Brain Research, 521 (1990) 15-22
15
Elsevier BRES 15621
Pre- and postsynaptic distribution of/x, 8 and r opioid receptors in the superficial layers of the cervical dorsal horn of the rat spinal cord D . B e s s e 1'2, M . C .
Lombard 1'2, J . M .
Z a j a c 3, B . P . R o q u e s 3 a n d J . M .
B e s s o n 1'2
ZUnitg de Recherche de Neurophysiologie Pharmacologique (I.N.S.E.R.M., Unit~ 161), 2Laboratoire de Physiopharmacologie de la Douleur, Ecole Pratique des Hautes Etudes, and 3Unitd de Recherche de Pharmacochimie Mol~culaire (1.N.S.E.R.M., UniM 266 and C.N.R.S. UA 498), Paris (France)
(Accepted 19 December 1989) Key words: Opioid receptor; Rat spinal cord; Superficial layer; Dorsal rhizotomy; Quantitative autoradiography
Highly selective tritiated ligands and quantitative autoradiography have been used to study/~, 6 and x binding sites in the dorsal horn of the rat spinal cord. We have measured the proportions of the 3 main types of opioid binding sites in the superficial layers of the cervical dorsal horn (laminae I and II). The proportions of/~, 6 and r sites were 70 + 4%, 23 + 2% and 7 + 1%, respectively, over the whole C4-T2 extent. Similar percentages were encountered at the level of each individual segment from Ca to T2. Eight days after a unilateral dorsal rhizotomy C4-T2, dramatic decreases were seen on the ipsilateral side to the lesion by comparison to the intact side. In the C7 segment, these decreases were 76 + 1%, 61 + 1% and 53 + 3% fork, di and r binding sites, respectively. The C7 segment can be considered as completely deafferented, so we attribute the residual values to postsynaptic binding whereas the decrease can be attributed to a loss of the presynaptic sites. These results are discussed with respect to the contribution of pre- and postsynaptic depressive effects of opiates on the transmission of noxious messages at the level of the dorsal horn. INTRODUCTION Both pre- and postsynaptic mechanisms have been proposed as a basis for the depressive effects of opioid agonists on the activity of nociceptive dorsal horn neurons 5A7'71. In a recent electrophysiological study, comparing the action of morphine on the activity of nociceptive dorsal horn neurons in the intact and the deafferented decerebrate rat 3s, we reported that the opioid was only half as potent in the absence of primary afferent fibers. These results are in good agreement with the fact that dorsal rhizotomy or peripheral nerve section induces a dramatic decrease (30-60%) in opioid binding sites at the level of the dorsal horn 12"28"32"44'45'67. In addition, these results relate to clinical observations claiming that the analgesic effect of morphine is more marked against pains due to an excess of nociception (activation of peripheral nociceptors in an intact nervous system) than against pains arising from deafferentation 1'6°. It is likely that each of the 3 main types of opioid receptors are located within the superficial dorsal horn; numerous studies, using receptor autoradiography, have visualized opioid receptors in the superficial layers of the dorsal horn of the rat 2'12'15'22'24'25'40'45-48'51'56,57,62,68,
-
-
primate 44'63 and human 18'54. As already discussed, a high Correspondence:
France.
proportion of these receptors are located on primary afferent fibers. Further evidence for this presynaptic location is provided by the demonstration of opiate binding in dorsal root ganglia 44'6s, and dorsal roots or nerves 2'3A9. Interestingly, a high level of opioid binding is found in the neuritic outgrowth from the dorsal root ganglia 26 in fetal mouse dorsal root ganglia and spinal cord tissue cultures. In addition, treatment of the neonatal rat with capsaicin suggests that these receptors are preferentially localized on thin afferent fibers 12'21'43. However, with the exception of a few studies T M previous studies did not include data on the relative proportions of the 3 main types of opioid receptors. In addition there is no quantitative data available concerning their pre- and postsynaptic location (see, however, ref. 67). From a general point of view, the data related to opioid receptors within the spinal cord are difficult to interpret since: - a large number of binding studies have been performed on membrane preparations where it is impossible to locate the binding sites; most of the ligands used were not selective and do not allow a clear distinction between each type of receptor; various types and extents of peripheral lesions were used.
