence of 1 pM levorphanol. At the end of the incubation, spinal cord sections were washed twice in 350 ml of ice-cold 50 mM TrisHC1 buffer (pH 7.4) for 10 min each, rinsed in ice-cold bidistillated water for 2 s and air-dried. For saturation experiments, spinal cord sections from C6 to C8 were successivelythaw-mounted onto gelatinized slides so that, for a given ligand, adjacent sections corresponded to different concentrations. Four sets of 8 slides were made corresponding to: total and non-specific [3H]DAMGO binding and total and non-specific [3H]DTLET binding. For both ligands, about 10 sections per rat were used for each of the 8 concentrations from 0.5 to 24 nM. Slides were kept at -80°C up to incubation. For incubation, slides at room temperature were put in incubation chambers. Spinal cord sections were incubated for 60 rain in 600 pl of 50 mM Tris-HCl buffer (pH 7.4) containing the tritiated ligand with (non-specific binding) or without (total binding) 1/~M levorphanol. At the end of the incubation, sections were washed twice in 350 ml of ice-cold 50 mM Tris-HCl buffer (pH 7.4) for 10 min each, rinsed in ice-cold bidistillated water for 2 s and air-dried. Labelled sections were then juxtaposed against tritium-sensitive films (Hyperfilm-3H, Amersham France, Les Ulis) and exposed for 15 weeks at 4°C. Autoradiograms were developed in Kodak D19 for 2-3 rain at 20°C, fixed for 5 min, washed for 20 min under running water arid dried. Autoradiograms were analysed by densitometry using a Biocom 200 analyser system (Biocom, France). According to Nissl poststaining sections, we measured binding in the area corresponding to laminae I and II of Rexed's classification as defined for the rat spinal cord26'38. Grey levels measured on autoradiograms were converted into binding site concentration values by reference to tritium standards (Amersham). Specific binding was calculated by subtracing, from total binding, the amount of binding measured from sections incubated for non-specific labeling. Except for the saturation study, measurements of receptor concentrations were performed every 160 pm, from C4 to T2 segments, on the intact side and on the rhizotomized side. This procedure gave a large number of values (7-10 in each spinal segment) and allowed detection of any differences in binding in successive spinal segments to be observed. For an initial analysis, results were expressed as specific binding concentrations (fmol/mg of tissue) on each side. In a second part of the study, results were expressed as ratios of binding measured in the rhizotomized side compared to the intact side. These ratios were compared between the different experimental situations. The 'time of half-decrease' (qr2) was defined as the time of 50% of the maximal decrease in binding on the rhizotomized side compared to the intact side. In saturation experiments, binding parameters at equilibrium (dissociation constant = Kd and specific binding capacity = Bmax) were determinated from the linear regression analysis of Scatchard. For each rat, each point relative to a given concentration corresponded to the mean of binding values obtained from about l0 spinal cord sections. Determination of successive spinal segments was made using an atlas constructed from Nissl-stained spinal cord sections. The statistical analysis was performed using either a Student t-test or a variance analysis (ANOVA), followed by the LSD multiple range test (Least Significant Difference).
