Expression of mu, kappa, and delta opioid receptor messenger RNA in the human CNS: a 33P in situ hybridization study

Pergamon

PII:

Neuroscience Vol. 88, No. 4, pp. 1093–1135, 1999 Copyright  1998 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/99 $19.00+0.00 S0306-4522(98)00251-6

EXPRESSION OF MU, KAPPA, AND DELTA OPIOID RECEPTOR MESSENGER RNA IN THE HUMAN CNS: A 33P IN SITU HYBRIDIZATION STUDY D. PECKYS and G. B. LANDWEHRMEYER* Department of Neurology, Albert-Ludwigs-University Freiburg, Neurozentrum, Breisacherstrasse 64, D-79106 Freiburg, Germany Abstract––The existence of at least three opioid receptor types, referred to as µ, ê, and ä, is well established. Complementary DNAs corresponding to the pharmacologically defined µ, ê, and ä opioid receptors have been isolated in various species including man. The expression patterns of opioid receptor transcripts in human brain has not been established with a cellular resolution, in part because of the low apparent abundance of opioid receptor messenger RNAs in human brain. To visualize opioid receptor messenger RNAs we developed a sensitive in situ hybridization histochemistry method using 33P-labelled RNA probes. In the present study we report the regional and cellular expression of µ, ê, and ä opioid receptor messenger RNAs in selected areas of the human brain. Hybridization of the different opioid receptor probes resulted in distinct labelling patterns. For the µ and ê opioid receptor probes, the most intense regional signals were observed in striatum, thalamus, hypothalamus, cerebral cortex, cerebellum and certain brainstem areas as well as the spinal cord. The most intense signals for the ä opioid receptor probe were found in cerebral cortex. Expression of opioid receptor transcripts was restricted to subpopulations of neurons within most regions studied demonstrating differences in the cellular expression patterns of µ, ê, and ä opioid receptor messenger RNAs in numerous brain regions. The messenger RNA distribution patterns for each opioid receptor corresponded in general to the distribution of opioid receptor binding sites as visualized by receptor autoradiography. However, some mismatches, for instance between µ opioid receptor receptor binding and µ opioid receptor messenger RNA expression in the anterior striatum, were observed. A comparison of the distribution patterns of opioid receptor messenger RNAs in the human brain and that reported for the rat suggests a homologous expression pattern in many regions. However, in the human brain, ê opioid receptor messenger RNA expression was more widely distributed than in rodents. The differential and region specific expression of opioid receptors may help to identify targets for receptor specific compounds in neuronal circuits involved in a variety of physiological functions including pain perception, neuroendocrine regulation, motor control and reward.  1998 IBRO. Published by Elsevier Science Ltd. Key words: brain, human, in situ hybridization, messenger RNA, opioid receptors, phosphorus radioisotopes.

Since their discovery in the 1970s101,116,138,163,175 the opioid receptors (ORs) have been intensively studied. Cloning of the ä41,88,201 and µ23,178,188 as well as ê100,201 OR cDNAs in the early 1990s confirmed that *To whom correspondence should be addressed. Abbreviations: Acc, accumbens nucleus; AVP, vasopressin; CRH, corticotropin-releasing hormone; DAMGO/ DAGO, (D-Ala2, N-Me-Phe4,glycinol5)-enkephalin; DG, dentate gyrus; EDTA, ethylenediaminetetra-acetate; GnRH, gonadotropin-releasing hormone; GP, globus pallidus; GPe, globus pallidus external part; GPi, globus pallidus internal part; 5-HT, 5-hydroxytryptamine or serotonin; ISHH, in situ hybridization histochemistry; LC, locus coeruleus; NBM, nucleus basalis of Meynert; OR, opioid receptor; PAG, periaqueductal gray; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PVN, paraventricular hypothalamic nucleus; RFM, reticular formation; SN, substantia nigra; SNpc, substantia nigra pars compacta; SNpr, substantia nigra pars reticulata; SON, supraoptic nucleus; SP, substance P; SSC, standard saline citrate; TRH, thyrotropin-releasing hormone.

they all belong to the superfamily of G-proteincoupled receptors. The most common cellular response of OR activation is depression of neuronal excitability through various mechanisms, among them inhibition of cyclic AMP formation, an increase in K+ channel and a decrease in Ca2+ channel conductances (for review see Ref. 158). Depending on whether they are located on inhibitory or excitatory neurons, the net effect of OR activation may be inhibition or disinhibition. It has become clear that the ORs play important roles in a broad range of functions and behaviours, such as sensory perception (particularly nociception), reinforcement and reward, neuroendocrine regulation, motor control, learning and memory (for reviews see Refs 124 and 133). Alterations of ORs or the endogenous opioid system have been implicated in the pathophysiology of disorders like Parkinson’s disease, Gilles de la Tourette syndrome,20,54,59 Alzheimer’s disease,6,66 schizophrenia and depression.11,45

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Autoradiographic studies in rodent and primate brain suggested striking species differences in the distribution of ORs.16,152,161,179,205 Following the cloning of the µ, ê and ä OR genes/cDNAs in rat, the cellular distribution of OR transcripts33,48,110 and the localization of OR immunoreactivity has been studied in detail.3,30,107,109,187 These studies confirmed the different distribution patterns of the µ, ê and ä ORs. Although the human homologues of the OR genes have been recently published,93,164,165 there is so far no detailed study establishing the regional and cellular distribution of OR transcripts in human brain. The regional distribution of human OR transcripts has been studied using tissue homogenates;145,164,165 there are two preliminary reports on in situ hybridization histochemistry (ISHH) with OR probes in human brain2,73 and one report on the expression of ê OR mRNA expression in dopaminergic cells of the substantia nigra.200 In the present study, we compare the expression of all three known OR transcripts in human brain at a regional and a cellular level. In view of opposed effects of µ and ê OR activation within the same neuronal system,22,89,95,126,168 we were particularly interested in the question, whether µ and ê OR mRNAs were differently expressed. EXPERIMENTAL PROCEDURES