D. Besse, Unit6 de Recherche de Neurophysiologie Pharmacologique (I.N.S.E.R.M., Unit6 161), 2 rue d'AMsia, 75014 Paris,
0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
16 The aim of the present study, using quantitative autoradiography and highly selective ligands was (1) to determine, at the level of the superficial dorsal horn (laminae I and II), the relative percentages of ~, 6 and 7c binding sites; and (2) to gauge their relative pre- and
multiple range test, LSD (least significant difference) for multiple comparison. Unpaired Student's t-test was used when comparison was limited to two groups of data.
RESULTS
postsynaptic locations. For this latter purpose we used a large unilateral dorsal rhizotomy (C4-T2) corresponding to the brachial plexus.
Relative proportions of ~, 6 and ~c opioid receptors in the superficial layers of the cervical dorsal horn
MATERIALS AND METHODS
In control rats, all 3 ligands labeled the superficial layers of the dorsal horn. Rough examination of the
Experiments were performed on male Sprague-Dawley albino rats, weighing 200-225 g. Three control animals were used for the measurement of the respective proportions of/~, 6 and r receptors and 3 others were submitted to dorsal rhizotomies C 4 - T 2. In this case, the surgical procedures were performed using ketamine (100 mg/kg) as a general and xylocaine (2%) as a local anesthetic. A dorsal hemilaminectomy of vertebrae cervical 3 (C3) to thoracic 3 (T3) was made on the right side and the 7 corresponding dorsal roots C4-Tz (Fig. 1A) 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. The rats were sacrificed by decapitation 8 days after the rhizotomy. The cervical enlargement C4-T2 was removed rapidly and frozen in isopentane (-40 °C) and preserved at -80 °C. The whole cervical enlargement C4-T2 was then cut (frontal section, 15 /~m thick) from C4 to T2 on a cryostat (-20 °C), thaw-mounted onto gelatinized slides and kept at -80 °C up to incubation. The sections were then brought back to room temperature and incubated in 50 mM Tris-HCl buffer (pH 7.4) at 25 °C for 60 min with one of 3 ligands. [3H]DAGO (Tyr-D-Ala-Gly-(Me)Phe-Gly-ol) (1.85 TBq/mmol; CEN Saclay) was used at a final concentration of 3 nM to label/~ sites. [3H]DTLET (Tyr-D-Thr-Gly-Phe-Leu-Thr) (2.22 TBq/mmol; CEN Saclay) was used at a final concentration of 3 nM to label 6 sites. [3H]Ethylketocyclazocine ([3H]EKC) (0.55 TBq/mmol; NEN) was used at a final concentration of 10 nM, in the presence of 100 nM of cold DAGO and 100 nM of cold DTLET, to label r sites. For these 3 ligands, the non-specific binding was evaluated under the same conditions in the presence of 10-6 M levorphanol (Hoffman-La Roche). At the end of the incubation the sections were washed twice in ice-cold buffer for 10 min, rinsed in water and air-dried. Labeled sections were brought into close contact with tritium-sensitive film (Amersham) and exposed for 15 weeks at 4 °C. The autoradiograms were developed in Kodak D19. Quantitative measurements were performed with a Biocom analyser system at the level of the superficial layers (laminae I and II) of the dorsal horn. Fig. IB illustrates the area of the measurement. The grey values were converted to binding site density values by reference to tritium standards (Amersham). The specific binding was calculated by subtracting from the total binding the amount of ligand bound in sections incubated for non-specific labeling. The quantity of sites were calculated as: Bma x =
B (1 + Ka/L)
with B corresponding to the amount of bound ligand for a concentration L of ligand and Kd its affinity. For each ligand, systematic measurements were performed every 120/~m, over the whole length of the C 4 - T 2 segments. This procedure allows firstly, a large number of measurements, and secondly, any differences in opioid binding sites over different spinal segments to be observed. Some spinal cord sections were Nissl stained using Cresyl violet for histological determination of anatomical boundaries. Statistical analysis was made using ANOVA followed by the
autoradiograms revealed that the binding due to [3H]DAGO was more p r o n o u n c e d than for [3H]DTLET and that the binding due to [3H]EKC was weak. From binding studies performed on rat spinal cord slices (unpublished results), the K o values of the ligands were found to be 4.6, 1.7 and 1.7 n M for D A G O , D T L E T and EKC, respectively. Thus the Bmax values of [3H]DAGO, [3H]DTLET and [3H]EKC were calculated to be 90 _+ 5, 30 + 2 and 9 +_ 2 fmol per mg of tissue, respectively, for the whole C 4 - T 2 enlargement. Consequently, the relative percentages of kt, 6 and ~c receptors are 70 _+ 4%, 23 -+ 2% and 7 + 1% (Table I), respectively. These mean values were obtained for each ligand with between 110 and 130 m e a s u r e m e n t s per rat for both dorsal horns (left and right). The distribution of the 3 types of opioid binding sites was homogenous, since similar percentages were encountered for each segment. For example the values obtained in the central segment (C7) which are illustrated in Table I and in Fig. 3A are not significantly different from those found for the entire C 4 - T 2 region.
