Ultrastructural study of δ-opioid receptors in the dorsal horn of the rat spinal cord using monoclonal anti-idiotypic antibodies

Iom'aalol

Chemical__ Neuroanatomy ELSEVI ER

Journal of Chemical Neuroanatomy 7 (1994) 159-170

Ultrastructural study of 6-opioid receptors in the dorsal horn of the rat spinal cord using monoclonal anti-idiotypic antibodies Fouzia Zeran" a, Djamila Zouaom" a, Myriam Gastard a, Emmanuelle --v °A"arqs" a Jacqueline Fischer a, Fabienne Herbrecht b, Anny Cupo b, Karine Cucumel b, Marie Conrath *a aDOpartement de Neurobiologie des Signaux Intercellulaires, Institut des Neurosciences, CNRS URA 1488, UniversitO Pierre et Marie Curie, CNRS UPA 1488, 7 Quai Saint Bernard, 75252 Paris Cedex 05, France b]nstilut de Pharmacologie, Molkculaire et Cellulaire du CNRS UPR 411, 660 Route des Lucioles, ,Sophia Antipolis, 06560 Valbonne Cedex, France

Accepted 22 February 1994

Abstract

The ultrastructural localization of 6-opioid receptors was studied using monoclonal anti-idiotypic antibody prepared with an anti-D-Ala2-D-LeuLenkephalin. Immunocytochemical techniques were used on vibratome sections from rats perfused with paraformaldehyde. A high density of immunoreactivity was observed in the dorsal horn of the spinal cord, particularly the two superficial layers, the dorsolateral funiculus and the area surrounding the central canal. The labelling was absent when the antibody was preincubated with the immunogen. Competition between the anti-idiotypic antibody and different ligands, ~5or/z, was controlled by preincubation of tissue sections with the ligand in the presence of peptidase inhibitors for 3-4 h before addition of the antiidiotypic antibody. Enkephalin, dermenkephalin and naltrindole induced disappearance of the labelling at 10-9 M while dermorphin or dermorphin Lys 7 were ineffective at the same concentration. Lamina II of the dorsal horn was studied by electron microscopy. The immunolabelling was mainly localized on cell membranes at appositions between two neurons. About one third were localized between an axon terminal and a dendrite, the same proportion of labellings were between two axon terminals. Labelling was occasionally observed at appositions between a glomerular terminal and a dendrite or a terminal or at axoglial appositions. Axosomatic localizations were rare. The presynaptic localization of the labelling is in favor of a presynaptic mechanism of action for 6-opioids in the spinal cord, providing that these receptors are functional. 6-Opioid peptides probably act non-synaptically since receptors were never localized on synaptic differentiations.

Keywords: Dorsal horn; Enkephalin receptor; Immunocytochemistry; Opioid peptide receptor; Substantia gelatinosa

1. Introduction

Several lines of evidence suggest that a spinal site o f action contributes to the analgesic effect o f opioids and opioid peptides. Morphine or opioid peptides administered intrathecally or systemically in spinally transected animals inhibit noxious-evoked dorsal horn neuronal activity (Yaksh, 1984). The microiontophoresis o f opioids onto dorsal horn neurons inhibits unit activity in a naloxone-reversible manner (Duggan et al., 1976; Zieglgansberger and Tulloch, 1979; Willcockson * Corresponding author. 0891-0618/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSD1 0891-0618(94)00013-X

et al., 1986). This effect is mediated by opioid receptors which are highly concentrated in the two superficial layers o f mammalian spinal cord (Atweh and Kuhar, 1977; Zajac et al., 1989; Besse et al., 1990; Gouarderes et al., 1991; Stevens et al. 1991), where most nociceptiveconducting afferent fibres terminate. Enkephalins (ENK), most c o m m o n opioid peptides in the spinal cord, are localized in numerous neurons (cell bodies and terminals) of the dorsal horn (H6kfelt et al., 1977; Bennett et al., 1982; Conrath et al., 1983; Glazer and Basbaum, 1983). E N K act on neurons preferentially through 6-opioid receptors. Both a presynaptic and a postsynaptic mechanism o f action have been proposed.