Post mortem specimens Human brains used in this study were obtained in collaboration with the Department of Neuropathology, University of Freiburg. Brains from eight human subjects (four male, four female; mean age 5313 years, range 26–69; mean interval death–autopsy 18.312 h, range 5.5–45) without known neurological or psychiatric disorder were examined. One hemisphere was fixed in 10% formalin for neuropathological examination. Neuropathological examination was within normal limits for all subjects studied. The other hemisphere and the brainstem including

the cerebellum was immediately dissected at autopsy into slices (1-cm-thick, coronal orientation), rapidly frozen with cooling devices (aluminium plates designed by Vonsattel et al.184) on dry ice and stored at
80C. Blocks were dissected out of the frozen slices and 20-µm-thick sections were cut using a microtome cryostat (Leica CM 3050). The sections were thaw-mounted on Poly--Lysine-coated slides, briefly dried at room temperature and stored at
80C until use. Generation of probes DNA templates for synthesizing RNA probes were generated using polymerase chain reaction (PCR) with cDNA, reverse transcribed from Poly(A)+-enriched human total brain mRNA (Stratagene). PCR was carried out with nested pairs of primers (synthesized by Pharmacia according to our specifications). Primer sequences were chosen from the published OR subtype mRNA sequences (µ OR,5 ê OR,165 ä OR164) using Oligo 4.0 software analysis (National Biosciences). For the localization of the primers and the templates see Fig. 1. Outer primers pairs were 20 mers specific for the targeted cDNA segment. Inner primer pairs were 40 mers, 20 bases of which were specific for the targeted cDNA. The remaining 20 bases included binding sequences for the RNA polymerases SP6 or T7, respectively. Amplification with outer and inner primer pairs was performed using standard protocols.155 Inner primer pair products yielded 0.6-kb (µ OR), 0.62-kb (ê OR) and 0.47-kb (ä OR) long fragments, which were purified using a DNA purification kit (Boehringer), vacuum-dried to a final concentration of 0.8 mg/ml and stored at
20C. RNA probes were synthesized by in vitro transcription of 1 mg of the PCR-generated templates using 80 units SP6 or T7 RNA polymerase (Promega) in a reaction mixture containing 6.5 µM unlabelled UTP, 8 µM á[33P]-UTP (1000–3000 Ci/ mmol, 20 mCi/ml, Amersham), 250 µM each of ATP, CTP and GTP, 40 units RNAsin (Promega), in transcription buffer (Promega, 40 mM Tris–HCl, pH 7.9, 6 mM MgCl2, 2 mM spermidine, and 10 mM NaCl) (total volume 20 µl). In order to obtain a sufficiently high concentration of total UTP (>10 µM of each nucleotide is thought to be necessary for full-length transcripts122) and a minimum of 50% radioactive UTP among the UTPs incorporated in the RNA probes, the á[33P]-UTP was vacuum-dried to half its volume. The reaction mixture was incubated for 1 h at 37C. Following in vitro transcription the template was digested

Abbreviations used in figures A17 Acc Am AO Cau CIN Cl CM cp DG DH DM DMHA F GPe GPi grL IC ic LD LHA LP PAG

Brodmann Area 17 accumbens nucleus nucleus ambiguus accessory olivary nucleus caudate nucleus central inferior collicular nucleus claustrum centromedian thalamic nucleus cerebral peduncle dentate gyrus dorsal horn dorsomedial thalamic nucleus dorsomedial hypothalamic area fornix globus pallidus external part globus pallidus internal part granular cell layer of the dentate gyrus inferior colliculus internal capsula laterodorsal thalamic nucleus lateral hypothalamic area lateroposterior thalamic nucleus periaqueductal gray

PEV PN PRF Put PVN PyD RET RFM scp SG SN SOL tCX V VH VL VMHA VPL VPM X XI ZI III Ve

periventricular hypothalamic nucleus pontine nuclei pontine reticular formation putamen paraventricular hypothalamic nucleus pyramidal decussation reticular thalamic nucleus reticular formation superior cerebral peduncle substantia gelatinosa substantia nigra solitary nucleus temporal cortex spinal trigeminal nerve nucleus ventral horn ventrolateral thalamic nucleus ventromedial hypothalamic area ventroposterior lateral thalamic nucleus ventroposterior medial thalamic nucleus dorsal vagal nerve nucleus accessory nerve nucleus zona incerta third ventricle.

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Fig. 1. Schematic representation of the three cloned human ORs cDNAs with the localization of the templates for both sets of RNA probes. by addition of 1 unit RQ1 DNase (Promega) and incubation at 37C for an additional 15 min. Limited alkaline hydrolysis was used to improve probe penetration: RNA products were cut to fragments of 200 bp by incubation at 60C for 31 min (ê and µ OR) or 27 min (ä OR), respectively, in a carbonate buffer (120 mM Na2CO3, 80 mM NaHCO4, pH 10.4).28 Following limited alkaline hydrolysis sodium acetate was added in order to achieve a neutral pH. Prior to ethanol precipitation yeast tRNA (10 mg/ml) was added as a carrier improving the visibility of the pellet.195 The RNA product precipitated in ethanol at
80C overnight and was pelleted the following day by two centrifugation steps (30 min and 10 min, respectively, 12,500 g, 0C), interrupted by a 70% ethanol wash. The pellet was resuspended in Tris–EDTA buffer with 40 units RNAsin. Incorporation of the radioactive label was assessed by liquid scintillation counting. The required amount of probe for the subsequent ISHH was calculated and immediately used. In addition, we attempted to confirm the results obtained with the probes described above using a second set of probes. The second set of probes was derived from partial human OR cDNAs, subcloned into Bluescript and was generously supplied by B. Kieffer and F. Simonin. These probes only partially overlapped the targeted mRNA sequences of the PCR-derived probes. For the localization of the templates see Fig. 1. The plasmids were linearized by SacI (Promega) digestion. Run off transcription using T7 RNA polymerase (Promega) resulted in antisense probes.

Transcription and ISHH procedures were the same for this second set of probes as for the first. A limited number of brain regions were hybridized with the second set of probes to allow for a comparison of hybridization signals at the regional and cellular level. In situ hybridization histochemistry Slide-mounted tissue sections were brought to room temperature, fixed in 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS), pH 7.4 (10 min), washed in three changes of PBS (5 min each), acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine pH 8.0 (10 min), rinsed in PBS (5 min), dehydrated through graded ethanol solutions (70%, 80%, 95%, 100%, 2 min each), delipidated in chloroform (10 min), rinsed in 100% and 95% ethanol and air dried. All solutions were made with 0.1% diethylpyrocarbonate-treated water. Sections were hybridized for 4 h at 60C in a buffer containing 50% formamide, 0.3 M NaCl, 10 mM Tris, 5 mM EDTA, 10% dextran sulphate, 1 Denhardt’s solution, 100 mM dithiothreitol, 0.1% sodium dodecyl sulphate, 4 mg/ml salmon sperm DNA, 10 mg/ml yeast tRNA, and 10 mg/ml yeast total RNA type XI. The quantity of radioactive probe was adjusted to achieve 100,000 c.p.m./ml hybridization buffer. Depending on the size of the brain sections, 130 to 200 µl hybridization buffer were applied on each section and covered with glass coverslips of an appropriate size. After hybridization, sections were briefly rinsed in 2 standard