Relative percentages of pre- and postsynaptic binding sites at the cervical level In order to measure the relative percentages of preand postsynaptic binding sites, we compared the lesioned side to the intact side 8 days after a unilateral dorsal rhizotomy C4-T 2. Compared to the intact side, the autoradiograms show a marked decrease in kt, 6 and ~¢ binding sites at all levels of the deafferented segments. A n individual example is shown for/~ sites in Fig. lB. As shown in Fig. 2 and Table II, we have quantified the decrease in sites over the entire C 4 - T 2 enlargement for all 3 ligands. For/~ and 6 binding sites, the lesion induced a highly significant decrease (P < 0.001) in each segment. The decrease in ~ binding sites was more p r o n o u n c e d than that of ~ sites. This difference which was of the order of 15% for the entire C4-T 2 enlargement, is highly significant (P < 0.001) and was systematically observed for each spinal segment. The comparisons, segment to segment, of the percentages of binding for [ 3 H ] D A G O and for [3H]DTLET reveal significant differences between only C4-C 5 (P
15
Elsevier BRES 15621
Pre- and postsynaptic distribution of/x, 8 and r opioid receptors in the superficial layers of the cervical dorsal horn of the rat spinal cord D . B e s s e 1'2, M . C .
Lombard 1'2, J . M .
Z a j a c 3, B . P . R o q u e s 3 a n d J . M .
B e s s o n 1'2
ZUnitg de Recherche de Neurophysiologie Pharmacologique (I.N.S.E.R.M., Unit~ 161), 2Laboratoire de Physiopharmacologie de la Douleur, Ecole Pratique des Hautes Etudes, and 3Unitd de Recherche de Pharmacochimie Mol~culaire (1.N.S.E.R.M., UniM 266 and C.N.R.S. UA 498), Paris (France)
(Accepted 19 December 1989) Key words: Opioid receptor; Rat spinal cord; Superficial layer; Dorsal rhizotomy; Quantitative autoradiography
Highly selective tritiated ligands and quantitative autoradiography have been used to study/~, 6 and x binding sites in the dorsal horn of the rat spinal cord. We have measured the proportions of the 3 main types of opioid binding sites in the superficial layers of the cervical dorsal horn (laminae I and II). The proportions of/~, 6 and r sites were 70 + 4%, 23 + 2% and 7 + 1%, respectively, over the whole C4-T2 extent. Similar percentages were encountered at the level of each individual segment from Ca to T2. Eight days after a unilateral dorsal rhizotomy C4-T2, dramatic decreases were seen on the ipsilateral side to the lesion by comparison to the intact side. In the C7 segment, these decreases were 76 + 1%, 61 + 1% and 53 + 3% fork, di and r binding sites, respectively. The C7 segment can be considered as completely deafferented, so we attribute the residual values to postsynaptic binding whereas the decrease can be attributed to a loss of the presynaptic sites. These results are discussed with respect to the contribution of pre- and postsynaptic depressive effects of opiates on the transmission of noxious messages at the level of the dorsal horn. INTRODUCTION Both pre- and postsynaptic mechanisms have been proposed as a basis for the depressive effects of opioid agonists on the activity of nociceptive dorsal horn neurons 5A7'71. In a recent electrophysiological study, comparing the action of morphine on the activity of nociceptive dorsal horn neurons in the intact and the deafferented decerebrate rat 3s, we reported that the opioid was only half as potent in the absence of primary afferent fibers. These results are in good agreement with the fact that dorsal rhizotomy or peripheral nerve section induces a dramatic decrease (30-60%) in opioid binding sites at the level of the dorsal horn 12"28"32"44'45'67. In addition, these results relate to clinical observations claiming that the analgesic effect of morphine is more marked against pains due to an excess of nociception (activation of peripheral nociceptors in an intact nervous system) than against pains arising from deafferentation 1'6°. It is likely that each of the 3 main types of opioid receptors are located within the superficial dorsal horn; numerous studies, using receptor autoradiography, have visualized opioid receptors in the superficial layers of the dorsal horn of the rat 2'12'15'22'24'25'40'45-48'51'56,57,62,68,
-
-
primate 44'63 and human 18'54. As already discussed, a high Correspondence:
France.