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The postsynaptic hypothesis is strongly supported by morphological data showing that ENK-immunoreactive axon terminals are presynaptic to dendrites and cell bodies of the dorsal horn (Bennett et al., 1982; Sumal et al., 1982; Glazer and Basbaum, 1983; Lamotte and DeLanerolle, 1983). Furthermore, it has been demonstrated that half of the spinal dorsal horn neurons that project to the thalamus are directly contacted by ENK varicosities (Ruda et al., 1984). The presynaptic hypothesis is supported by electrophysiological data and opioid receptor binding studies. It has been shown that dorsal rhizotomy leads to a 40-75% decrease in #- and 8-opioid receptors in the dorsal horn (Hiller et al., 1978; Zajac et al., 1989; Besse et al., 1990; Gouarderes et al., 1991; Stevens et al. 1991) suggesting that part of opioid receptors are localized on the primary afferent terminals. Further evidence for this presynaptic localization is provided by the demonstration of opioid binding in dorsal root ganglia and dorsal roots (Fields et al., 1980, Young et al., 1980; Ninkovic et al., 1982). Moreover, the release of substance P, potential transmitter of fine unmyelinated afferents, is inhibited by ENK (Jessel and Iversen, 1977; Mauborgne et al., 1987; Pohl et al., 1989). The pre.synaptic hypothesis has never been confirmed by electron microscopy since axoaxonic contacts between primary afferent terminals and ENK immunoreactive varicosities are inexistent (Hunt et al., 1980; Aronin et al., 1981; Sumal et al., 1982; Glazer and Basbaum, 1983). To gain further insights into this question - - to identify target cells - - the ultrastructural localization of opioid receptors in the dorsal horn of the spinal cord needs to be known. Beaudet and co-workers proposed the use of a synthetic Met-enkephalin analogue for electron microscopic autoradiographic visualization of ~binding sites in the striatum (Hamel and Beaudet, 1984a,b, 1986). More recently, they developed the ultrastructural visualization of g- and 8-receptors in the striatum (Jomary et al., 1992; Pasquini et al., 1992) still using autoradiography. However, resolution of the precise sub-cellular localization of opioid receptors has been limited owing to problems inherent in the autoradiographic method, such as radiation scatter and diffusion. These limitations do not apply to immunocytochemical methods, which are now feasible thanks to the development of antibodies against peptide receptors. However, since 6-opioid receptors have been cloned only recently (Evans et al., 1992; Kieffer et al., 1992), to our knowledge no direct anti-receptor antibody is yet available. Several groups have thus developed an anti-idiotypic approach to obtain monoclonal (Gramsh et al., 1988; Coscia et al., 1991) or polyclonal (Ornatowska et al., 1992) anti-opioid receptors antibodies and shown that they may recognise opioid receptors on cell cultures (Ornatowska et al., 1992) or rat brain (Hassan et al., 1989). We have produced four monoclonal anti-idiotypic anti-

bodies (anti-Id mAb) prepared from an anti-D-Ala 2D-LeuS-enkephalin (DADLE) antibody and shown that they can be used in immunocytochemistry at both light and electron microscope levels (Conrath et al., 1989; Cupo et al., 1992). In this paper we give a detailed analysis of the immunolabelling obtained with one of these monoclonal anti-idiotypic antibodies in the two superficial layers of the rat spinal cord at the ultrastructural level, focusing on plasma membrane labelling to investigate the synaptic organization of 8-opioid systems. We also show that the anti-idiotypic approach allows specificity to be controlled on tissue sections by direct competition between different ligands and the antibody. 2. Mate~als and methods

2.1. Production of monoclonal anti-idiotypic antibodies Rat monoclonal anti-idiotypic antibodies were raised against the 8-opioid receptor (Cupo et al., 1992). Briefly antibodies (abl) directed against the opioid agonist DADLE were used as immunogen. The rats were then immunized with purified anti-ligand antibodies and cell fusion was performed. Selected hybridomas have to express both an anti-idiotypic activity (inhibition of ligand/anti-ligand interaction) and an anti-opioid receptor activity (inhibition of 8-agonist ligand/binding site interaction). On the basis of these two activities, four different monoclonal anti-idiotypic antibodies were selected. Clone 5 used in this study totally inhibited the ligand/abl interaction. It was able to bind to the NG 108-15 cell line which provided a homogeneous source of 6-opioid receptor. It also inhibited the 3H-DADLE binding to NG 108-15 cells and precipitated a 52 500 Da molecule after cell surface iodation of the NG 108-15 cells.

2.2. Immunocytochemistry Under deep chloral hydrate anesthesia (35 rag/100 g), 40 adult Wistar rats were perfused via the ascending aorta with 100 ml of 0.9% NaCI containing 0.1% sodium nitrite as washing solution followed by 500-800 ml of 4% paraformaldehyde in 0.1 M Strensen buffer, pH 7.4, as fixative. The spinal cord was then dissected out and post fixed in the same fixative for 2 h. After extensive washings in the buffer, blocks of the cervical spinal cord were cut in 50-~,m sections using a vibratome. Sections were preincubated in 3% normal sheep serum diluted in 0.15 M S6rensen buffer for 30-60 rain. The following incubation baths were prepared using the same medium (normal sheep serum in S6rensen buffer). Sections were incubated in a 1/200 dilution of the anti-Id mAb. An Ig fraction purified by precipitation with ammonium sulfate (8.1 mg/ml) was used. Incubations were carried out for 2 h at room temperature or overnight at 4°C. After extensive washings in the buffer, sections were incubated for 90 min in a 1/250 dilution of anti-rat immunoglo-

F. Zerari et al./J. Chem. Neuroanat. 7 (1994) 159-170

bulin coupled to horseradish peroxidase (Pasteur Production). Alternatively, the avidin-biotin-peroxidase method was used (Kit Vektastain, Vector Lab.). After washing, the peroxidase activity was revealed by 3,3'diaminobenzidine (Sigma). The sections were first rinsed in Tris buffer pH 7.2 for 15 min and then incubated in the 3,3'-diaminobenzidine (15 mg/50 ml Tris) supplemented with 0.01% hydrogen peroxide. After 10-20 rain of incubation, the sections were rinsed in Tris buffer then in S6rensen buffer. For light microscopy, sections were mounted on glass slides with glycerol/ S6rensen as mounting medium. For electron microscopy, the dorsal horns were dissected out, postfixed in 2% osmium tetroxide in 0.15 M S6rensen, pH 7.4, washed, dehydrated and then embedded in araldite or epon.

