Down-regulation of μ-opioid receptors in rat and monkey dorsal root ganglion neurons and spinal cord after peripheral axotomy

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Neuroscience Vol. 82, No. 1, pp. 223–240, 1998 Copyright ? 1997 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/98 $19.00+0.00 S0306-4522(97)00240-6

DOWN-REGULATION OF µ-OPIOID RECEPTORS IN RAT AND MONKEY DORSAL ROOT GANGLION NEURONS AND SPINAL CORD AFTER PERIPHERAL AXOTOMY X. ZHANG,*† L. BAO,*†‡ T.-J. SHI,* G. JU,† R. ELDE§ and T. HO } KFELT*¶ *Department of Neuroscience, Karolinska Institute, Stockholm 171 77, Sweden †Department of Neurobiology, Institute of Neurosciences, 4th Military Medical University, Xian 710032, P.R. China ‡Department of Aviation Physiology, 4th Military Medical University, Xian 710032, P.R. China §Department of Cell Biology and Neuroanatomy, University of Minnesota, Minneapolis, MN 55406, U.S.A. Abstract––To understand the role of opioids and their receptors in chronic pain following peripheral nerve injury, we have studied the µ-opioid receptor in rat and monkey lumbar 4 and 5 dorsal root ganglion neurons and the superficial dorsal horn of the spinal cord under normal circumstances and after peripheral axotomy. Our results show that many small neurons in rat and monkey dorsal root ganglia, and some medium-sized and large neurons in rat dorsal root ganglia, express µ-opioid receptor-like immunoreactivity. Most of these neurons contain calcitonin gene-related peptide. The µ-opioid receptor was closely associated with the somatic plasmalemma of the dorsal root ganglion neurons. Both µ-opioid receptor-immunoreactive nerve fibers and cell bodies were observed in lamina II of the dorsal horn. The highest intensity of µ-opioid receptor-like immunoreactivity was observed in the deep part of lamina II. Most µ-opioid receptor-like immunoreactivity in the dorsal horn originated from spinal neurons. A few µ-opioid receptor-positive peripheral afferent terminals in the rat and monkey dorsal horn were calcitonin gene-related peptide-immunoreactive. In addition to pre- and post-junctional receptors in rat and monkey dorsal horn neurons, µ-opioid receptors were localized on the presynaptic membrane of some synapses of primary afferent terminals in the monkey dorsal horn. Peripheral axotomy caused a reduction in the number and intensity of µ-opioid receptor-positive neurons in the rat and monkey dorsal root ganglia, and of µ-opioid receptor-like immunoreactivity in the dorsal horn of the spinal cord. The decrease in µ-opioid receptor-like immunoreactivity was more pronounced in the monkey than in the rat dorsal root ganglia and spinal cord. It is probable that there was a parallel trans-synaptic down-regulation of µ-opioid-like immunoreactivity in local dorsal horn neurons of the monkey. These data suggest that one factor underlying the well known insensitivity of neuropathic pain to opioid analgesics could be due to a marked reduction in the number of µ-opioid receptors in the axotomized sensory neurons and in interneurons in the dorsal horn of the spinal cord. ? 1997 IBRO. Published by Elsevier Science Ltd. Key words: endorphins, morphine, nerve injury, pain, sensory neurons.

The three opioid receptors, the µ-, ä- and ê-opioid receptors,15,20,50,60,75,76 have a wide distribution in the nervous system and are involved in numerous regulatory functions (see Refs 8, 34, 48 and 71). Using autoradiographic ligand binding methodology, evidence has been obtained that all three receptor subtypes are present in the dorsal horn of the spinal cord.6,9–12,27,30,31,38,46,78 This region contains the terminations of nociceptive primary afferents (see Refs 13 and 87) and, in fact, there was early evidence from ligand binding studies for the existence of opioid ¶To whom correspondence should be addressed. Abbreviations: CCK, cholecystokinin; CGRP, calcitonin gene-related peptide; DRG, dorsal root ganglion; L, lumbar; LDCV, large dense-core vesicle; LI, like immunoreactivity; PB, phosphate buffer; PBS, phospatebuffered saline.

receptors on primary sensory neurons.29,69,70,90 This is in agreement with a decrease in all three types of opioid binding sites in the dorsal horn after transection of the dorsal roots.11,28 Recently, all three opioid receptors have been cloned, that is the ä-opioid receptor,14,25,44 the µ-opioid receptor16,68,80,83 and the ê-opioid receptor.17,62,67,68,89 This has permitted localization of receptor mRNA at the cellular level with in situ hybridization,51,52,56–59,65–67,74 and of the receptor protein using immunohistochemistry and antibodies raised against peptide sequences of the receptor.3–5,21,35,37,41,53,55,93 Some of these studies focus on the expression of opioid receptors in dorsal root ganglia (DRGs) and spinal cord, i.e. on the first- and second-order neurons in the processing of pain sensation. Thus, both immunohistochemical and

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in situ hybridization studies have provided evidence for the existence of all three receptor types both in the superficial layers of the spinal cord as well as in DRGs.3–5,18,21,37,41,51–54,56,57,65,67 Earlier studies have described plasticity of opioid receptor binding at the spinal level after inflammatory pain.12,38,43,63,64,77 In a recent immunohistochemical study on rat, it was shown that carrageenan-induced inflammation causes an increase in the percentage of µ-opioid receptorpositive neurons and a decrease in ä-opioid receptorand ê-opioid receptor-positive neurons in DRGs.41 Moreover, the expression of µ-opioid receptor and ê-opioid receptor mRNAs, but not ä-opioid receptor mRNA, is increased in the dorsal horn of arthritic rats.51 Peripheral nerve injury (axotomy) in the rat is followed by a massive discharge in damaged sensory fibers, the so-called injury discharge first described by Wall and Gutnick,82 and spontaneous activity will subsequently arise in the neuroma and continue for several weeks.32 Such ongoing discharges may play a role in the development of neuropathic pain (see Ref. 73). Peripheral nerve injury is also known to markedly change expression of both peptides and peptide receptors in DRG neurons.36 It is well known that, in contrast to inflammatory pain, neuropathic pain is comparatively insensitive to opioid treatment.1,2,24,45,79 This is also true for the rat.88 This may be partly due to a reduction in expression of opioid receptors after nerve injury.10,28,40,49,77 We have now analysed the expression of µ-opioid receptors in DRG neurons and in the dorsal horn of the spinal cord of rat and monkey after peripheral nerve injury using immunohistochemistry and antibodies raised to peptide sequences of this opioid receptor,4 as well as in situ hybridization and oligonucleotide probes complementary to sequences of this opiate receptor (for references see above). EXPERIMENTAL PROCEDURES