proportion of these receptors are located on primary afferent fibers. Further evidence for this presynaptic location is provided by the demonstration of opiate binding in dorsal root ganglia 44'6s, and dorsal roots or nerves 2'3A9. Interestingly, a high level of opioid binding is found in the neuritic outgrowth from the dorsal root ganglia 26 in fetal mouse dorsal root ganglia and spinal cord tissue cultures. In addition, treatment of the neonatal rat with capsaicin suggests that these receptors are preferentially localized on thin afferent fibers 12'21'43. However, with the exception of a few studies T M previous studies did not include data on the relative proportions of the 3 main types of opioid receptors. In addition there is no quantitative data available concerning their pre- and postsynaptic location (see, however, ref. 67). From a general point of view, the data related to opioid receptors within the spinal cord are difficult to interpret since: - a large number of binding studies have been performed on membrane preparations where it is impossible to locate the binding sites; most of the ligands used were not selective and do not allow a clear distinction between each type of receptor; various types and extents of peripheral lesions were used.
D. Besse, Unit6 de Recherche de Neurophysiologie Pharmacologique (I.N.S.E.R.M., Unit6 161), 2 rue d'AMsia, 75014 Paris,
0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
16 The aim of the present study, using quantitative autoradiography and highly selective ligands was (1) to determine, at the level of the superficial dorsal horn (laminae I and II), the relative percentages of ~, 6 and 7c binding sites; and (2) to gauge their relative pre- and
multiple range test, LSD (least significant difference) for multiple comparison. Unpaired Student's t-test was used when comparison was limited to two groups of data.
RESULTS
postsynaptic locations. For this latter purpose we used a large unilateral dorsal rhizotomy (C4-T2) corresponding to the brachial plexus.
Relative proportions of ~, 6 and ~c opioid receptors in the superficial layers of the cervical dorsal horn
MATERIALS AND METHODS
In control rats, all 3 ligands labeled the superficial layers of the dorsal horn. Rough examination of the
Experiments were performed on male Sprague-Dawley albino rats, weighing 200-225 g. Three control animals were used for the measurement of the respective proportions of/~, 6 and r receptors and 3 others were submitted to dorsal rhizotomies C 4 - T 2. In this case, the surgical procedures were performed using ketamine (100 mg/kg) as a general and xylocaine (2%) as a local anesthetic. A dorsal hemilaminectomy of vertebrae cervical 3 (C3) to thoracic 3 (T3) was made on the right side and the 7 corresponding dorsal roots C4-Tz (Fig. 1A) 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. The rats were sacrificed by decapitation 8 days after the rhizotomy. The cervical enlargement C4-T2 was removed rapidly and frozen in isopentane (-40 °C) and preserved at -80 °C. The whole cervical enlargement C4-T2 was then cut (frontal section, 15 /~m thick) from C4 to T2 on a cryostat (-20 °C), thaw-mounted onto gelatinized slides and kept at -80 °C up to incubation. The sections were then brought back to room temperature and incubated in 50 mM Tris-HCl buffer (pH 7.4) at 25 °C for 60 min with one of 3 ligands. [3H]DAGO (Tyr-D-Ala-Gly-(Me)Phe-Gly-ol) (1.85 TBq/mmol; CEN Saclay) was used at a final concentration of 3 nM to label/~ sites. [3H]DTLET (Tyr-D-Thr-Gly-Phe-Leu-Thr) (2.22 TBq/mmol; CEN Saclay) was used at a final concentration of 3 nM to label 6 sites. [3H]Ethylketocyclazocine ([3H]EKC) (0.55 TBq/mmol; NEN) was used at a final concentration of 10 nM, in the presence of 100 nM of cold DAGO and 100 