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U l t r a t h i n sections were cut a n d stained with lead citrate before observation with a Philips E M 300 or Jeol electron microscope. Serial u l t r a t h i n sections were cut from four blocks from different animals a n d collected on the same grid. The p r o p o r t i o n of each type of labelling was counted on micrographs at sufficient magnification to clearly interpret the morphology. 2.3. Control experiments (1) In order to evaluate b a c k g r o u n d due to the second antibody, the specific anti-Id m A b , was omitted in the first i n c u b a t i o n step. (2) T o ascertain whether the imm u n o s t a i n i n g obtained was specifically due t o . a n t i - I d , the anti-Id m A b (1/200) was p r e i n c u b a t e d for 2 h at room temperature with the a n t i - D A D L E a n t i b o d y

~:iip

Fig. 1. 1: dorsal horn (dh) in the cervical (CVII-CVIII) segment. Note the intense reaction in the superficial layers and the dorsolateral funiculus (dlf). Scale bar: 200 #m. 2: high magnification of the dorsal horn showing that the immunoreactivityis restricted to laminae I and I1. Dark points of precipitate are uniformiy distributed in the tissue. Inner layers are almost totally devoid of immunoreactivity. Scale bar: '200 gm. 3: high magnificationof the dorsolateral funiculus showing long immunoreactivefibers (arrows). Magnificationas in Fig. 2. 4: region surrounding the central canal (cc) at the same level (CVII-CVIII). The immunoreactivity is regularly distributed in lamina X. Magnification as in 2. 5: dorsal horn (dh) of the lumbar region (LI). The immunoreactivity is intense in the two superficial layers and in the dorsolateral funiculus. Note also the immunoreactivityof Lissauer's tract. Magnificationas in 1.6: immunoreactivityis absent when the antibody is preincubated with 1/2000anti-DADLE antiserum (used as immunogen for the preparation of the anti-idiotypic antibody) before addition of the section. Magnification as in 1.

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(1/2000) which was used as immunogen for obtaining the anti-Id mAb. (3) A preimmune rat serum was tested. (4) Competition between the anti-Id mAb and different opioid ligands was tested by preincubation of tissue sections in a ligand at concentrations ranging from 10-5 to 10-11 M in the presence of a cocktail of peptidase inhibitors (20 /~M bestatin, 10 t~M thiorphan, 20 #M kelathorphan, 20/zM bacitracin) for 3-4 h at room temperature before addition of the anti-Id mAb (1/200). In most cases, the mixture thiorpharffkelathorphan was judged sufficient to prevent the degradation of ligands used. 6-Specific agonists, e.g., enkephalin, DADLE, dermenkephalin, or antagonists, e.g., naltrindole, were tested. ~-Agonists like dermorphin (and its inactive enantiomer L-dermorphin) or dermorphin-Lys 7 were also tested. (Ligands and peptidase inhibitors were purchased from Sigma or Neosystem, L-dermorphin was a generous gift from P. Nicolas, IRBM, Paris.) 3. Results

3.1. Light microscopy In the cervical spinal cord, the most intense immunoreaction was observed in the dorsal horn (Fig. 1,1) particularly in the two superficial layers and the dorsolateral funiculus. In the dorsal horn, the labelling appeared as small randomly distributed dark points. It was intense in laminae I and II, while laminae III and IV were almost totally devoid of labelling (Fig. 1,2). The reticular part of lamina V showed a low immunoreactivity (not shown). The dorsolateral funiculus was intensely labelled, long immunoreactive fibres could be followed (Fig. 1,3). The area surrounding the central canal exhibited an intense immunoreaction in lamina X extending to lamina VII (Fig. 1,4). In the ventral horn, immunoreactivity was sparse except in lamina IX where it was concentrated around large neurons, presumably motoneurons (not shown). In other regions of the spinal cord the distribution of immunoreactivity was similar to that seen in the cervical segment as shown in segment L1 (Fig. 1,5). In the tract of Lissauer a moderate immunoreaetivity was observed. When the specific antibody, anti-Id mAb, was omitted in the first incubation bath, no immunoreaction was seen in the spinal cord. When anti-Id mAb was preincubated with the anti-DADLE antibodies (1/2000) used as immunogen for obtaining the anti-Id mAb, it resulted in total absence of immunoreactivity (Fig. 1,6). The same result was observed when a preimmune serum was used instead of anti-Id mAb. When tissue sections were preincubated in/~-ligands before addition of the anti-Id mAb, the labelling was never modified at 10-9 M dermorphin or dermorphin Lys 7 (Fig. 2,2 with ligand, Fig. 2,1 without ligand). By contrast, preincubation of tissue sections in 6-1igands at about the same concentration led to a dramatic decrease

as seen here with DADLE (Fig. 2,3) or disappearance of the labelling as seen with dermenkephalin (Fig. 2,4), enkephalin (Fig. 2,5) or naltrindole (Fig. 2,6). Note that in these experiments, the background labelling (i.e., on the border of the section) was not affected. Labelling in the dorsal horn reappeared when tissue sections were incubated in 10 -II M for ~-ligands and 10 -9 M for/~ligands (not shown). In contrast with dermorphin, the inactive enantiomer L-dermorphin was without effect whatever the concentration tested (10 -6 to 10 -9 M.