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Fig. 2. Phosphorimager pictures of adjacent sections of the frontal cortex demonstrating riboprobe specificity. Hybridization with the ê OR antisense probe (A) produced specific hybridization signals. No signals were observed following hybridization with the antisense probe after RNase A pretreatment (100 µg/ml, 30 min) (B) or following hybridization with the ê OR sense probe (C). Scale bars=2 mm. saline citrate (SSC) at room temperature, washed in 0.1 SSC at 70C for 30 min, treated with RNase A (100 µg/ml in 0.5 M NaCl, 10 mM Tris–HCl, 1 mM EDTA, pH 7.2) for 30 min at 37C, rinsed in RNase buffer for 15 min, washed two more times in 0.1 SSC at 70C for 30 min each, briefly rinsed in 100% and 95% ethanol for 2 min each, and air dried. Dried sections were apposed to phosphorimager plates (Fuji) for five days. Plates were read (Fuji BAS 5000) and converted into digitized images with a resolution of 25 µm (TINA software). Slides were then dipped in Kodak NBT2 emulsion diluted 2:1 with 0.6 M NaAc, stored at 4C for six weeks, developed with D19 developer and counterstained. Dipped slides were viewed using a light microscope (Leica DMRB). Photomicrographs were taken through a camera mounted on the microscope using colour reversal film (Kodak Ektachrome 160 Tungsten). Selected photomicrographs were later converted into digitized pictures. Both, pictures from phosphorimager plates and digitized micrographs, were further edited using Adobe Photoshop 3.0. Experimental controls included hybridization of sections using RNA probes in the sense orientation and treatment with RNase A (100 µg/ml in 0.5 M NaCl, 10 mM Tris–HCl, 1 mM EDTA, pH 7.2) for 30 min at 37C prior to hybridization. RESULTS

General observations Hybridization with the sense strand RNA probes or following pretreatment with RNase A produced no detectable signal in any region of the brain examined. Figure 2 shows results from control experiments for the ê OR probe; similar results were obtained with the µ and ä OR probes. There was some variation in signal intensity between the eight subjects examined. However, the distribution patterns produced by the

µ, ê and ä OR probes were identical in all brain specimens included in this study. Comparison of hybridization signals obtained with probes derived from PCR-generated templates and probes from linearized cDNAs demonstrated identical signal distribution for each receptor in all areas studied. Signals obtained with the latter probes tended to be somewhat less intense both at the cellular and regional level. In the following paragraphs we therefore use signals obtained from PCR-derived templates to illustrate our findings. µ, ê and ä OR antisense RNA probes resulted in distribution patterns distinct for each receptor. The pattern characteristic for hybridization with µ receptor probes consisted of strong signals in the cerebral cortex with a thin line of intense signal in the upper part of layer V, weak scattered signals in the hippocampal formation, patchy signals in the striatum, medium intense labelling of thalamic nuclei, hypothalamus and cerebellum, and low or moderate labelling in substantia nigra, some brainstem nuclei and spinal cord. ê OR probes resulted in hybridization signals in similar regions. However, with the ê OR probes more intense and more widely distributed signals were observed in the striatum, in lower cortical layers and in the claustrum. In some regions there was no overlap in µ and ê OR distribution: the reticular nucleus of the thalamus or the globus pallidus exhibited only µ OR signals. With the ä OR probes intense signals were found in the cerebral cortex, moderate signals in hippocampus and pontine nuclei and weak regional signals in striatum;

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Fig. 3. Phosphorimager pictures of sections of the prefrontal cortex (Brodmann Area 6) (A–C) and occipital cortex (including Brodmann Area 17) (D–F), hybridized with the µ (A, D), ê (B, E) or ä (C, F) OR probes. Scale bars: (A–C)=2 mm, (D–F)=5 mm.

thalamus, hypothalamus and spinal cord appeared unlabelled. At the microscopic level, OR signals were in general associated with cellular profiles of a morphology suggesting neurons. In some instances scattered small cellular profiles of a distribution and morphology reminiscent of glial cells were labelled, suggesting that the expression of ORs is not restricted to neurons in human brain (data not shown). Signal intensities associated with these presumptive glial cells were low. Microscopic

inspection demonstrated that as a rule only a subpopulation of neurons within most brain regions appeared to express OR mRNA. In addition, in some regions each specific OR mRNA seemed to be restricted to a separate subpopulation of neurons suggesting little co-expression of the ORs in individual cells; e.g., in striatum and substantia nigra, where transcripts of all three ORs were present, ä receptor signals were found in different cell populations than µ and ê signals.

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D. Peckys and G. B. Landwehrmeyer Table 1. Distribution of opioid receptor messenger RNA in the telencephalon* µ

Region Prefrontal cortex Layer I Layer II Layer III Layer IV Layer V Layer VI Occipital cortex, area 17 Layer I Layer II Layer III Layer IV Layer V Layer VI Hippocampus Dentate gyrus CA1 CA2 CA3 CA4 Striatum Accumbens nucleus Putamen anterior part Putamen posterior part Caudate nucleus anterior part Caudate nucleus posterior part Ventral pallidum Globus pallidus external Globus pallidus internal Claustrum Basal nucleus of Meynert

ê

ä

Density of labelled neurons

Grain density per labelled neuron

Density of labelled neurons

Grain density per labelled neuron

Density of labelled neurons

Grain density per labelled neuron

0 0 ++ ++ +++ ++

0 0 ++ ++ ++ +–+++

0 ++ +–++ 0 +++ +++

0 +–++ +–++ 0 ++–+++ ++

0 ++–+++ ++–+++ +++ +–++ +

0 + +–++ +–++ + +–++

0 + + + 0 0

0 + + + 0 0

0 + + 0 ++–+++ ++

0 +–++ + 0 +–++ +

0 +++ +++ +++ +–++ +–++

0 + + + +–++ +–++

+–++ +–++ ++ ++ +++

+ ++–+++ ++–+++ +++ +–++

+++ + + ++ +

++ ++ ++ ++ ++

+++ + ++ + +

++–+++ +–++ ++ ++ +

+++ +++ ++ +++ ++ +++ ++ + + +++

++–+++ ++–+++ +–++ ++ +–++ ++–+++ ++–+++ ++ + ++–+++

+++ +++ ++ +++ ++ ++ 0 0 ++++ 0

++ +–++ +–++ +–++ +–++ + 0 0 +++–++++ 0

+++† ++† ++† ++† ++† ++ +–++ 0 0 0

+++ +++ +++ +++ +++ +++ ++ 0 0 0

*Density of labelled neurons: 0, no labelled cells; +, few labelled cells; ++, scattered cells; +++, numerous labelled cells; ++++, nearly all cells labelled. Grain density per labelled neuron: 0, no labelling; +, light labelling; ++, moderate labelling; +++, dense labelling; ++++, very dense labelling. † Only large-sized neurons.