Immunohistochemistry Fifteen male Sprague–Dawley rats (body wt 250–300 g, ALAB, Stockholm, Sweden) and three male monkeys (Macaca mulatta; 3–4 kg) were anesthetized with sodium pentobarbital (Mebumal; 60 mg/kg, i.p.), and the left sciatic nerve was transected above trochanter major. In all cases a 3–5 mm portion of the nerve was resected. The experiments on rats had been approved by a local ethical committee (Stockholms norra djurfo¨rso¨ksetiska na¨mnd). The operation on monkeys was carried out in the Department of Neurobiology, Institute of Neurosciences, Xian, Shannxi, P.R. China. The experiments had been approved by the Chinese National Committee on the Use of Experimental Animals for Medical Purposes, Shaanxi branch. The rats were allowed to survive for two, seven, 14, 21 and 28 days (monkeys only 14 days). Operated animals, as well as 10 rats and three male monkeys used as controls, were deeply anesthetized and perfused transcardially with warm (37)C), Ca2+-free Tyrode’s solution followed by a fixative containing 4% paraformaldehyde and 0.2% picric acid in 0.16 M phosphate buffer (PB) for 6 min.91 The L4 and L5 spinal cord segments and the L4 and L5 DRGs were quickly

dissected out, immersed in the same fixative for 90 min, and then in 10% sucrose buffer solution containing 0.01% sodium azide and 0.02% bacitracin (Bayer, Leverkusen, Germany) for at least 24 h. Before sectioning, the experimental and control tissues were always mounted on the same block and fused with 10% sucrose. The tissues were cut into 14-µm-thick sections in a cryostat (Microm, Heidelberg, Germany). The ganglia were cut longitudinally. The sections were processed according to the indirect immunofluorescence technique (see Ref. 19) or the immunoperoxidase method. For the immunofluorescence staining, the sections were incubated in a humid chamber with rabbit antiserum against the µ-opioid receptor (1:4000),4 or a mixture of rabbit antiserum against µ-opioid receptor and mouse monoclonal antibodies against calcitonin generelated peptide (CGRP; 1:400; Celltech Limited, Slough, Berkshire, U.K.) for 24 h at 4)C, rinsed, incubated with fluorescein isothiocyanate-conjugated goat anti-rabbit antibodies (1:80; Boehringer Mannheim Scandinavia, Stockholm, Sweden) or a mixture of fluorescein isothiocyanate-labeled donkey anti-rabbit antibodies (Jackson ImmunoResearch, West Grove, PA, U.S.A.) and lissamine rhodamine-labeled donkey anti-mouse antibodies (Jackson ImmunoResearch) for 30 min in a humid atmosphere at 37)C, mounted in a mixture of glycerol/ phospate-buffered saline (PBS) (3:1) containing 0.1% paraphenylenediamine.42,72 The sections were examined in a Nikon Microphot-FX microscope equipped with epifluorescence and proper filter combinations or a Bio-Rad MRC600 laser scanning confocal imaging system. For the immunoperoxidase staining, the sections were incubated with µ-opioid receptor antibody (1:8000) for 48 h at 4)C, followed by biotinylated goat anti-rabbit immunoglobulin G (1:200) for 3 h at 22)C and then avidin–biotin–peroxidase complex (1:100; Vector ABC kit, Vector, Burlingame, CA, U.S.A.) for 3 h at 22)C. The sections were incubated for 5–10 min in a medium containing 60 mg of 3,3*diaminobenzidine and 330 µl of 30% H2O2 in 0.01 M PBS, rinsed with PBS, dried in air, dehydrated in increasing concentrations of ethanol, cleared in xylene and mounted with coverslips. The sections were examined in a Nikon Microphot-FX microscope. Electron microscopy Four male Sprague–Dawley rats (body wt 250 g; ALAB) and two male monkeys (M. mulatta; 3–4 kg) were anesthetized with sodium pentobarbital and perfused transcardially with warm (37)C) saline followed by a fixative containing 4% paraformaldehyde, 0.05% glutaraldehyde and 0.02% picric acid in 0.1 M PB for 15 min. The L4 and L5 spinal cord segments, the L4 and L5 DRGs, and the L4 and L5 dorsal roots were quickly dissected out and immersed in the same fixative for 150 min. The tissues were cut into 50-µmthick sections with a Vibratome>. The sections were immersed in 20% sucrose and subjected to freeze–thaw treatment. The sections were processed according to the immunoperoxidase method or immunogold–silver enhancement method. All sections were incubated in a humid chamber with rabbit antiserum against the µ-opioid receptor (1:8000) for 48 h at 4)C and rinsed. Some sections were incubated with biotinylated goat anti-rabbit immunoglobulin G (1:200) followed by avidin–biotin–peroxidase complex (1:100; Vector ABC kit). The sections were incubated for 10–15 min in a medium containing 60 mg of 3,3*diaminobenzidine and 330 µl of 30% H2O2 in 0.01 M PBS. Other sections were incubated with goat anti-rabbit immunoglobulin G conjugated with gold particles which were 1.4 nm in diameter (1:100; Nanoprobes Inc., Stony Brook, NY, U.S.A.) for 2 h at 22)C, then rinsed with PBS for 15 min and fixed in 1% glutaraldehyde for 15 min. The sections were rinsed in deionized water and incubated with the silver enhancement solution (HQ Silver=, Nanoprobes) in a dark room for 4–6 min at 22)C. All sections were rinsed