3.2. Electron microscopy The ultrastructural study concerned exclusively lamina II of the dorsal horn in the cervical rogion. The most striking observation was the predominance of plasma membrane-linked immunoreaction. However, a small proportion of immunoreactivity was intra~tlular. It was observed in cell bodies, axons (myelinated or not), dendrites or perikarya. This intracellular reaction was generally weak and restricted to discrete patches localized on ribosomes, endoplasmic reticulum, nuclei or synaptic vesicles. No intracellular labelling was observed in glial processes. Membrane labelling consisted of very small (< 300 nm) dark patches of dense precipitate. It was localized at appositions or interfaces between two neurites (occasionally between a neurite and a glial process). Usually it was not possible to determine to which particular apposed membrane the immunoprccApitat¢ was linked. About one third of membrane labelling was localized at axo-dendritic appositions. Another third was localized between two axons, in this case one of the axons could be preterminal (and did not contain synaptic vesicles). The last third was heterogeneous comprising axoglial, axodendritic or axosomatic appositions. It also contained appositions where one of the element was not clearly identifiable. When the labelling was localized at axodendritic appositions (Figs. 3 and 4) the precipitate of the reaction filled the intermcrabranous space betweon the axon terminal and the dondrit¢. A synaptic differentiation was not always observed between the axon terminal and the dendrite (Fig. 3,1). But when a synaptic differentiation was present, the immunoraaction was never localized on it (Figs. 3,2 and 3 and Fig. 4) but just near it. Often the labelling was in continuity with the synaptic differentiation, and it extended over 100-150 nm, i.e., equal to or less than the length of the synaptic differentiation itself. The neighboring synaptic differentiations can be symmetrical (Fig. 3,2) or asymmetrical (Fig. 3,3; Fig. 4). Axon terminals could be the C-terminal of a glomerulus (Figs. 4 and 5). In these cases, they were large (about 1/zm) and contacted several post-synaptic dendrites (Fig. 4). Here also the labelling was localized next to synaptic differentiations, but never directly on the differentiation itself.

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l

1

DRM

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3

DADLE

4

DREK

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6

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Fig. 2. Control sections of competition between the anti-Id mAb and opioid ligands. Tissue sections were incubated in different ligands in the presence of peptidase inhibitors before addition of the anti-Id mAb. (1) no ligand, (2) 10-9 M dermorphin (DRM), (3) 10 -8 M DADLE, (4) l0 -9 M dcrmenkcphalin (DREK), (5) 10-9 M Met-enkephalin (ENK), (6) 10-9 M naltrindole (NAL). Note that the labelling is not modified by preincubation with the ligand DRM but is greatly decreased by preincubation with DADLE and totally absent after preincubation with DREK, ENK and NAL. Background labelling is however present in the border of sections probably corresponding to non-specific absorption in the depth of the section. Scale bar = 200 #m.

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Fig. 3. Electron micrograph of immunoreactivity obtained with the anti-ld mAb in the two superficial layers of the cervical dorsal horn. The immunopn~cipitate (arrowheads) is localized between an axon (A) and a dendrite (D) either apposing (I) or synapsing (2,3). Note in 2 and 3 that the immunoreaction is localized in the vicinity of the synaptic differentiations but never directly on them. Note also the presence of gap junctions (small arrows) in 3. In 3, another immunoreaction is observed at an apposition between an axon and glial process (G). The star in A indicates intracellular labelling. Scale bars = 100 nm.

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Fig. 4. A glomerularC terminal (C), presumably of primary afferent origin, presents two immunoreactivezones (arrowheads). These reactions are in direct contact with or in the vicinity of synaptic differentiations (arrows) between the C terminal and two dendrites (D). The star bottom left of the micrograph indicates intracellular labelling. Scale bars = 200 nm.

Since single sections did not reveal labelling on synaptic specializations, serial sections were made. They showed that when labelling was observed at apposed membranes, no synaptic differentiation was seen on serial sections (Fig. 5). In addition, they showed that labellings were thin since they were generally observed only on two consecutive sections (140 nm) but not on the third one. Membrane labellings at axoglial appositions were rare. Nevertheless they were occasionally found (Fig. 6,1; see also Fig. 3,3 and Fig. 5,bl and 2). As noted above, apposing membranes from two axon terminals accounted for about one third of total membrane labelling. In these cases, the extent of the reaction seemed greater than when labellings were localized at axo-dendritic appositions (Fig. 6,2 to 4). Generally one of the two axon terminals established synaptic contact

with neighbouring dendrites (Fig. 6,2 and 3). In contrast to axodendritic labelling, the immunoreaction was never observed in the vicinity of synaptic differentiations. Axon terminals involved in these labelled axoaxonic appositions were of heterogeneous morphological types. They contained either flattened vesicles (Fig. 6,2), in these cases they could be vesicle-containing dendrites or round vesicles (Fig. 6,3 and 4). Large granular vesicles were rarely observed in these terminals. 4. Discussion 4.1. Validation o f the method