Telencephalon Inspection of the cerebral cortex at the macroscopic level showed that all three OR probes generated detectable signals in all regions examined (frontal, temporal and occipital lobe, cingulate gyrus). Each OR exhibited characteristic and distinct expression patterns in the cortical layers (Fig. 3). Signals generated by the probe targeting the µ OR mRNA were present in laminae III to VI. Superficial parts of lamina V were slightly stronger labelled (Fig. 3A). Signals obtained with the ê OR probe were most prominent in laminae V and VI; weaker signals were apparent in lamina II and III. Lamina IV was devoid of hybridization signals (Fig. 3B). With the ä OR probe, signals were observed in laminae II to VI; in laminae II to IV signals were slightly more intense (Fig. 3C; Table 1). Inspection of emulsion-coated sections demonstrated that distinct subpopulations of cells were labelled by the three OR probes. The µ OR probe produced no signals over neurons in lamina II (Fig. 4B), whereas in lamina III (Fig. 4C) moderate signals appeared over a subpopulation of pyramidal cells

and in lamina IV (Fig. 4D) numerous small- or medium-sized neurons were labelled. In lamina V (Fig. 4E) neuronal profiles of different shapes exhibited grains of variable densities. The macroscopic intense labelling within the superficial layer V was due to a higher ratio of labelled pyramidal cells. Labelled neurons in lamina VI (Fig. 4F) were rare, but some of the smaller neurons displayed rather high grain densities. In cortical sections hybridized with the ê OR probe (Fig. 4G–L), the majority of labelled neurons was of small size and situated between the larger pyramidal cells. A few pyramidal cells were also labelled, but with weaker signal intensities. In laminae II and III, subpopulations of small neurons were labelled (Fig. 4G, H). No labelled cells were found in lamina IV (Fig. 4J). The strong staining of laminae V/VI at the macroscopic level was due to hybridization signals over numerous cellular profiles of variable morphology, consistent with principal neurons and interneurons (Fig. 4K, L). Cellular grain densities were of moderate or high intensity. Inspection of ä OR dipped slides revealed labelling of different types of cell bodies in all cortical laminae

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Fig. 4A–F (caption on p. 1101).

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Fig. 4G–Q.

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Fig. 5. Phosphorimager pictures of hippocampal sections hybridized with the µ (A), ê (B) and ä (C) OR probes. Scale bars=5 mm.

(Fig. 4M–Q). Grain densities per neuron were weak to moderate. Small neurons in lamina II appeared weakly labelled (Fig. 4M). A small number of the pyramidal cells in laminae III was weakly labelled. Moderate labelling was found over large populations of non-pyramidal cells in laminae III and IV (Fig. 4N, O). Only few moderately-labelled neurons of small or medium size were seen in lamina V (Fig. 4P, Q). There were minor regional variations in these laminar labelling patterns and in signal intensity in all neocortical areas examined and in cingulate cortex, with the exception of the occipital cortex (Fig. 3D– F). Macroscopically very weak µ OR hybridization signals were found in the occipital cortex; in area 17 signal intensity decreased to background levels. Microscopically only very few small-sized cells were weakly labelled in laminae II to IV in the occipital cortex. ä OR signals in area 17 in contrast were particularly prominent with strong signals in a heterogeneous cell population of laminae II to IV. Here, the majority of cells was moderately labelled and contributed, together with a particularly high packing density of neurons, to the strong macroscopic signal.

In the hippocampus, macroscopic examination disclosed no signals in the granular cell layer of the dentate gyrus (DG) in slides hybridized with the µ OR probe, but showed a marked hybridization signal with the ê and ä OR probes (Fig. 5). Low signal intensities for all three OR subtype mRNAs were found in the pyramidal cell layer showing different distribution patterns in the CA1–4 subregions for µ, ê and ä OR probes. Microscopic study of µ OR slides (Table 1) revealed grain densities slightly above background levels over the granular cells of the DG. The majority of pyramidal cells in the CA subregions displayed no or weak signals. More densely-labelled cells of variable morphology and size were scattered throughout CA1–4 (Fig. 6A). The distribution of cells labelled by the ê OR probe in the hippocampus was different: granular cells of the DG were moderately labelled, in the CA4 region a small subpopulation of pyramidal cells was sparsely labelled (Fig. 6B); their number and cellular labelling intensity increased in CA3. Slightly stronger signals were observed on small neurons scattered in the stratum oriens and lacunosum-moleculare. Signals on similar cells, probably representing interneurons, could be found in the CA1 region and in the subiculum.

Fig. 4. Nissl-stained section of the prefrontal cortex (A) and micrographs of prefrontal cortex sections hybridized with the µ (B–F), ê (G–L) and ä (M–Q) OR probes. With the µ OR probe no signals were seen over neurons in lamina II (B). Signals were present over a subset of pyramidal cells in lamina III (C) and over smaller neurons in lamina IV (D). In lamina V (E), neurons of different shapes were labelled: arrowheads point to labelled pyramidal cells, whereas the arrow indicate a smaller labelled neuron. In lamina VI (F), labelled neurons were rare. ê OR-labelled neurons were found in lamina II to III (see arrows in G and H), but were absent in lamina IV (J). Labelling of many small- to medium-sized neurons was found especially in laminae V (K) and often over similar-sized neurons in lamina VI (L). Micrographs from sections of the prefrontal (M, N, P, Q) and occipital (A17) (O) cortices, hybridized with the ä OR probe. In panel M, arrowheads indicate small labelled neurons in the deeper parts of lamina II. In lamina III (N), a large subpopulation of cells with similar profiles (see arrowheads) were labelled. In lamina IV of the visual cortex (N), where neurons were more densely packed, numerous small-sized neurons (arrowheads) were found to be labelled. Only a few small- to medium-sized neurons (arrowheads) were labelled in laminae V (P) and VI (Q). Scale bars: (A)=200 µm, (B–Q)=50 µm.

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Fig. 6. Micrographs from hippocampal sections hybridized with the µ (A), ê (B) and ä (C) OR probes. Panel A shows a typical small labelled neuron, situated among unlabelled or weakly-labelled pyramidal cells in CA2, panel B shows the CA4 region: the arrow points to a small labelled neuron lying between unlabelled and weakly-labelled pyramidal cells (arrowheads) and panel C shows an isolated neuron in the stratum oriens. Scale bars=50 µm.