µ-Opioid receptor in sensory and dorsal horn neurons in deionized water and 0.1 M PB, and fixed in 1% osmium tetroxide for 30 min. Dehydration was curried out in increasing concentrations of ethanol. After passing through propylene oxide and incubation in Epon 812, the sections were flat embedded in Epon 812 between two sheets of plastic film. The superficial dorsal horn containing µ-opioid receptor-like immunoreactivity (LI) was selected under the light microscope and mounted on blank resin stubs. Ultrathin sections were cut on an LKB III ultratome and counterstained with uranyl acetate and lead citrate. The sections were examined in a Jeol-1200 electron microscope. Control for immunohistochemistry The specificity of the CGRP or µ-opioid receptor antisera was tested by absorption with synthetic CGRP (Peninsula, Belmont, CA, U.S.A.) or synthetic µ-opioid receptor peptide4 at a concentration of 1 µM for 24 h at 4)C. Control experiments were also carried out by omission of primary antisera or substitution of primary antisera with normal rabbit or mouse serum. In situ hybridization Twenty adult male Sprague–Dawley rats (body wt 200– 250 g; ALAB) were deeply anesthetized with sodium pentobarbital (Mebumal; 60 mg/kg, i.p.), and the left sciatic nerve was transected above trochanter major. A 3–5 mm portion of the nerve was resected. The animals were allowed to survive for two, seven, 14, 21 and 28 days (four animals in each group). Lesioned animals and four normal control animals were deeply anesthetized (as above) and perfused via the aorta with 50 ml warm (37)C) saline to clear the blood, followed by rapid dissection and freezing of L4 and L5 DRGs. Before sectioning, experimental and normal DRGs were fused by saline on the same blocks, so that all groups could be processed on the same slide. Sections (14 µm) were cut in a cryostat (Microm, Heidelberg, Germany) and thawed on to ‘‘Probe On’’ slides (Fisher Scientific, Pittsburgh, PA, U.S.A.) and stored in sealed boxes at "20)C until hybridization. Three oligonucleotide probes complementary to nucleotides 270–317, 536–583 and 1422–1469 of the µ-opioid receptor16 were labeled at the 3* end with á-[35S]dATP (New England Nuclear, Boston, MA, U.S.A.) using terminal deoxynucleotidyl transferase (Amersham, Amersham, U.K.) in a buffer containing 10 mM CoCl2, 1 mM dithiothreitol, 300 mM Tris base and 1.4 M potassium cacodylate (pH 7.2). Subsequently, the labeled probe was purified through an Nensorb-20 column (New England Nuclear) and dithiothreitol was added to a final concentration of 10 mM. The specific activities obtained ranged from 1 to 4#106 c.p.m./ng oligonucleotide. Our procedure followed previously published protocols.22 Sections were hybridized after thawing without pretreatment for 16–18 h at 42)C in humidified boxes with 0.5 ng of labeled probe per 100 µl hybridization cocktail. After hybridization, the sections were rinsed repeatedly (4#15 min) in 1#standard saline citrate at 55)C, then brought to room temperature over 30 min while in the final rinse, dipped twice in distilled water, dehydrated through 60% and 95% ethanol, and dried in air. The slides were dipped in NTB2 nuclear track emulsion (Kodak, Rochester, NY, U.S.A.) diluted 1:1 with distilled water, exposed in the dark at "20)C for six weeks, developed in D-19 (Kodak) for 3 min, fixed in Kodak 3000A&B for 6 min and rinsed for 30 min in running water. Developed slides were mounted with glycerol and coverslipped for analysis in a Nikon Microphot-FX microscope equipped with a dark-field condenser or stained with Toluidine Blue, mounted with Entellan (Merck, Darmstadt, Germany) and a coverslip for viewing under bright field.

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For control, hybridizations were carried out with an excess of cold probe (100-fold), together with the labeled probe. Quantification To determine the percentage of labeled neuron profiles, counts were done on sections processed for the immunoperoxidase method and on sections processed for in situ hybridization which were stained with Toluidine Blue. For each method, every fourth section of a series of sections, five to eight sections in total for each L5 DRG, from three control rats and three injured rats at each time-point were selected. The counts for µ-opioid receptor-positive neuron profiles in monkey DRGs were carried out on the sections processed for the immunoperoxidase method. In total, five sections at different levels of the L5 DRG from three control monkeys and three axotomized monkeys were analysed. The sections were examined under bright field using a #20 objective lens. In the immunostained sections, the neurons with immunoreaction product in the cytoplasm, especially in the perinuclear region, and along the somatic plasmalemma were counted. In the hybridized sections, the neurons with three times more grains than background grain densities were counted. Mean background grain densities (two to six grains per 100 µm2) were determined by averaging grain counts over defined areas of the neuropil devoid of positively labeled cell bodies. Five-hundred and fifty-three to 1177 neurons from the immunostained sections of each DRG, and 720–900 neurons from the hybridized sections of each DRG were analysed. The total number of labeled neuron profiles was divided by the total number of neuron profiles. The size of neuronal profiles in the DRGs and the density of fiber network in the dorsal horn were measured on a Macintosh IIx computer equipped with a Quick Capture frame grabber board (DATA Translation, Marlboro, MA, U.S.A.) and a Dage-MTI 72 CCD camera (DAGE-MTI, Michigan City, IN, U.S.A.) connected to a Nikon microscope. Image processing was performed with NIH-image software (courtesy of W. Rasband, NIMH). The sizes of 200 neuron profiles containing µ-opioid receptor-LI and 400 neuron profiles with or without µ-opioid receptor-LI from both control and ipsilateral DRGs 14 days after unilateral peripheral axotomy were measured. Neuron profiles with an area less than 1000 µm2 and a nucleus, and neuron profiles with area larger than 1000 µm2 and with or without a nucleus were analysed. The relative density levels of immunostaining were measured in the medial half of the superficial dorsal horn of the rat spinal cord. Each image was digitized with 256 gray levels for each picture element. Ten sections of each spinal cord from three control rats, or three rats killed seven or 14 days after unilateral sciatic nerve transection were analysed. The data were assessed with the analysis of variance (ANOVA) test and the unpaired two-tailed t-test. All data are presented as mean&S.E.M. RESULTS