Control experiments showed that the second antibodies, biotinylated or peroxidase-coupled anti-rat IgG, give a negligible background. In addition, preincubation

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Fig. 5. Serial sections. On the central panel (al/bl) a glomerular terminal can he seen, it is divided in two parts al and bl which can be followed in two serial sections in the left-hand panel of the micrograph (a2 and a3) and right-hand panel (b2 and b3). Arrowheads indicate immunoreactive zones at appmitions between the 81omerular terminal and another profile. Large arrows in bl indicate an immunoreactivity probably on a glial cell membrane. Note that imunoreactivity is clearly visible only on one section, on the following section it is faint or absent. Moreover, when membrane labelling is obeerved at apposed membranes, no synaptic differentiation could he observed in serial sections. Labelling in synaptic vesicles (small arrows) is observed in a small subpopulation of synaptic vesicles of the terminal. Scale bar = 150 nm.

of the anti-Id mAb with the immunogen rabbit antiDADLE resulted in an absence of immunoreaction indicating that the labelling is due to the s ~ i f i c antibody. Indeed, a preimmune rat serum did not label spinal cord sections. More importantly, preincubation of tissue sections with ~-specific ligands totally prevented the labelling. This competition between ligands and the anti-Id mAb for binding to the receptor site occurred despite tissue fLxation with aldehyde. That is probably why relatively high concentrations of ligands, compared to their affinity on fresh tissue, were needed in our case. This competition is possible only because our antibody is specifically directed against the receptor binding site. It would of course be impossible in the case of antipeptide antibody obtained with a sequence of the cloned receptor, unless this anti-peptide antibody recognized the binding domain on the receptor. However, the likelihood of obtaining antibodies that recognize the receptor binding site in this way is very low since this region is poorly known and probably corresponds to the conformational rather than sequential epitope. We have shown that the minimal concentration of enkq~mlin, dermenkephalin or naltrindole needed to abolish the immunostaining in the spinal cord is 10-9 M. Surprisingly the concentration is the same for the three ligands, whereas their affinity and specificity for the 6-site are not identical. In addition their sensitivity to endogenous peptidases is unlikely to be similar since dermenkephalin possesses a D amino acid and naltrindole is non-peptidic. We have indeed demonstrated that enkephalin degradation is largely prevented by a cocktail of peptidase inhibitors. We have also shown that DADLE is less potent in competing with our antibody than the previously cited ligands. This may be accounted for by the poor specificity of DADLE for

6-sites. It is possible also that small differences between these ligands may not be revealed by our method which is not quantitative. However, we have observed that/~specific ligands (dermorphin and dermorphin Lys 7) compete less effectively with the anti-Id mAb since a concentration of 10-7 M is necessary to abolish the labelling. These results indicate that the anti-Id mAb has a hundred-fold more affinity for ~-sites than for/~-sites. Moreover it recognizes the agonist as well as the antagonist 6-sites. It should be noted that preincubation with 8-1igands led to total disappearance of the immunolabeUing in the spinal grey matter, where 6-1igands classically bind, but had no effect on labelling at the edge of sections, which probably corresponds to an artefact due to penetration of the antibody into the thickness of the sections. These control experiments show the specificity of our anti-Id mAb for ~-sites, at least in this immunocytochemical study. They also emphasise the interest of the anti-idiotypic approach for labelling peptide receptors. As stated in the introduction, anti-idiotypic antibodies have already been used to label opioid receptors by immunocytochemical methods in the central nervous system (Hassan et al., 1989); however, the antibody used in that study does not allow distinction between ~- and 8binding sites.

4.2. Distribution of immunoreactivity in spinal cord The regional distribution of the immunoreactivity using the anti-Id mAb is quite similar to that of opioid binding sites obtained with autoradiography (Atweh and Kuhar, 1977; Zajac et al., 1989; Besse et at., 1990; Gouarderes et al., 1991; Stevens et al., 1991). The two superficial layers of the dorsal horn are highly immunereactive while the density of the labelling is lower in the

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Fig. 6. In I, the reaction is localized on an axon apposed to a glial process (G). In 2, 3 and 4, the reaction is localized on appositions between two axon terminals (AI and A2). No synaptic differentiation can be seen between these axons. In 2 and 3, axons A1 contact dendrites as indicated by synaptic differentiations (arrows). Note the extent of the immunoreaction compared to that seen in the preceding figures (axodendritic). Scale bars = 250 nm.

deeper layers. We have previously observed in other regions of the spinal cord a distribution o f immunoreactivity quite similar to data obtained with autoradiography, in particular a high density o f the labelling in lamina X around the central canal and a r o u n d large neurons in the ventral horn (Cupo et al., 1992). It appears, however, that the resolution o f our immunocytochemical method is better than that obtained by conventional autoradiography. Under the electron microscope, some immunoreactivity was observed in cell bodies. This is not surprising since tS-receptors have to be synthesized, stored and

transported before being exposed to the cell membrane. In addition it is quite possible that the ligand/receptor complex would internalize. This p h e n o m e n o n has already been demonstrated for 6-opioid receptors in the N G 108-15 cell line (Blanchard et al., 1983; Belcheva et al., 1992) and suggested for several other peptides. Moreover, intracellular immunoreactivity has also been observed in the central nervous system with antibodies directed against opioid receptors (Hassan et al., 1989). Using a photoaffinity-labelled 6-1igand to localize 6receptors at the electron microscopic level, Pasquini et al. (1992) have shown that about 40% of binding is