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Moderate signals generated with the ä OR probe were found on granular cells of the DG and in CA1–2 sub-regions on scattered neurons of moderate size. In addition, signals from the ä OR probe were found on a few isolated cells of the stratum oriens (Fig. 6C). In the striatum, signals were obtained with probes for all three ORs. µ OR signals appeared macroscopically heterogeneous and patch-like (Fig. 7A). Shape and size of these patches varied considerably between individual brains and patches were more or less prominent in different subjects. In the nucleus accumbens (Acc), µ OR signals were intense. Shell and core divisions of the Acc (delineated in closeby sections by autoradiographic labelling with [3H]DAMGO; data not shown) did not differ in signal intensity. Staining intensity decreased in the dorsal parts of the anterior striatum; in the dorsal regions patches became smaller. There was a clear anteriorposterior decrease of signal intensity, particularly in dorsal parts of the putamen. In the posterior putamen (at the level of the globus pallidus [GP]) patches were barely visible. In addition, ventral parts of the putamen displayed stronger signals than dorsal parts. Macroscopically, signals obtained with the ê OR probe were more widely distributed in the anterior striatum than those seen with the µ OR probe and extended throughout the anterior striatum (Fig. 7B). Higher signal intensities were seen in medioventral parts, including the Acc. Shell and core divisions of the Acc appeared not to be differentially labelled. A heterogeneous patchy ê OR probe staining pattern, already visible in the anterior striatum, but less marked than the µ OR patches, became more apparent in posterior parts of the putamen. In the rostrocaudal direction a decrease in signal intensity was visible; the staining of ventromedial parts was slightly stronger than of dorsal parts (Fig. 7C). Macroscopic staining produced by the ä OR probe was punctate but rather weak in all parts of the striatum. The internal and external parts of the globus pallidus (GPi and GPe) appeared macroscopically unlabelled by the µ, ê (Fig. 7C) and ä OR probes. On microscopic examination, the three different OR probes appeared to label distinct populations of striatal neurons, although there seemed to be some overlap between neurons labelled by µ and ê OR probes. Inspection of slides hybridized with the µ probe showed clusters (Fig. 8A) of strongly labelled medium-sized cells (Fig. 8B) surrounded by almost unlabelled cells. This arrangement prevailed in the anterior striatum, particularly in the caudate nucleus. In caudal parts, clusters were less apparent and consisted of fewer and weaker labelled cells. Large striatal neurons appeared to be unlabelled. The µ OR probe gave rise to cellular hybridization signals over some cells in both parts of the GP (Fig. 9A) as well as over few large cells found in the capsula interna and lamina medullaris lateralis. µ OR labelled cells, sometimes arranged into groups, could also be seen in the ventral pallidum.

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Microscopic examination of slides hybridized with the ê OR probe revealed that a large number of cells in the anterior striatum, probably the majority, was labelled. The intensity of the cellular signal varied. Moderate to high cellular signals were associated with medium-sized cells, arranged into clusters. Low cellular signals were found over medium-sized cells which were more mixed with unlabelled neurons and surrounded the clusters forming a loose matrix (Fig. 10A, B). Probably for this reason ê OR clusters appeared less distinct than the clusters seen with the µ OR probe. Large-sized neurons appeared to be unlabelled. Cell clusters occupied most of the area of the Acc; the highest hybridization signals were found in and around the Acc. Progressing in the rostrocaudal direction of the striatum, the number of labelled cells decreased. In the posterior striatum the caudate nucleus tended to contain more labelled cells than the putamen. Few large cells displaying ê OR signals were detected in white matter tracts lining the different segments of GP and at the border to the internal capsula. Within the GP no ê OR labelled cells were detected. Cellular hybridization signals for the ä OR mRNA were only seen over large, polygonal cells, scattered throughout the entire striatum, probably representing cholinergic striatal interneurons (Fig. 11A). Labelling intensity per cell was high, but due to the low density of labelled cells, the signal intensity at the macroscopic level was low. In the ventral striatum, these labelled cells were encountered more frequently, sometimes forming loosely clustered groups. Signals from the ä OR probe in the striatum were restricted to this distinct cell population, which displayed no labelling with the µ or ê OR probes: no other striatal cell population was labelled with the ä OR probe. Few labelled cell were found in the GPe (Fig. 11B) and in the surrounding white matter tracts. The GPi did not contain ä OR labelled cells. In the nucleus basalis Meynert (NBM) clusters of large neurons displayed µ OR signals. About 50% of the cells were labelled at medium intensity (Fig. 11C). Hybridization with the ê or ä OR probe resulted in no signals in neurons in the region of the NBM. However, we observed clusters of large ê OR-labelled neurons in the white matter tracts lining the GP including the ventral internal capsula, which might represent a subpopulation of cholinergic neurons. The claustrum, in contrast, showed no macroscopic labelling with the µ or with the ä probe, but very high hybridization signals with the ê OR probe (Fig. 7C). Microscopic inspection confirmed a strong cellular ê OR labelling (Fig. 13D) and revealed low µ OR signals over some neurons. Diencephalon In most thalamic and hypothalamic nuclei, intense signals were obtained with µ as well as with ê OR probes (Fig. 12A–D). In no diencephalic structure

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Fig. 7. Phosphorimager pictures of the anterior (A, B) and posterior striatum (C) demonstrating the different distribution patterns produced with the µ (A) and ê (B, C) OR probes. Scale bars=5 mm.

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Fig. 8. Micrographs of µ OR mRNA distribution in the Acc. (A) Dark-field micrograph demonstrating the typical patch-like clustering of µ OR-labelled cells. (B) Bright-field micrograph at higher magnification showing a set of strongly-labelled medium-sized striatal neurons within a ‘‘patch’’, arrowheads point to unlabelled neurons at the border of the cell cluster. Scale bars: (A)=400 µm, (B)=50 µm.

studied could signals indicating ä OR mRNA expression be detected (Table 2). Macroscopic inspection of thalamic slides, incubated with the µ OR probe, revealed a strong labelling of the anterior and medial nuclear group, of the laterodorsal nucleus, the dorsal aspects of the lateral nuclear group, the intralaminar nuclei including the zona incerta, and of the reticular nucleus. Within the lateral nucleus signals in the ventroposterior lateral and ventroposterior medial nuclei were comparatively weak. The centromedian nucleus appeared unlabelled (Fig. 12A). ê OR signals could be detected macroscopically over the anterior and medial nuclear group as well as over the lateral dorsal nuclei with a less apparent ventrally decreasing gradient in the lateral nuclear group. The centromedian

and the reticular nucleus and the zona incerta were unlabelled (Fig. 12B). Microscopic examination of the µ OR dipped slides revealed that, within any given macroscopically labelled thalamic nucleus, most neurons displayed moderate to high grain densities (Fig. 13A, B). In the reticular nucleus, all neurons showed very high grain densities (Fig. 13B). In general, a moderate signal intensity was found in most neurons of macroscopically labelled nuclei. The macroscopic absence of ê OR signals in the centromedian, the reticular nucleus and in the zona incerta was confirmed by microscopic inspection; no labelled cells were found in these nuclei. Neurons in the ventroposterior lateral nucleus were found to be moderately labelled (Fig. 13C).

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Fig. 9. Micrographs representing µ OR distribution at the cellular level in the GPe (A) and in the subthalamic nucleus (B). One of the few, but strongly µ OR-labelled neurons in the GPe is shown in panel A. In the subthalamic nucleus, numerous neurons displayed moderate grain densities (B). Scale bars=50 µm.