Distribution of µ-opioid receptors and µ-opioid receptor messenger RNA in normal dorsal root ganglion neurons Rat. About 50% of the rat DRG neuron profiles were µ-opioid receptor-immunoreactive (Figs 1a, c, 2). They were mainly small and medium-sized (Figs 1, 3a). Most (81%) µ-opioid receptor-positive neuron profiles contained CGRP-LI (of 237 counted neuron profiles, 191 profiles contained both µ-opioid receptor- and CGRP-LIs, 13 only µ-opioid receptor and 33 only CGRP-LI) as seen after double-

Fig. 1. (a–k) Bright-field (a, b), confocal (c, k), fluorescence (d, e, i, j) and dark-field (f–h) micrographs (c, k) of normal rat (a, c–f) and monkey (i, k) L5 DRGs, and of ipsilateral DRGs of rat (b, g) and monkey (j), and contralateral DRG of rat (h) 14 days after unilateral sciatic nerve-cut. The sections have been processed for the immunoperoxidase method (a, b), single (c, i–k) or double (d, e) labeling immunofluorescence histochemistry using µ-opioid receptor antiserum, or a mixture of µ-opioid receptor and CGRP antisera, or in situ hybridization (f–h) with a mixture of probes for µ-opioid receptor mRNA. (a) In normal rat DRGs, many small and some large neurons (arrowhead) contain µ-opioid receptor-LI. (b) The number of µ-opioid receptor-positive neurons (arrowheads) is reduced in the ipsilateral DRGs after peripheral axotomy. (c) µ-Opioid receptor-LI is located along the somatic plasmalemma (arrowheads) and in the perinuclear region of rat DRG neurons. (d, e) µ-Opioid receptor- and CGRP-LIs are co-localized in rat DRG neurons (arrows). Stars mark neurons containing only CGRP-LI. (f, g) Many small DRG neurons are µ-opioid receptor mRNA-positive in normal (f) and contralateral (h) rat DRGs. Their number is decreased on the ipsilateral side (g). (i–k) µ-Opioid receptor-LI is seen in monkey DRG neurons, especially along the somatic plasmalemma (i, k), with a marked reduction in number ipsilaterally after peripheral axotomy (j). Scale bars=100 µm (a, b, f–h, i, j), 10 µm (c, k), 50 µm (d, e).

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Fig. 2. Percentage of µ-opioid receptor-immunoreactive neuron profiles in rat DRGs of control rat and in ipsi- (Ipsi) and contralateral (Contra) DRGs two, seven, 14 and 28 days after unilateral sciatic nerve-cut. The number of µ-opioid receptor-positive neuron profiles in ipsilateral DRGs is decreased. The data are presented as mean&S.E.M. **P<0.01 as compared to the control.

immunostaining (Fig. 1d, e). In situ hybridization showed that a similar, but much smaller, subpopulation of DRG neurons expressed detectable levels of µ-opioid receptor mRNA (Fig. 1f). µ-Opioid receptor-LI was observed near the somatic plasmalemma and in the perinuclear region under the confocal microscope (Fig. 1c). Localization of µ-opioid receptor-LI in the Golgi complex and in the somatic plasmalemma was revealed by immunoelectron microscopy (Fig. 4a, b). However, no certain subcellular structures could be distinctly associated with µ-opioid receptor-LI after its ‘‘release’’ from the Golgi complex. Monkey. Eighteen percent (18.3&1.2%) of the neuron profiles counted in monkey DRGs were µ-opioid receptor-immunoreactive (Figs 1i, k, 2b); they were mainly of small size (Fig. 3c). µ-Opioid receptor-LI was co-localized with CGRP-LI in these neurons. Of 379 counted neuron profiles, 107 profiles (or 28%) contained both µ-opioid receptor- and CGRP-LIs, and 272 only CGRP-LI. The striking feature of these neurons was a very strong µ-opioid receptor-LI along the somatic plasmalemma (Fig. 1i, k). The immunostaining in the perinuclear region was moderate. At the ultrastructural level, an intense immunolabeling for µ-opioid receptor was seen under the somatic plasmalemma which formed many invaginations, and in the cytoplasm nearby (Fig. 4c). These neuronal invaginations were often closely apposed by processes of satellite cells (Fig. 4d). A strong immunolabeling for µ-opioid receptor was also observed in the axoplasm and on the plasmalemma of unmyelinated nerve fibers in the monkey dorsal roots (Fig. 4e).

Distribution of µ-opioid receptors in the dorsal horn of the spinal cord Rat. In laminae I and II of rat L4 and L5 spinal cord segments, µ-opioid receptor-LI was seen with the highest intensity in the deeper part of lamina II, where µ-opioid receptor-positive structures often formed patches (Fig. 5a). Many µ-opioid receptorimmunoreactive neuronal cell bodies were observed in deep lamina II (Fig. 5a). The distribution of µ-opioid receptor-LI in laminae I and II was very similar to that of CGRP-LI (Fig. 5a, b), but only a few nerve fibers could be observed to contain with certainty both µ-opioid receptor- and CGRP-LIs. Under the electron microscope, lamina I was characterized by a high density of thin, myelinated fibers, whereas lamina II had only low numbers of such fibers, which again became abundant in lamina III. In lamina II, only a few glomeruli were weakly labeled for µ-opioid receptor-LI, with the reaction product often close to the plasmalemma (Fig. 6a). The labeled glomeruli contained only a few large dense-core vesicles (LDCVs). In contrast, glomeruli containing many LDCVs were not labeled for µ-opioid receptor-LI. There were many strongly stained dendrites in lamina II, especially in its deep part (Fig. 6b). Synaptic contacts between µ-opioid receptor-positive dendrites and glomeruli were often encountered (Fig. 6b). µ-Opioid receptor-LI was localized on the somatic plasmalemma of neurons which had a thin rim of dense cytoplasm (Fig. 6c, d). µ-Opioid receptor-immunoreactive cell bodies often received synapses from axonal terminals (Fig. 6e). Some small µ-opioid receptor-positive axonal terminals formed synapses on dendrites (Fig. 6f).