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localized in cell bodies in the striatum. It remains to be determined whether the proportion of intracetlular /~receptors varies depending on the region in the central nervous system or depending on the method used to reveal the receptors (immunocytochemistry or autoradiography). However, previous studies by Beaudet group have shown that intracellular #- and ~-opioid binding sites also revealed by autoradiography are markedly less abundant (Hamel and Beaudet, 1984a,b, 1986; Jomary et al., 1992). Membrane labelling was localized at non-junctional interfaces between two neuronal processes. This result agrees with that of Pasquini et al. (1992) in the rat striatum. In their study, however, because of limitations inherent in the autoradiographic technique, it was not possible to determine a precise localization of the labelling. Statistical analysis was necessary to determine the probability of the 'real' localization of receptors. The immunocytochemical technique used here shows clearly that 8-opioid receptors in the spinal cord are never localized on the synaptic differentiation itself. Moreover, serial sections confirm this observation since they do not reveal synaptic specialization. When the immunoreaction is localized on axo-dendritic appositions, it can be seen next to the synaptic differentiation and sometimes in continuity with it. This suggests that opioid peptides may indirectly influence or regulate (or be influenced or regulated by) another system acting directly at these synapses. #-Receptors also seem to be localized nonsynaptically (Hamel and Beaudet, 1984a,b, 1986) since only 7% of binding sites were associated with synaptic junctions in the striatum, the same result was obtained for the neurotensin receptor in the ventral tegmental area and interfascicular nucleus (Dana et al., 1989). However, the non-synaptic localization is not a general rule for peptidergic or even opioid receptors since it has recently been shown that 23% of K-opioid receptors are associated with synaptic junctions in the striatum (Jomary et al., 1992). Several data exist concerning other types of receptors in the rat spinal cord, for example glycinergic receptors have been demonstrated to be associated with synaptic differentiations (Triller et ai., 1985; Van den Pol and Gorcs, 1988). But previously nothing was known about the ultrastructural distribution of peptidergic receptors in the spinal cord. One third of membrane labelling is localized on axon terminals since it is observed at axoaxonic appositions. This presynaptic localization is in favor of a presynaptic mechanism of action for opioid peptides in the dorsal horn of the rat spinal cord. However, this proportion seems to be low compared to results in the literature. Decreases of up to 60-75% in opioid binding sites have been described after complete deafferentation by dorsal rhizotomy of seven consecutive roots (Besse et al., 1990), suggesting that this proportion of opioid receptors is

localized on primary afferent terminals. In addition, it has been suggested that endogenous opioids can exert an inhibitory influence on their own release through presynaptic autoreceptors (Bourgoin et al., 1991). To know the proportion of presynaptic versus postsynaptic localizations, it needs to be determined to which particular apposed membrane the receptor is linked. Indeed this is the great limitation of our method, due to the extracellular localization of the epitope recognized by the antibody. In addition, target cells are not identified. In particular it cannot totally be ruled out that some axon terminals observed in axoaxonic appositions may represent vesicle-containing dendrites (Gobel et al., 1980; Todd, 1988). However such presynaptic dendrites preferentially contact other dendrite or preterminal axons which do not contain synaptic vesicles. About one third of 8-opioid receptors are localized at axodendritic appositions. This would confirm the postsynaptic effect of opioids in the dorsal horn, provided that the receptor is localized on the dendritic side. Whether the axons involved in labelled appositions contain an opioid peptide will have to be verified. It is possible that axon terminals involved in axodendritic appositions contain such peptides since opioid-containing varicosities (namely enkephalin) have been found presynaptic to spinothalamic neurons in lamina V (Ruda et al., 1984), cells which send dendrites to lamina II. In contrast, it is highly improbable that axon terminals involved in axoaxonic appositions contain enkephalin, since opioid containing varicosities do not contact axon terminals (Hunt et al., 1980; Aronin et al., 1981; Sumat et al., 1982; Glazer and Basbaum, 1983). In addition the axon terminals observed here generally do not contain large granular vesicles, which are the classical vehicle of neuropcptidesl These results suggest that enkephalins, if they are the ligand of 6-receptors are released at a distance from their target cells. References Aronin, N., Difliglia, M., Liotta, A.S. and Martin, J.B. (1981) Ultrastructural localization and biochemical features of immunoreactive leuenkephalin in monkey dorsal horn. J. Neurosci. 1,561-577; Atweh, S.F. and Kuhar, M.J. (1977) Autoradiographic localization of opiate receptors rn the rat brain. I Spinal cord and lower medulla. Brain Res. 124, 53-67. Belcheva, M., Barg, J., Glocckner, C.A., Gao, X.M., Chuang, D~M. and Coscia, C.J. (1992) Antagonist induced transient downregulation of opioid receptor in NG 108-15 neurohybrid cells. J. Neurosci. 13, 104-114. Btanchard, S.G., Chang, K.J. and Cuatrecasas, P. (1983) Characterisation of the association of tritiated enkephalin with neuroblastoma cells under conditions optimal for receptor downregulation. J. Biol. Chem. 258, 1092-1097. Bennett, G.J., Ruda, M.A., Gobel, S. and Dubner, R. (1982) Enkephalin immunoreactive stalked cells and lamina lib islet cells in cat substantia g©latinosa. Brain Res. 240, 162-167.