In the subthalamic nucleus, numerous cells moderately labelled with the µ OR probe were detected (Fig. 9B). Hybridization with the ê and ä OR probes resulted in no definitive cellular labelling. The hypothalamus was studied at the tuberal region and the mammillary region. Signals were obtained with µ and ê, but not with ä OR probes. Macroscopically, strong µ OR signals appeared over the paraventricular nucleus (PVN) and extended further in dorsolateral regions. (Fig. 12C). Signals from the periventricular, ventromedial, dorsomedial and lateral hypothalamic areas were weaker. The ê OR probe produced strong and more uniform signals over these hypothalamic regions. In the mammillary body, neither at the macroscopic, nor at the micro-

scopic level, were signals detected using any OR probe. Microscopically, moderate to strong cellular hybridization signals were obtained with the µ OR probe in about 40% of the parvocellular neurons of the PVN. Within the magnocellular population of the PVN a small number of neurons was moderately labelled. In contrast, in the supraoptic nucleus (SON) almost every third neuron was labelled by the µ OR probe, but cellular signal intensity was lower than in PVN. The morphology of labelled neurons in the periventricular, ventromedial, dorsomedial and lateral hypothalamic areas were quite diverse. The proportion of labelled neurons was lower than in the PVN, grain densities were moderate

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Fig. 10. Micrographs of ê OR mRNA expression signals at the cellular level in the Acc. A typical ê OR cell cluster composed of medium-sized neurons is shown in panel A. In panel B, some medium-sized neurons in the Acc are shown at higher magnification demonstrating unlabelled neurons mixed with labelled neurons. Scale bars: (A)=50 µm, (B)=20 µm.

to strong. The signal intensity and number of labelled neurons in the dorsolateral area was similar to the PVN. The number of ê OR labelled parvo- and magnocellular neurons in the PVN was similar to those in sections hybridized with the µ OR probe. In the SON, no ê OR signals could be found. In the periventricular, ventromedial, dorsomedial dorsolateral and lateral hypohalamic areas around half of the neurons were moderately to densely labelled. With the ä OR probe no signals or labelled cells could be detected in the regions of the hypothalamus studied (Table 2). Mesencephalon Phosphorimager pictures demonstrated moderate signals for the µ OR subtype mRNA in the substantia nigra (SN) (Fig. 15A). The ê OR probe produced a

moderate hybridization signal within the SN (Fig. 15B) whereas ä OR probe signals were not detectable. Macroscopically, none of the three OR probes labelled the red nucleus or the central inferior collicular nucleus. At the microscopic level, there were marked differences in the distribution patterns of the OR subtypes in the SN. In the µ OR slides, no signals could be detected over neuromelanin-containing cell bodies (probably dopaminergic neurons). A moderate to strong labelling was seen over many nonpigmented cells within the area of the pigmented SN pars compacta (SNpc) neurons, and extending in the lateral and dorsal direction (Fig. 16A). In the medial dorsal part of the red nucleus, a subpopulation of medium-sized cells was moderately labelled by the µ OR probe. Moderate ê OR probe signals were seen over the typical large, pigmented cell bodies of the SNpc (Fig. 16B) and few non-pigmented cells

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Fig. 11. Micrographs depicting ä OR mRNA distribution at the cellular level in the caudate nucleus (A) and GPe (B) and µ OR mRNA distribution in the NBM (C). A typical ä OR-labelled striatal cell of polygonal shape and large size is shown in panel A. Few cells in the GPe (B) were found to be labelled by the ä OR probe. In panel C µ OR-labelled neurons of the NBM are shown. Scale bars: (A, B)=50 µm, (C)=100 µm.

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Fig. 12. Phosphorimager pictures of the thalamus (A, B) and hypothalamus (C, D) demonstrating the different distribution patterns generated with the µ (A, C) and ê (B, D) OR probes. Scale bars=2 mm.

scattered in between. The ä OR probe produced no unequivocal signals in SN. In the central inferior collicular nucleus, scattered neurons showed low to moderate µ OR signals. In the periaqueductal gray (PAG), µ and ê OR scattered labelled cells with moderate to strong hybridization signals were found (Fig. 18C).

Met- and myencephalon The pontine nuclei were clearly labelled by the ê and ä OR probes (Fig. 15B, C). With the µ OR probe macroscopic signals were not detected. Within the large group of brainstem nuclei, the detection of macroscopically visible signals of the µ OR probe was

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D. Peckys and G. B. Landwehrmeyer Table 2. Distribution of opioid receptor messenger RNA in the diencephalon* µ

Region Subthalamic nucleus Zona incerta Thalamus Anterior nuclear group Medial nuclear group Lateral nuclear group Ventral posterior lateral nucleus Ventral posterior medial nucleus Lateral dorsal nucleus Intralaminar nuclei Reticular nucleus Centromedian nucleus Hypothalamus Supraoptic nucleus Paraventricular nucleus Periventricular nucleus Dorsomedial area Ventromedial area Lateral area Mammillary body

ê

Density of Grain density labelled per labelled neurons neuron

Density of labelled neurons

ä

Grain density Density of Grain density per labelled labelled per labelled neuron neurons neuron

++–+++ ++

++–+++ +++

0 0

0 0

0 0

0 0

++++ ++++

+++ ++

+++–++++ +++

+–++ +

0 0

0 0

0 + ++++ ++++ ++++ 0

0 ++ ++ +++ +++ 0

++–+++ +++ ++ ++++ 0 0

+–++ +–++ +–++ ++ 0 0

0 0 0 0 0 0

0 0 0 0 0 0

+++ +++ ++ ++–+++ ++–+++ ++–+++ 0

++–+++ ++–+++ ++–+++ +++ +++ +++ 0

0 +++ ++–+++ +++ +++ +++–++++ 0

0 ++–+++ ++–+++ +++ +++ +++–++++ 0

0 0 0 0 0 0 0

0 0 0 0 0 0 0

*See Table 1 for legend.

restricted to parts of the reticular formation (RFM), to the spinal trigeminal nerve (V) and to the accessory olivary nucleus (Fig. 17A, B). Moderate ê OR signals were restricted to the arcuate and nucleus ambiguus. At the macroscopic level, the locus coeruleus (LC) and the raphe nuclei appeared to be unlabelled. Microscopically, most cells in all parts of the pontine nuclei were labelled by the ê OR probe, but cellular labelling intensity was weak to moderate. Labelling with the ä OR probe produced hybridization signals over all neurons throughout the pontine nuclei with moderate cellular grain densities. No labelled cells were found in the pontine nuclei with the µ OR probe. Microscopic inspection of the raphe nuclei showed moderate to high grain densities over scattered neurons in sections hybridized with the µ (Fig. 18A) and ê OR probes. The LC revealed moderate signals with the µ OR probe over many non-pigmented neurons of the LC pars alpha, lying just adjacent to the large, pigmented cells of the LC (Fig. 18B). These pigmented cells were not labelled by any of the three OR probes. The ä OR probe produced no signals in this areas. Distinct hybridization signals obtained with the µ, ê and the ä OR probes were found in numerous other brainstem nuclei (see Table 3). In all instances not every neuron in the nuclei studied was labelled. Signals with the µ OR probe, but not with any other OR probe, were found in the tegmental pedunculopontine nucleus, parabrachial and paralemniscal nuclei and in the inferior olivary nucleus. For the ê OR probe, exclusive signals were obtained in the gigantocellular (Fig. 19B), trochlear and arcuate