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Fig. 4. (a–e) Electron micrographs of neurons in normal rat (a, b) and monkey (c, d) DRGs and monkey dorsal root (e) processed for pre-embedding immunogold labeling with silver enhancement (a–c, e) or the immunoperoxidase method (d). (a, b) µ-Opioid receptor-LI is localized in the Golgi complex (a), and along the somatic plasmalemma (arrowheads) next to a satellite cell (Sc; star indicates cell body) (b). (c) µ-Opioid receptor-LI is concentrated along the invaginations of the surface of the cell body (star) in a monkey DRG. (d) Numerous gold–silver particles for µ-opioid receptor-LI are seen in the cytoplasm near the somatic plasmalemma of a monkey DRG neuron (star), and many of them are on the plasmalemma of the invaginations (arrowheads). (e) Numerous gold–silver particles for µ-opioid receptor-LI are seen in an unmyelinated fiber in the L5 dorsal root, and many of them are located on the plasmalemma (arrowheads). Scale bars=200 nm (a, b), 250 nm (c–e).

Immunogold–silver staining showed that µ-opioid receptor-LI was localized on the plasmalemma of the dendrites outside the postsynaptic membrane specializations (Fig. 6e, g). Monkey. In lamina I and outer lamina II of the monkey spinal cord, the µ-opioid receptor-LI was less intense than that seen in rat, but a strong µ-opioid receptor-LI was seen in deep lamina II (Fig.

7a, d). The distribution of µ-opioid receptor-LI in deep lamina II was similar to the CGRP-LI in this region (Fig. 7a, b), but only some µ-opioid receptorpositive nerve fibers seemed to contain CGRP-LI. The µ-opioid receptor-positive structures formed distinct patches. Some µ-opioid receptor-positive cell bodies and many fibers were visualized in these patches with the confocal microscope (Fig. 7c, d). Under the electron microscope, µ-opioid receptor-

Fig. 3. (a–c) Size range of µ-opioid receptor-immunoreactive neuron profiles in normal (a) and axotomized (b) rat and normal monkey (c) DRGs. Two hundred positive neuron profiles and 400 neuron profiles with or without µ-opioid receptor-LI were measured in the normal and ipsilateral rat DRGs. One hundred positive neuron profiles and 400 neuron profiles were measured in the normal monkey DRGs. Most µ-opioid receptor-positive profiles range below 900 µm2 in both rat (a) and monkey (c) DRGs. There is no significant change in the size range of µ-opioid receptor-positive neuron profiles in the ipsilateral DRGs (cf. b with a).

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Fig. 5. (a, b) Fluorescence micrographs of normal rat dorsal horn processed for doubleimmunofluorescence for µ-opioid receptor- (a) and CGRP-LI (b). µ-Opioid receptor-immunoreactive structures are observed in laminae I and II with a higher density in deep lamina II, where positive cell bodies are seen (arrows in a). CGRP-positive nerve fibers are seen in laminae I and II. µ-Opioid receptorand CGRP-LIs have a similar distribution, but only single CGRP-positive nerve fibers (arrowheads) are clearly shown to contain µ-opioid receptor-LI (b). Scale bars=50 µm (a, b).

immunoreactive dendrites, axons and axonal terminals were observed in lamina II. Among 985 random µ-opioid receptor-positive structures analysed in lamina II, 90.4% were dendrites, 8.3% axons and 1.3% glomeruli. In lamina II, 25% of all glomeruli were µ-opioid receptor-positive (100 glomeruli analysed), and 69.2% of the µ-opioid receptorpositive glomeruli contained many LDCVs. Conversely, 39.1% of the glomeruli containing many LDCVs were µ-opioid receptor-positive. In deep lamina II, µ-opioid receptor-immunoreactive cell bodies had a thin rim of translucent cytoplasm, and axosomatic synapses were observed (Fig. 8a). Many µ-opioid receptor-negative glomeruli were surrounded by µ-opioid receptor-immunoreactive dendrites (Fig. 8b, d). The strongly labeled glomeruli usually did not form synaptic contacts with µ-opioid receptor-positive dendrites (Fig. 8c), but could receive synapses from µ-opioid receptor-negative glomeruli (Fig. 8d). Immunogold–silver staining revealed that the µ-opioid receptor-LI was mainly localized on the plasmalemma of the axonal terminals and dendrites (Fig. 9a–f). µ-Opioid receptor-LI was localized outside of the pre- and postsynaptic membrane specializations in axonal terminals and dendrites, respectively (Fig. 9a–c). However, localization of µ-opioid receptor-LI on the presynaptic mem-

brane was sometimes observed at certain synapses of some glomeruli, while other presynaptic membrane segments of the same glomeruli did not have any detectable µ-opioid receptor-LI (Fig. 9d–f). Reduction of µ-opioid receptor-like immunoreactivity and µ-opioid receptor messenger RNA in dorsal root ganglion neurons and spinal dorsal horn after peripheral axotomy Rat. In the rat, unilateral sciatic nerve transection caused about 30% reduction in the number of µ-opioid receptor-immunoreactive neurons in the ipsilateral DRGs (Fig. 1b, Fig. 2). The decrease in the number of µ-opioid receptor-positive neurons was already seen two days after axotomy. µ-Opioid receptor-immunoreactive DRG neurons remaining in the ipsilateral DRGs were mainly small in size (Fig. 3b). There were no significant changes in the number of µ-opioid receptor-immunoreactive neurons in the contralateral DRGs (Fig. 2). In situ hybridization also showed a 50% decrease in the number of µ-opioid receptor mRNA-positive neuron profiles in the ipsilateral DRGs (Fig. 1f–h; 10% of all counted neuron profiles were µ-opioid receptor mRNApositive in controls, and 5% in the ipsilateral DRGs after axotomy).