RESULTS In intact rats, autoradiograms reveal [ 3 H ] D A M G O and [3H]DTLET specific binding mainly concentrated in the superficial layers of the dorsal horn (Fig. 1). At this level, /~ binding sites are p r e d o m i n a n t compared to 6 binding sites. For [ 3 H ] D A M G O as well as for [3H]DTLET, binding is similar in both right and left sides (see Table I for the C7 segment). The non-specific binding is constant all along the C4-T 2 extent and corresponds to about 10 and 20% of the total binding for [ 3 H ] D A M G O and [3H]DTLET, respectively. In rhizotomized rats, autoradiograms revealed a loss in ~ and 6 binding on the rhizotomized side compared to the intact side (Fig. 1). The decrease in specific binding becomes more and more p r o n o u n c e d until 8 days postlesion (P.L.). For longer delays (15-90 days), there is no further significant decrease in either ~ or 6 binding. Specific binding values for the central C7 segment are reported in Table I. As regards specific binding values, on the intact side of rhizotomized rats, /~-specific binding for when considering the nificant variation in p
there is a tendency of decrease in some particular delays. However, delays altogether, there is no sigas well as 6 specific binding on the
intact side ( A N O V A : F7.16 = 1.10 for [ 3 H ] D A M G O and F7a6 = 0,90 for [3H]DTLET). O n the rhizotomized side, a significant decrease in binding is observed when compared to values of intact rats ( A N O V A : F7.16 ~-- 19.71 for [ 3 H ] D A M G O and F7,16 = 11.26 for [3H]DTLET). In fact, for [ 3 H ] D A M G O as well as for [3H]DTLET, a comparison of binding values for each group with that of intact rats reveals significant decreases after 2 days P.L. Considering ratios, a significant effect is revealed as early as the first day. For both ligands, decreases in the ratio are related to the postlesion delay until 8 days. After this time, no further loss of binding is observed up to 90 days P.L. Fig. 2 illustrates the time course o f p and 3 binding ratios in the central Cv segment. For short survival delays (1-3 days), the time-courses are similar for both p and 6 opioid receptors, However, a noticeable difference between the decreases in p and 6 receptors appears in the 3 - 8 days interval. Thus, the decrease in 6 receptors reaches a minimal ratio of about 0.40 while the decrease in p receptors is more marked, the minimal ratio being about 0.28. Delays corresponding to the 50% decrease of both /~ and 6 sites (see Materials and Methods) are ttr2 = 2.5 days. From 8 to 90 days, the proportions of remaining p receptors (0.26-0.29) persist at lower levels than those of 6 receptors (0.390.43). The m e a n difference between the two types of receptors is 13% (P < 0.001, Student-t = 7.7, df = 22) in the C7 segment.
•,~l!suop ~u!pu!q jo uo!le.ue^ olq!s!^ ~oqlanj ou s! oa~q~ "q'd s,~ep 06 pue s,~ep 8 u~a~lo~t '(1~/) uaoq les~op ~q~ jo (II-I ~eu!m~l) sa~'~el Ie!~9 a~dns ~ql u! ae~l~ s! l~JJ~ ~q~ 'SletU!u~ loelu.l ol p~aedtuo2) '/~tuo~oz!q~ leS.~op z / _ ~ ) ~q~ o~ ~p!s
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118 TABLE I Specific binding values for 3 nM [3H]DAMGO and 3 nM [3H]DTLET in laminae l-II of the C7 segment according to various delays following a C4-T2 right dorsal rhizotomy Values are expressed as mean concentration (fmol/rng tissue) + S.E.M. (n = 3 rats). Ratios correspond to rhizotomized side/intact side binding values. Intact
[3H]DAMGO Left Right Ratio [3HIDTLET Left Right Ratio
Rhizotomized 1
2
4
8
15
30
90days PL.
35.7±4.9 35.5±5.0
35.5±3.0 31.9+2.2
33.4+5.6 24.5+3.9 a
37.9±2.4 17.5±1.4 b
27.4+4.1 7.9-+2.0 c
25.5+1.6 7.4±0.4¢
31.9±7.1 7.4+0.3 c
25.2+6.6 6.9±0-8c
1.00+0.02
0.90+0.03 a
0.74+0.01 c
0.46+0.01 c
0.28-+0.03c
0.29+0.02 ¢
0.26+0.05 c
0.28+0.04¢
11.0+1.9 11.0+1.9
14.2+0.7 13.1+0.7
9.9+1.3 6.6+0.9 b
10.0±1.1 5.5±0.6¢
8.5+0.3 3.2+0.2 ¢
12.2+2.3 4.8-+1.1 c
9.1±1.1 3.6+0.3 c
10.6+1.1 4.6-+0.6 c
1.00+0.05
0.89+0.02 b
0.68+0.01 c
0.55+0.04 ¢
0.39+0.04 c
0.39±0.02 c
0.40+0.02 ¢
0-43+0.01¢
ap < 0.05, bp < 0.01 and eP < 0.001 as compared to data obtained in intact rats, for left (intact) side, right (rhizotomized) side and ratios (ANOVA followed by LSD test).