F. Zerari et al. /J. Chem. Neuroanat. 7 (1994) 159-170

Besse, D., Lombard, M.C., Zajac, J.M., Roques, B.P. and Besson, J.M. (1990) Pre and postsynaptic distribution of mu, delta and kappa opioid receptors in the superficial layers of the cervical dorsal horn of the rat spinal cord. Brain Res. 521, 15-22. Bourgoin, S., Collin, E., Benoliel, J.J., Chantrel, D., Mauborgne, A., Pohl, M., Hamon, M. and Cesselin, F. (I 991) Opioid control of the release of met-enkephalin-like material from the rat spinal cord. Brain Res, 551, 178-184. Conrath, M. and Cupo, A. (1989) Immunocytochemical demonstration of brain delta opiate receptors using anti-idiotypic antibodies. Adv. Biosci. 75, 273-275. Conrath, M., Dietl, M., Arluison, M., Cesselin, F. and Hamon, M. (1983) Localization of met-enkephalin-like immunoreactivity within pain related nuclei of cervical spinal cord and midbrain in the cat. Brain Res. Bull. 1I, 587-604. Coseia, C.J., Sziics, M., Barg, J., Belcheva, M.M., Bem, W.T., Khoobehi, K., Donnigan, T.A., Juszczak, R., McHale, R.I., Hanley, M.R. and Barnard, E.A. (1991) A monocional antiidiotypic antibody to mu and delta receptors. Mol. Brain Res. 9, 299-306. Cupo, A., Conrath, M., Eybalin, M., Fourier, O., Zouaoui, D., Caidy, P. and Herbrecht, F. (1992) Monoclonal anti-idiotypic antibodies against delta opioid receptor as an electron microscopy probe. Eur. J. Cell Biol. 57, 273-284. Dana, C., Vial, M., Leonard, K., Beauregard, A., Kitabgi, P., Vincent, J.P., Rostene, W. and Beaudet, A. (1989) Electron microscopic localization of neurotensin binding sites in the midbrain tegrnentum of the rat. I. Ventral tegrnental area and interfascicular nucleus. J. Neurosci. 9, 2247-2257. Duggan, A.W., Hall, J.G. and Headly, P.M. (1976) Morphine, enkephalin and substantia gelatinosa. Nature 264, 456-458. Evans, C.J., Keith, D.E., Morrisson, H. Magendzo, K. and Edwards, R.H. (1992) Cloning of a delta opioid receptor by functional expression. Science 258, 1963-1955. Fields, H.L., Emson, P.C., Leigh, B.K., Gilbert, R.F.T. and Iversen, L.L. (1980) Multiple opiate receptor sites on primary afferent fibres. Nature 284, 351-353. Glazer, E.J. and Basbaum, A.I. (1983) Opioid neurons and pain modulation: an ultrastructural analysis of enkephalin in cat superficial dorsal horn. Neuroscience 10, 357-376. Gobel, S., Fails, W.M., Bennett, G.J., Abdelmoumene, M., Hayashi, H. and Humphrey, E. (1980) An EM analysis of the synaptic connections of horseradish peroxidase-filled stalked cells and islet cells in the substantia gelatinosa of adult cat spinal cord. J. Comp. Neurol. 194, 781-807. Gouarderes, C., Beaudet, A., Zajac, J.M., Cros, J. and Quirion, R. (1991) High resolution radioautographic localization of 125I-FK33-824 labelled mu opioid receptors in the spinal cord of normal and deafferented rats. Neurosci. 43, 197-209. Gramsch, C., Shultz, R., Kosin, S. and Herz, A. (1988) Monoclonal anti-idiotypie antibodies to opioid receptors. Biol. Chem. 263, 5853-5859. Hamel, E. and Beaudet, A. (1984a) Electron microscopic autoradiographic localization of opioid receptors in rat neostriatum. Nature 312, 155-157. Hamel, E. and Beaudet, A. (1984b) Localization of opioid binding sites in rat brain by electron microscopic radioautography. J. Electron Microsc. Tech. 1,317-329. Hamel, E. and Beaudet, A. (1986) Opioid receptors in rat neostriatum: radioautographic distribution at the electron microscopic level. Brain Res. 401,239-257. Hassan, A.H.S., Almeida, O.F.X., Gramsch, Ch. and Herz, A. (1989) Immunocytochemical demonstration of opioid receptors in selected rat brain areas and neuroblastoma-glioma hybrid (NG 108-