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Fig. 6. (a–g) Electron micrographs of µ-opioid receptor-LI in lamina II of the rat spinal cord, processed with pre-embedding immunoperoxidase (a–d, f) or immunogold labeling with silver enhancement (e, g). (a) A µ-opioid receptor-immunoreactive axonal terminal (star) makes synaptic contacts (arrows) with three µ-opioid receptor-negative dendrites. The immunoreactivity has a patchy localization close to the plasmalemma (arrowheads). (b) Three µ-opioid receptor-positive dendrites (small stars) and a negative one form synapses with an axon terminal (big star). (c) µ-Opioid receptor-LI is located along the somatic plasmalemma (arrowheads) of a neuron which has a thin rim of dense cytoplasm. (d) High magnification of the boxed area in (c). Note µ-opioid receptor-LI in patches close to the plasmalemma. (e) An axonal terminal (star) makes a synaptic contact with a neuron (open star). µ-Opioid receptor-LI is localized on the somatic plasmalemma (small arrowheads) outside the postsynaptic membrane (arrows), also close to the postsynaptic zone (arrowheads). (f) A small µ-opioid receptor-positive axonal terminal (star) synapses on a dendrite (arrow). An axonal terminal (star) makes synaptic contacts (arrows) with two dendrites. (g) µ-Opioid receptor-LI (arrowhead) is seen on the plasmalemma outside the postsynaptic density of a dendrite. Scale bars=250 nm (a, b, d–g), 500 nm (c).

µ-Opioid receptor-LI in the superficial dorsal horn was reduced after peripheral axotomy. The intensity of µ-opioid receptor-LI in the medial half of the ipsilateral dorsal horn was reduced to 57.9&3.1% (P<0.05) of that of the controlateral side seven days after axotomy, and to 48.1&5.1% (P<0.05) 14 days after axotomy. No significant change was detected between contralateral side and control dorsal horn.

Monkey. In the monkey, only single µ-opioid receptor-immunoreactive neurons were observed in the ipsilateral DRGs 14 days after unilateral sciatic nerve-cut (Fig. 1b; 0.5&0.2% of counted neuron profiles; P<0.01 as compared to the control). No significant change in µ-opioid receptor-LI was seen in the contralateral DRGs (17.7&1.3% of counted neuron profiles vs 18.3&1.2% in the normal ganglia).

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Fig. 7. (a–e) Immunofluorescence micrographs (a, b, d, e) and a confocal micrograph (c) of sections of the superficial dorsal horn of normal monkey (Con; a–d) and 14 days after unilateral sciatic nerve cut (e). (a, b) On the same section, µ-opioid receptor-immunoreactive structures are mainly observed in deep lamina II of monkey spinal cord. (b) However, CGRP-positive nerve fibers distribute in laminae I and II. Some nerve fibers contain both µ-opioid receptor- and CGRP-LIs (arrowheads) (cf. a with b). (c) µ-Opioid receptor-LI is located along the somatic plasmalemma (arrowhead) of a cell body among many fine, µ-opioid receptor-positive fibers in deep lamina II. (e) Fourteen days after axotomy, µ-opioid receptor-LI is markedly decreased in lamina II of the ipsilateral dorsal horn. Scale bars=100 µm (a, b, d, e), 10 µm (c).

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Fig. 8. (a–d) Electron micrographs of lamina II of monkey spinal cord processed with the pre-embedding immunoperoxidase method. (a) A µ-opioid receptor-negative axonal terminal makes synaptic contact (arrow) with a µ-opioid receptor-positive cell body. (b) A µ-opioid receptor-negative axonal terminal (star) forms synapses (arrows) with four µ-opioid receptor-positive dendrites (small stars) and a µ-opioid receptor-negative dendrite (open star). Arrowheads point to synapses on a µ-opioid receptor-positive dendrite. (c) Two big µ-opioid receptor-immunoreactive axonal terminals contain many LDCVs (1 and 2) and form synapses with several µ-opioid receptor-negative dendrites (arrows). (d) A µ-opioid receptornegative nerve terminal (open star) makes synapses (arrows) with a µ-opioid receptor-positive (star), a µ-opioid receptor-negative (small open star) and a big µ-opioid receptor-positive terminal (star), which in turn forms a synapse (arrowhead) with a µ-opioid receptor-negative dendrite. Scale bars=500 nm in all micrographs.

A marked reduction in µ-opioid receptor-positive nerve fibers was seen in the ipsilateral dorsal horn after axotomy (Fig. 7e), whereas no marked changes were seen in the contralateral dorsal horn. Controls The control experiments showed that absorption of the antisera at their working dilution with the appropriate peptides resulted in abolition of the immunostainings. Omission of primary antibodies or substitutions of primary antibodies with normal rabbit or mouse serum resulted in disappearance of the immunostainings. In the in situ hybridization experiments an excess of cold probe added to the incubation cocktail abolished the signals described above.

DISCUSSION

Control dorsal root ganglia The present results demonstrate expression of µ-opioid receptors in both rat and monkey DRG neurons using immunohistochemistry, whereby around 50% of all DRG neuron profiles are positive in the rat vs somewhat less than 20% in the monkey. In both species, the majority of neurons are of small size, but in the rat there are also some medium-sized and large neurons. Whether the differences in number and size distribution represent true species differences or a lower sensitivity of our technique for monkey tissue remains to be shown. The apparent difference should be treated with caution in view of the low number of animals studied, and in view of the fact

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Fig. 9. (a–f) Two sets of electron micrographs of two nerve terminals (a–c and d–f) labeled with immunogold–silver particles for µ-opioid receptor-LI in lamina II of monkey spinal cord. (a–c) Three adjacent sections show µ-opioid receptor-LI on the plasmalemma outside the presynaptic membrane. Arrowheads indicate the postsynaptic zone and star the postsynaptic dendrite. (d–f) Immunogold–silver particles for µ-opioid receptor-LI are located on the plasmalemma of a nerve terminal (star) which makes synaptic contact with three dendrites (D1–D3). There is no µ-opioid receptor-LI on the presynaptic membrane at the synapses on dendrites D1 and D3. However, the presynaptic membrane at the synapse on dendrite D2 is distinctly labeled for µ-opioid receptor-LI (arrows). Postsynaptic zones are indicated by arrows. Scale bars=250 nm in all micrographs.