In order to detect any variation along the rostrocaudal axis of the spinal cord of either [ 3 H ] D A M G O or [3H]DTLET specific binding, we performed a detailed analysis over the C4-T 2 segments (Fig. 3). Considering each survival time, a progressive decrease in binding is
1.00
-
-7-
DTLET DAMGO
though quite similar effects are found in C6 and C s. Indeed, statistical analysis of ratios corresponding to C 6, C 7 and Ca segments does not reveal significant variation at any survival delays. In order to check whether the observed decrease in binding is due to a loss of sites or to a change in their affinity, saturation experiments were performed for intact and rhizotomized rats at 8 days E L . Binding characteristics, obtained in C6-C 8 segments (Table II), demonstrate that: (i) there is no significant variation in either
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observed over all the 7 segments. As expected, the C 7 segment corresponds to the level of maximal effect, al-
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0.40-
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# or 6 receptor affinity after unilateral C4-T 2 rhizotomy, (ii) specific binding capacities on the intact side of rhizotomized rats are similar to those of intact rats, (iii) specific binding capacities on the rhizotomized side are 62 and 55% l o w e r ' t h a n on the intact side for/a and 6 binding sites, respectively.
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DISCUSSION
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Earlier investigations2'8A5'22'29'45 have shown a reduc0.00
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post-lesion delay (days) Fig. 2. Diagram showing the time-related decrease of 3 nM [3H]DAMGO and 3 nM [3H]DTLET specific binding on the rhizotomized side compared to the intact side. Measurements were performed at the C7 segment level, in laminae I-II of the dorsal horn, after a unilateral C4-T2 dorsal rhizotomy. Note that the maximal decrease for both [3H]DAMGO and [3H]DTLET binding is obtained after 8 days and that the half maximum effect titz is observed after 2.5 days. A significant difference between ratios of [3H]DAMGO and [3H]DTLET binding is observed from 4 to 90 days EL.
tion of opioid receptors in the dorsal horn after dorsal rhizotomy. The aim of the present study was to analyse the time-course of the decreases in # and 6 binding sites after a C4-T2 dorsal rhizotomy. From the present study several points arise, some of them being comparable to those obtained with [125I]FK-33-824 U-selective ligand) after a less extensive lesion in the rat 15. These points are: (a) both # and 6 receptor decreases begin as early as the first day postrhizotomy, (b) up to 3 days, the curves of /z and 6 receptor decreases related to the survival time are superimposable, (c) both/~ and 6 receptor losses are
119
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SPINAL SEGMENTS
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Fig. 3. Curves showing the rhizotomized vs. intact side ratios measured every 160/tm along the C4-T 2 spinal segments, following 1, 2, 4 and 8 days after a unilateral C4-T 2 dorsal rhizotomy. For [3H]DAMGO as well as for [3H]DTLET, each curve represents the mean values for 3
rats.
maximal at 8 days and remain stable at least over the analysed period (90 days), (d) 50% decreases in binding are obtained at tl/2 = 2.5 days for both [ 3 H ] D A M G O and [3H]DTLET, (e) the loss of/~ binding sites (71-74%) is significantly more pronounced than the loss of 6 binding sites (57-62%) and (f) affinities of postsynaptic/~ and receptors were found to be similar to those of the to-
TABLE II Binding characteristics at equilibrium for [3H]DAMGO and [3H]DTLET in laminae 1-I1 of intact rats and rats with a unilateral (right) C4--T2 dorsal rhizotomy
Data were measured from sections obtained from C6-C8 spinal segments which correspond to the center of the lesion in C4-T 2 rhizotomized rats. They were obtained by the linear regression analysis of Scatchard. Degree of correlations are comprised inbetween r = -0.948 and r = -0.999. Postlesion delay is 8 days. Results are expressed as mean + S.E.M. (n = 6 for intact and n = 8 for rhizotomized rats).