nucleus. ä OR probe signals were present in scattered cells in the gracile nucleus. Both µ and ê OR probes produced signals over subpopulations of neurons in the reticular pontine nuclei (Fig. 19A) medial accessory olivary nucleus, solitary tract nucleus and dorsal vagal nerve nucleus (Fig. 20A, B). Hybridization signals from the ê and ä OR probes were observed in the arcuate nucleus and in the cuneate nucleus (Fig. 20C). Signals from all three OR probes were detected in the spinal trigeminal nerve nucleus (Fig. 19C) and in the nucleus ambiguus. In both nuclei most, if not all neurons, were found to be labelled with all three OR probes. Spinal cord Slides of cervical spinal cord hybridized with the µ and ê OR probes produced macroscopically visible signals in the outer laminae of the dorsal horn (Fig. 17C, Table 4). In addition, the µ OR probe produced weak hybridization signals in the zona intermedia and scattered signals in the ventral horn. No signals were detected using the ä OR probe. The caudal parts of the accessory nerve nucleus, descending into the first segments of the cervical spinal cord, were macroscopically labelled by the µ OR probe. At the microscopic level, a subpopulation of cells in the accessory nerve nucleus displayed moderate to strong µ OR labelling, but no labelling with the two other OR probes. Weak to moderate µ OR signals were seen over numerous small cells in the substantia gelatinosa (Fig. 21A). Some scattered medium-sized neurons in the zona intermedia and very few cells in the ventral horn were weakly labelled (Fig. 21B). In

Fig. 13. Micrographs of µ (A, B) and ê (C, D) OR mRNA distribution in the thalamus (A–C) and claustrum (D). The dark-field micrograph (A) demonstrates the expression of µ OR mRNA in neurons of the lateral dorsal nucleus. The bright-field micrograph (B) shows µ OR-labelled neurons of the reticular nucleus. A subpopulation of neurons in the ventroposterior lateral nucleus displayed ê OR signals (C). In the claustrum (D), nearly all neurons exhibited high ê OR signals. Scale bars: (A)=400 µm, (B–D)=100 µm.

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Fig. 14. Micrographs of hypothalamic sections, hybridized with the µ (A) and ê (B, C) OR probe. A dark-field micrograph (A) of µ OR-labelled cells in the dorsomedial hypothalamic area. High magnification bright-field micrographs of the PVN demonstrating signals over µ (B) and ê OR (C)-labelled magno- (arrowheads) and parvocellular neurons (arrows). Scale bars: (A)=100 µm, (B, C)=50 µm.

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Fig. 15. Phosphorimager pictures of brain sections at the caudal level of the mesencephalon (A, B) and the rostral pons (C), hybridized with µ (A), ê (B) and ä OR (C) probes. Scale bars=5 mm.

sections hybridized with the ê OR probe fewer cells in the substantia gelatinosa were labelled (Fig. 21C). Compared to sections hybridized with the µ OR probe, ê OR mRNA-positive cells were found to extend more ventrally into dorsal horn. In the intermediate and ventral parts, scattered weakly ê ORlabelled neurons were observed (Fig. 21D). There was no evidence for ä OR-labelled cells at microscopic inspection. Cerebellum In the cerebellum, the granular cell layer showed, at the macroscopic level, intense hybridization signals for the µ and ê OR probes and very weak signals for the ä probes (Fig. 22A–C). Microscopic inspection confirmed a moderate µ and ê OR labelling of the granular cells. ä OR signals over granular cells were not above background levels. Subpopulations of Golgi cells were moderately labelled by all three OR probes (Fig. 23A, C). The fraction of Golgi cells displaying µ OR signals was equal to the one labelled by the ê or ä OR probes. In addition, a number of Purkinje cells exhibited weak ê OR signals (Fig. 23B). Signals over Purkinje cells from µ and ä OR probes were close to background levels. No OR mRNA signals were detected in the dentate nucleus of the cerebellum. DISCUSSION

In the present study we report on the regional and the cellular localization of mRNAs encoding µ, ê and ä ORs in the normal human brain. To our

knowledge this is the first description of the expression of all three known OR genes in human brain allowing a comparison of their distribution. Our findings complement previous receptor binding studies demonstrating a differential distribution of the µ, ê and ä OR binding sites in the human brain44,64,66,74,102,119,141,143,144,183,185 and clarify the cellular localization of the OR subtype mRNAs. In many regions, ISHH disclosed that the expression of OR mRNA was restricted to subpopulations of neurons. Specificity of probes and hybridization signals The specificity of the in situ hybridization signals obtained was assessed in several ways. No signals were seen in sections treated with RNase A prior to hybridization suggesting that the signals observed resulted from binding to single-stranded RNA. Hybridization with OR sense probes did not result in any signals thus arguing against a non-specific interaction of the RNA probes with other RNA species. Taking into account nucleic acid homologies of about 60% between the cloned µ, ê and ä mRNAs,92,158 there is the possibility that probes intended to recognize one OR specifically may crosshybridize with other OR mRNAs. Hybridization with the three PCR-derived OR RNA probes resulted in signals with three unique distribution patterns distinctly different from one another, therefore suggesting, under the conditions used, specific hybridization signals. Furthermore, hybridization with a second set of OR riboprobes, derived directly from the cloned human cDNAs and only partially

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Fig. 16. Micrographs of cellular µ (A) and ê (B) OR mRNA expression signals, found in SNpc. The large and dark pigmented cell bodies, at the right border (A), were characteristically unlabelled with the µ OR probe, but smaller neurons, often located close to these large cells, were labelled. Hybridization with the ê OR probe produced labelling of the large pigmented neurons (B). Scale bars=100 µm.

overlapping with our probes, resulted in identical distribution patterns for each OR subtype. In addition, there is in general a good correspondence to OR binding studies. We are therefore confident that probes and conditions used allow the selective visualization of µ, ê and ä OR mRNAs in the human brain. Given that all three OR genes contain multiple introns and that splice variants have been described5,46,204 it is possible that each probe recognizes multiple OR mRNA isoforms. The probes were not designed to discriminate between different splice variants and the important question of a selective expression of alternatively spliced forms has to be addressed in future studies using isoform-selective probes. Pharmacological and receptor binding studies in rodent and human brain suggest subclasses

of µ,40,134 ê90,152,205 and ä ORs.65 Presently, however, there is no evidence for the existence of additional OR genes or for OR splice variants in humans with pharmacological properties characteristic for the proposed OR subclasses. The pharmacologically defined subtypes may therefore represent products of differential post-translational processing or may reflect different G-protein binding.149 If this reasoning is correct, our probes would be able to detect the expression of all OR transcripts. Our study is limited by the low apparent levels of OR transcripts in human brain and the fact that there is a perimortal reduction and degradation of mRNA in human postmortal tissue.7,19 The absence of signals in any region or cell population has therefore to be interpreted with caution: the absence of a detectable