that objective stereological techniques33 were not applied. Furthermore, it cannot be excluded that the post-transcriptional modification differs between rat and monkey. In an earlier study using the same µ-opioid receptor antiserum, we observed µ-opioid receptor-LI in somewhat more than 20% of all DRG neuron profiles in the rat.41 The present figure of 50% is the result of improving our histochemical technique and is more

in line with the in situ hybridization study of Minami et al.,65 who reported µ-opioid receptor mRNA in about 60% of all DRG neurons. In contrast, our in situ hybridization results only show µ-opioid receptor mRNA in about 10% of all DRG neuron profiles, presumably due to our use of oligonucleotide probes vs the more sensitive riboprobe system used by Minami et al.65 We were not successful in detecting mRNA in the monkey DRGs with

µ-Opioid receptor in sensory and dorsal horn neurons

in situ hybridization, maybe due to species differences in the nucleotide sequences of the receptors. The results are in good agreement with autoradiographic studies showing that DRG neurons contain µ binding sites.10,12,28,92 Expression of µ-opioid receptors has also been demonstrated in DRG neurons by other groups. Thus, Mansour et al.56,57 found µ-opioid receptor mRNA mainly in medium- and large-diameter ganglion cells, but did not provide any quantitative information. Maekawa et al.52 demonstrated µ-opioid receptor mRNA in many DRG neurons, and the quantification by Minami et al.65 revealed, as mentioned above, that µ-opioid receptor mRNA is expressed at a high level in 55% and at low level in 5% of all DRG neurons. Around 40% of the neurons expressing high levels of µ-opioid receptor mRNA were positive for preprotachykinin mRNA, and conversely almost all preprotachykinin mRNApositive neurons expressed µ-opioid receptor mRNA.65 The fact that almost all substance P DRG neurons, which are known to be mainly small and some medium-sized neurons, express µ-opioid receptor mRNA is in agreement with the presence of µ-opioid receptor-LI mainly in small neurons41 (and present results). In agreement, Arvidsson et al.4 showed that the majority of µ-opioid receptorpositive neurons do not stain for RT97, a marker for large myelinated primary afferents.47 A substantial proportion of the µ-opioid receptorpositive DRG neurons contained CGRP-LI, both in rat (81%) and monkey (28%). However, in the dorsal horn it was only possible to detect co-existence of µ-opioid receptor- and CGRP-LI in few fibers. This may be due to the high density of these fibers, but also to low levels of µ-opioid receptor-LI present in the central afferent branches, perhaps reflecting a low rate of centrifugal transport. In fact, both in rat and monkey, confocal and electron microscopic analyses showed that µ-opioid receptor-LI is located abundantly on the somatic plasmalemma of the DRG neurons, indicating an important role for µ-opioid receptors at the somatic level, i.e. in the DRGs. Especially in the monkey DRG neurons, the accumulation of µ-opioid receptor-LI close to the membrane of extensive invaginations of the cell surface and in the nearby cytoplasm was seen, suggesting that a large pool of µ-opioid receptors is accessible in the cell bodies of the monkey DRG neurons under normal circumstances, i.e. receptor protein is not only built into the membrane, but is accumulated in apparently large amounts in the adjacent cytoplasm. Control dorsal horn As also shown in earlier immunohistochemical studies, there is a dense network of µ-opioid receptorpositive fibers in the superficial layers of the dorsal horn.4,41 Dorsal rhizotomy partially reduced this immunoreactivity, especially in the most superficial layers of the dorsal horn, whereas a dense band

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remained in lamina II.4 Thus, µ-opioid receptors may be transported from DRG neurons to the superficial layers of the dorsal horn. In agreement, µ-opioid receptor-LI accumulates mainly proximal to a ligation of the sciatic nerve.41,94 However, the rhizotomy experiments4 and the immunohistochemical analysis of the dorsal horn, revealing small µ-opioid receptorimmunoreactive cell bodies in lamina II4 (and present results), as well as the demonstration of µ-opioid receptor mRNA in the dorsal horn,52 clearly demonstrate the existence of µ-opioid receptor-positive local neurons. In the monkey there is a dense fiber plexus in the superficial layers, but in lamina I and outer lamina II the fibers are only weakly fluorescent, whereas there is an intensely fluorescent band in lamina II. Confocal microscopic analysis revealed the presence of µ-opioid receptor-immunoreactive cell bodies and many fibers in the latter band. It is well established that primary afferent terminals form glomeruli, composed of a central terminal and several postsynaptic elements, in lamina II of the spinal cord (see Refs 61 and 87). The results of the electron microscopic study have been summarized in Fig. 10 and show that the detectable µ-opioid receptor-LI in lamina II of rat and monkey spinal cord mostly originates from local neurons. Only a limited number of primary afferent terminals contain detectable µ-opioid receptor-LI, thus representing prejunctional/presynaptic receptors. They are found more frequently in monkey than in rat, and the µ-opioid receptor-LI is mostly localized outside the synapses, but can occasionally be seen on the presynaptic membrane opposite to the postsynaptic density. In monkey the terminals are mostly characterized by content of many LDCVs, indicating storage of peptides. This is in agreement with the fact that µ-opioid receptor-positive neurons in the DRGs are small and express CGRP. However, the dendrites making synaptic contact with the axon of glomeruli always have µ-opioid receptor-LI outside the postsynaptic thickening. These µ-opioid receptor-positive dendrites are often found on µ-opioid receptornegative primary afferent terminals. Taken together, these findings suggest that the main sites of action of opioid peptides and opiates via µ-opioid receptors are on dendrites, especially in the rat. This is surprising in view of the fact that more than 50% of all DRG neurons express µ-opioid receptors65 (and present study). It may therefore be that µ-opioid receptors are only transported to a limited extent to the dorsal horn, and our techniques may not be sufficiently sensitive to detect them. In agreement, only few CGRP-positive fibers in the dorsal horn co-localize µ-opioid receptor-LI, and the µ-opioid receptor-LI in lamina I and superficial lamina II is weak. On the other hand, both local dorsal horn neuron cell bodies and, as mentioned above, dendrites are frequently µ-opioid receptor-positive. In fact, the somatic cell membrane of local neurons is often strongly