f H]DAMGO (0.5-24 nM)
[3H]DTLET (0.5-24 nM)
K~ (nM) Bma,
g~ (ng)
(fmol/mg) Intact Rhizotomized Left Right
B.~
(fmol/mg)
1.3+0.2
67.4+4.1
2.3+0.3
40.6+3.5
1.4+0.2 1.6+0.2
64.8+2.0 24.5+2.0 c
2.0+0.2 1.7+0.4
40.7+1.0 18.2+2.3 c
c p < 0.001 compared to values from intact rats.
tal receptor population in the superficial layers of the dorsal horn. So, it can be assumed that pre- and postsynaptic opioid binding sites have similar affinities for their own ligands. Although performed only for one P.L. delay (8 days), we think that this latter result can be extended to other P.L. delays since, at 8 days P.L., the maximal binding decrease is reached. If the decrease in binding was due to a modification of receptor affinity, it would be certainly detected at 8 days P.L. So, we can reasonably assume that the decreases in/~ and 6 binding observed in laminae I - I I are due to real losses of/~ and 6 binding sites rather than differences in receptor affinities. Two possible mechanisms can explain these decreases in ~ and 6 binding sites on the lesioned side as compared to the intact side: degeneration of neuronal elements associated with these binding sites or receptor regulation not directly linked to morphological changes. Unfortunately, our approach has not a sufficient accuracy to detect a modification of opioid receptor density on neuronal membranes. In the literature, there is no evidence for the existence of an alteration in opioid receptor regulation following dorsal rhizotomy. The only means to assess this phenomenon would be to visualize by electron microscopy the exact localization of opioid binding sites on neuronal membranes and to quantify their density. Although we cannot exclude a possible regulation of
120 opioid binding sites not directly linked to morphologic changes, the explanation of our results by the degeneration of primary afferent fibers is supported by various pieces of experimental data: (a) several studies show that presynaptic opioid receptors are associated with fine diameter primary afferent fibers, (b) the time courses of/~ and 6 receptor decreases are comparable to the time course of primary afferent fiber degeneration and (c) from 8 to 90 days P.L., there is no modification of/t and 6 binding sites in the central segments totally deafferented by the large C4-T 2 dorsal rhizotomy. With regard to the loss of presynaptic opioid receptors, several pieces of evidence favor their location on primary afferent fibers 9'17'44 and specially on fine diameter fibers 11'28. Since opioid receptors are membranebound glycoproteins, the intensity of the loss in opioid binding can be considered as a clue of the degree of disorganization of membrane structures occurring when fibers degenerate. In this respect, results obtained with both [3H]DAMGO and [3H]DTLET are in good agreement with most of the studies describing degeneration ~6' 21,25 and ultrastructural alterations 5,6,16,t9,2°,25.27.31-33 of dorsal root afferents following dorsal rhizotomies. Indeed, according to these various anatomical studies, fine diameter primary afferent fibers (C and A6 fibers) projecting in laminae I and II appear to degenerate as early as the first day postrhizotomy 5'6'16, the degeneration process being maximal at 2-3 days P.L. 6'16'20'27'32. The delay corresponding to the total disappearance of degenerative primary afferent terminals in laminae I and II is more controversial. Differences can be attributed to species and to the nature (myelinated or unmyelinated) of afferent fibers2°'32. However, with only a few exceptions 6'21'32, all fine diameter primary afferent fibers have degenerated after 1 week 5'16'2°. Interestingly, the timecourse in the decrease in p and 6 binding sites in laminae I and II is similar to that observed for degeneration of fine diameter primary afferent fibers at this level. These findings, which are relevant to the hypothesis of presynaptic opioid receptors located on fine diameter primary afferent fibers, suggest that 50% of fine diameter primary afferent fibers have degenerated in the central segment at 2.5 days after a large dorsal rhizotomy and that virtually all of them have disappeared after 8
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Acknowledgements. We thank Dr D.A. Dickenson for English correction and E. Dehausse and E Morain for drawings and photography.
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