169

15) cells using a monoclonal anti-idiotypic antibody. Neuroscience 32, 269-278. Hiller, J.M., Simon, E.J., Crain, S.M. and Peterson, E.J. (1978) Opiate receptors in culture of fetal mouse dorsal root ganglia (DRG) and spinal cord: predominance in DRG neurites. Brain Res. 154, 396-400. Hrkfelt, T., Ljundahl, A., Terenius, L., Elde, R. and Nilsson, C. (1977) Immunohistochemical analysis of peptide pathways possibly related to pain and analgesia: enkephalin and substance P. Proc. Natl. Acad. Sci. USA 74, 3081-3085. Hunt, S.P., Kelly, J.S. and Emson, P.C. (1980) The electron microscopic localization of methionine-enkephalin within the superficial layers (I and II) of the spinal cord. Neuroscience 5, 1871-1890. Jessel, T. and Iversen, L.L. (1977) Opiate analgesics inhibit substance P release from rat trigeminal nucleus. Nature 268, 549-551. Jomary, C., Gairin, J.E. and Beaudet, A. (1992) Synaptic localization of kappa opioid receptors in guinea pig neostriatum. Proc. Natl. Acad Sci. USA 89, 564-568. Kieffer, B.L., Befort, K., Gazveriaux-Ruff, C. and Hirth, C.G. (1992) The/i-opioid receptor: isolation of a cDNA by expression cloning and pharmacological characterization. Proc. Natl. Acad. Sci. USA 89, 12048-12052. LaMotte, C.C. and De Lanerolle, N.C. (1983) Ultrastructure of chemic,ally defined neuron systems in the dorsal horn of the monkey. I. Methionine-enkephalin immunoreactivity. Brain Res. 274, 51-63. Mauborgue, A., Lutz, O., Legrand, J.C., Hamon, M. and Cesselin, F. (1987) Opposite effects of mu and delta opioid receptor agonists on the in vitro release of substance P-like materiel from the rat spinal cord. J. Neurochem. 48, 529-537. Ninkovic, M., Hunt, S.F. and Gleave, J.R.W. (1982) Localization of opiate and histamine HI-receptors in the primate sensory ganglia and spinal cord. Brain Res. 241, 197-206. Ornatowska, M. and Glazel, J.A. (1992) Two- and three-dimensional distributions of opioid receptors on NG 108-15 cells visualized with the aid of fluorescence confocal microscopy and anti-idiotypic antibodies. J. Chem. Neuroanat. 5, 95-106. Pasquini, F., Bochet, P., Garbay-Jaureguiberry, C., Roques, B.P., Rossier, J. and Beaudet, A. (1992) Electron microscopic localization of photoaffinity labelled delta opioid receptors in the neostriatum of the rat. J. Comp. Neurol. 326, 229-244. Pohi, M., Mauborgue, A., Bourgoin, S., Benoliel, J.J., Hamon, M. and Cesselin, F. (1989) Neonatal capsaicin treatment abolishes modulations by opioids of substance P release from rat cord slices. Neurosci. Lett. 96, 102-107. Ruda, M.A., Coffield, J. and Dubner, R. (1984) Demonstration of postsynaptic opioid modulation of thalamic projection neurons by the combined techniques of retrograde horseradish peroxidase and enkephalin immunocytochemistry. J. Neurosci. 4, 2117-2132. Stevens, C.W., Lacey, C.B., Miller, K.E., Elde, R.P. and Seybold, V.S. ( 1991) Biochemical characterization and regional quantification of mu, delta and kappa opioid binding sites in rat spinal cord. Brain Res. 550, 77-85. Sumal, K.K., Pickel, V.M., Miller, R.J. and Reis, D.J. (1982) Enkephalin containing neurons in substantia gelatinosa of spinal trigeminal complex: ultrastructural and synaptic interaction with primary sensory afferents. Brain Res. 248, 223-236. Todd, A.J. (1988) An electron microscope study of Golgi-stained cells in lamina II of the rat spinal dorsal horn. J. Comp. Neurol. 275, 145-157. Triller, A., Cluzeaud, F., Pfeiffer, F., Betz, H. and Korn, H. (1985) Distribution of glycine receptors at central synapses: an immunoelectron microscopy study. J. Cell Biol. 101,683-688. Van den Pol, A.N. and Gores, T. (1988) Glycine and glycine receptor

170

F. Zerari et al./J. Chem. Neuroanat. 7 (1994) 159-170

immunoreactivity in brain and spinal cord. J. Neurosci. 8, 472-492. Willcockson, W.M.S., Kim, H., Shin, H.K., Chung, J.M. and Willis, W.D. (1986) Actions of opioids on primate spinothalnmic tract neurons. J. Neurosci. 6, 2509-2520. Yaksh, T.L. (1984) Multiple opiate receptor systems in brain and spinal cord: Part II. Fur. J. Anaesthesiol. 1,201-243. Young, W.S., III, Wamsley, J.K.., Zarbin, M.A. and Kuhar, M.J. (1980) Opioid receptors undergo axonal flow. Science 210, 76-78.

Zajac, J.M., Lombard, M.C., Peschanski, M., Besson, J.M. and Roques, B.P. (1989) Autorndiosraphic study of mu and delta opioid binding sites and neutral endopeptidase 24-11 in rat after dorsal root rhizotomy. Brain Res. 477, 400-403. Zieglgansberger, W. and Tulloch, L.H. (1979) The effects of methionine enkephalin and leucine-enkephalin on spinal neurons of the cat. Brain Res. 236, 77-84.