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Fig. 10. (a–d) Schematic illustration of the localization of the µ-opioid receptor-LI in lamina II of the dorsal horn of the rat and monkey spinal cord as revealed in this paper. (a) Although the majority of µ-opioid receptor-positive structures in lamina II are dendrites, µ-opioid receptor-LI is also observed in some primary afferent terminals, forming glomeruli with several µ-opioid receptor-negative dendrites (D2–D4) and occasionally a µ-opioid receptor-positive dendrite (D1). In the monkey spinal cord, more µ-opioid receptor-positive primary afferent terminals are seen than in the rat spinal cord, and they often contain many LDCVs. The µ-opioid receptor-LI is located on the plasmalemma outside of the presynaptic zone (prejunctional µ-opioid receptors), but sometimes it can be located on the membrane within the presynaptic zone (presynaptic µ-opioid receptors). (b) More frequently, we encountered µ-opioid receptor-negative primary afferent terminals making synapses with several µ-opioid receptor-positive dendrites (D1, D2 and D4) and a few µ-opioid receptor-negative dendrites (D3). (c) µ-Opioid receptorpositive neurons in lamina II often receive axosomatic synapses involving µ-opioid receptor-negative axonal terminals. The µ-opioid receptor-LI is located on the plasmalemma outside the postsynaptic zone (postjunctional µ-opioid receptors). (d) A µ-opioid receptor-positive axonal terminal presumably originating from a local neuron makes synaptic contact with a dendrite.

decorated with µ-opioid receptor-LI, suggesting a direct influence of opioids and opiates on cell bodies. Finally, our results indicate prejunctional µ-opioid receptors also on axonal terminals of local neurons. Effect of peripheral nerve injury The main result of the present study is that peripheral nerve injury (axotomy) causes a down-regulation of µ-opioid receptor expression. Thus, nerve injury induces a decrease in µ-opioid receptor-LI and

µ-opioid receptor mRNA levels, i.e. changes which are opposite to that seen after inflammation, where µ-opioid receptor-LI is up-regulated.41 Here we show a decrease in the number of µ-opioid receptorimmunoreactive cell bodies in DRG neurons 14 days after injury in rat and monkey, as well as a decrease in mRNA levels in the rat. In fact, a significant decrease can be seen in the rat already after two days. It is interesting that in the rat there is a 30% decrease in the number of µ-opioid receptor-positive DRG neuron profiles when compared to the contralateral

µ-Opioid receptor in sensory and dorsal horn neurons

side, whereas almost all µ-opioid receptor-LI disappears in the monkey. Moreover, there is a distinct decrease in µ-opioid receptor-LI in the monkey dorsal horn; in particular, the strongly fluorescent band in lamina II in the monkey decreased dramatically in intensity. However, only a small effect could be observed in the rat dorsal horn. This in agreement with the fact that the down-regulation in the DRGs is much stronger in monkey than in rat. With regard to the monkey dorsal horn, it is not possible to definitely decide whether the decrease seen reflects a decrease in receptor protein in the primary afferent fibers, or represents a secondary change in local dorsal horn neurons, or both. However, it is highly likely that there is a trans-synaptic down-regulation, since the strong band in lamina II mainly seems to reflect local neurons and their processes. Taken together, these findings suggest that the insensitivity to opioids observed in neuropathic pain1,2,24,45,79 may at least partly be explained by a decreased expression of µ-opioid receptors, i.e. the primary targets for morphine. Moreover, these results indicate that opiate insensitivity may be particularly pronounced in primates. Clearly there are other factors that may contribute to opioid insensitivity after nerve injury. For example, after nerve injury there is a marked increase in cholecystokinin B (CCKB) receptor mRNA in DRG neurons95 and there is also an up-regulation of CCK peptide.81 This may enhance CCKergic mechanisms in the dorsal horn, which may be of significance for pain treatment, since it has been shown that CCK is an endogenous inhibitor of opioid-induced analgesia.7,23,26,39,84–86 Thus, the present results indi-

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cate that treatment of neuropathic pain with CCKB antagonists may not be as efficient as was hoped, since endogenous CCK exerts its effect via counteracting opioid-induced analgesia, presumably via µ-opioid receptors. If, as shown here in monkey, the µ-opioid receptors are also down-regulated in man, the algesic effect of CCK should be markedly attenuated, as should the analgesic effect of a CCKB antagonist. We conclude that many small neurons and some medium-sized neurons in lumbar DRGs express µ-opioid receptors both in rat and monkey. These neurons can also contain substance P- and CGRPLIs. µ-Opioid receptor-LI is also observed in the superficial dorsal horn in both species, and it is present both on some primary afferents and on local neurons in deep lamina II. Peripheral axotomy induces a marked decrease in expression of µ-opioid receptors in DRG neurons and, presumably, transsynaptically in local dorsal horn neurons, and this effect is especially pronounced in the monkey spinal cord. Thus, the insensitivity to opioid analgesics in neurogenic pain, often a result of nerve injury, is perhaps mainly due to the down-regulation of expression of µ-opioid receptors.

Acknowledgements—This work was supported by the Swedish Medical Research Council (04X-2887), Marianne and Marcus Wallenberg’s Stiftelse, Gustav V’s and Drottning Victorias Stiftelse, Astra Pain Control AB, the Nature Science Foundation of China (Grants 39525010 and 39500045) and by grants from the National Institute on Drug Abuse, National Institute of Health (U.S.A.).

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