nM of cold DTLET, to label r sites. For these 3 ligands, the non-specific binding was evaluated under the same conditions in the presence of 10-6 M levorphanol (Hoffman-La Roche). At the end of the incubation the sections were washed twice in ice-cold buffer for 10 min, rinsed in water and air-dried. Labeled sections were brought into close contact with tritium-sensitive film (Amersham) and exposed for 15 weeks at 4 °C. The autoradiograms were developed in Kodak D19. Quantitative measurements were performed with a Biocom analyser system at the level of the superficial layers (laminae I and II) of the dorsal horn. Fig. IB illustrates the area of the measurement. The grey values were converted to binding site density values by reference to tritium standards (Amersham). The specific binding was calculated by subtracting from the total binding the amount of ligand bound in sections incubated for non-specific labeling. The quantity of sites were calculated as: Bma x =
B (1 + Ka/L)
with B corresponding to the amount of bound ligand for a concentration L of ligand and Kd its affinity. For each ligand, systematic measurements were performed every 120/~m, over the whole length of the C 4 - T 2 segments. This procedure allows firstly, a large number of measurements, and secondly, any differences in opioid binding sites over different spinal segments to be observed. Some spinal cord sections were Nissl stained using Cresyl violet for histological determination of anatomical boundaries. Statistical analysis was made using ANOVA followed by the
autoradiograms revealed that the binding due to [3H]DAGO was more p r o n o u n c e d than for [3H]DTLET and that the binding due to [3H]EKC was weak. From binding studies performed on rat spinal cord slices (unpublished results), the K o values of the ligands were found to be 4.6, 1.7 and 1.7 n M for D A G O , D T L E T and EKC, respectively. Thus the Bmax values of [3H]DAGO, [3H]DTLET and [3H]EKC were calculated to be 90 _+ 5, 30 + 2 and 9 +_ 2 fmol per mg of tissue, respectively, for the whole C 4 - T 2 enlargement. Consequently, the relative percentages of kt, 6 and ~c receptors are 70 _+ 4%, 23 -+ 2% and 7 + 1% (Table I), respectively. These mean values were obtained for each ligand with between 110 and 130 m e a s u r e m e n t s per rat for both dorsal horns (left and right). The distribution of the 3 types of opioid binding sites was homogenous, since similar percentages were encountered for each segment. For example the values obtained in the central segment (C7) which are illustrated in Table I and in Fig. 3A are not significantly different from those found for the entire C 4 - T 2 region.
Relative percentages of pre- and postsynaptic binding sites at the cervical level In order to measure the relative percentages of preand postsynaptic binding sites, we compared the lesioned side to the intact side 8 days after a unilateral dorsal rhizotomy C4-T 2. Compared to the intact side, the autoradiograms show a marked decrease in kt, 6 and ~¢ binding sites at all levels of the deafferented segments. A n individual example is shown for/~ sites in Fig. lB. As shown in Fig. 2 and Table II, we have quantified the decrease in sites over the entire C 4 - T 2 enlargement for all 3 ligands. For/~ and 6 binding sites, the lesion induced a highly significant decrease (P < 0.001) in each segment. The decrease in ~ binding sites was more p r o n o u n c e d than that of ~ sites. This difference which was of the order of 15% for the entire C4-T 2 enlargement, is highly significant (P < 0.001) and was systematically observed for each spinal segment. The comparisons, segment to segment, of the percentages of binding for [ 3 H ] D A G O and for [3H]DTLET reveal significant differences between only C4-C 5 (P