Opioid receptor mRNAs in human brain

Fig. 17. Phosphorimager pictures of µ OR-hybridized sections of the medulla oblongata at the level of the vagal nerve (A), the level of the pyramidal decussation (B) and a section of the cervical spinal cord (C). Scale bars=5 mm.

signal does not necessarily imply the absence of mRNA for a given OR but indicates at least low levels of expression. This caveat applies in particular to our results obtained with the ä probe since this probe was shorter than the µ and ê probes. It is interesting, however, to note that the comparatively low levels of expression of ä OR corresponds to low levels of ä OR binding sites in human brain.143 Cerebral cortex In the cerebral cortex, hybridization with OR probes resulted macroscopically in laminar signals highly characteristic and distinct for each OR. µ OR mRNA was present in laminae III to VI and appeared to be enriched in superficial parts of lamina V. Microscopically the intense labelling of the super-

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ficial part of layer V was due to a high density of labelled pyramidal neurons. µ OR binding is present in layers I–VI, more dense in layer IIIa and less dense in lower cortical layers suggesting a somatodendritic expression of µ ORs in the cerebral cortex.64 Aside from polymorph neurons of medium to small size (probably interneurons), larger neurons with a pyramidal morphology (most likely projection neurons) also expressed µ OR mRNA. It is conceivable, therefore, that µ ORs are expressed presynaptically on terminals of projection neurons and contribute to µ OR binding in layer I and target regions of corticofugal fibres e.g., striatum and thalamus, regions with intense µ OR binding185 where some mismatch between the distribution of µ OR binding and mRNA exist. Signals obtained with the ê OR probe were most prominent in laminae V and VI; weaker signals were apparent in lamina II and III. Neurons in lamina IV were unlabelled. The more intense signals in lower cortical layers are in agreement with previous findings using cRNA probes.2 Autoradiographic receptor binding studies of the human cortex64,102,143,144 have demonstrated strong ê OR binding in the deeper laminae V and VI, matching the distribution of ê OR mRNA signals. A predominantly local synthesis and expression of ê OR in cerebral cortex is therefore likely and consistent with the labelling of numerous putative interneurons with the ê OR probe. A local role of ê OR in deeper cortical layers is further suggested by the expression of prodynorphin mRNA in layer V neurons throughout the human cortex.69 In addition, some pyramidal cells, particularly in layer V and VI, were labelled and may give rise to ê OR signals in the main projection regions of these cells, namely the striatum121 and the thalamus,77,192 where strong ê OR binding can be seen.183 With the ä OR probe, signals were observed in laminae II to VI; in laminae II to IV signals were more intense than in deeper layers. Peak levels of ä OR binding were seen in laminae I to IIIa,64 only partially corresponding to ä OR mRNA peak levels. The additional ä OR binding sites in lamina I may indicate an expression on cortico-cortical terminals consistent with the weak ä OR hybridization signals over pyramidal cells. Moderate to intense labelling was found over large populations of non-pyramidal cells in the upper cortical layers. Proenkephalin mRNA signals, indicating the expression of the endogenous ligand for ä ORs, are predominant in the superficial layers69 supporting an intrinsic functional role of ä ORs. Hybridization signals were similar in all cortical areas studied with the exception of occipital cortex, where µ OR mRNA signals declined to background levels, especially in area 17, the primary visual cortex. ä OR mRNA signals in contrast, were more intense and more spread out in cortical layers of area 17. These findings are in agreement with OR binding studies of human occipital cortex64 showing similar

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Fig. 18. Micrographs of cells from various brainstem nuclei, labelled by the µ (A, B) and the ê OR (C) probes. The raphe nuclei contained many labelled neurons (A). None of the three OR probes labelled the large pigmented, presumably noradrenergic neurons of the LC depicted in panel B. However, some small neurons in the region of the LC, indicated by an arrow, were strongly labelled. In the PAG (C), scattered neurons displayed hybridization signals. Scale bars=100 µm.

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Table 3. Distribution of opioid receptor messenger RNA in the mes-, met- and myencephalon* µ

Region Substantia nigra Pars compacta Pars reticulata Central inferior collicular nucleus Periaqueductal gray Trochlear nerve nucleus Pontine nuclei Tegmental pedunculopontine nucleus Locus coeruleus Pigmented neurons Pars alpha Reticular formation Gigantocellular nucleus Reticular pontine nuclei Lateral lemniscal nucleus Raphe nuclei Parabrachial nucleus Paralemniscal nucleus Dorsal vagal nerve nucleus Solitary tract nucleus Gracile nucleus Cuneate nucleus Spinal tract trigeminal nerve nucleus Ambiguus nucleus Retroambiguus nucleus Inferior olivary nucleus Medial accessory olivary nucleus Arcuate nucleus Supraspinal nucleus Accessory nucleus Cerebellum Granular layer Golgi cells Purkinje cells

ê

ä

Density of Grain density Density of Grain density Density of Grain density labelled per labelled labelled per labelled labelled per labelled neurons neuron neurons neuron neurons neuron ++† + ++–+++ +++ 0 0 ++–+++

+++ +–++ ++–+++ ++–+++ 0 0 ++–+++

+++‡ + 0 ++–+++ + ++++ 0

++–+++ + 0 +++ ++ ++ 0

0 0 0 0 0 ++++ 0

0 0 0 0 0 ++ 0

0 ++§

0 +++

0 +¶

0 +++

0 0

0 0

0 +++ ++ ++ +++ ++–+++ ++–+++ +++ 0 0 ++++ ++++ ++ + + 0 0 ++

0 +++ +++–++++ +++ +++ ++–+++ ++–+++ ++–+++ 0 0 +++–++++ +++ +++ +–++ +–++ 0 0 ++–+++

+–++ + ++ +++ 0 0 +–++ ++ + 0 ++++ ++++ 0 0 0 ++++ ++ 0

+–++ + ++ ++–+++ 0 0 ++ +++ + 0 +++ +++ 0 0 0 ++–+++ +++ 0

0 0 0 0 0 0 + + + + +++ + 0 0 0 ++++ 0 0

0 0 0 0 0 0 + + + ++ ++ + 0 0 0 ++–+++ 0 0

++++ ++–+++ 0

++ ++–+++ 0

+++ ++–+++ ++

++ ++–+++ +

0 ++–+++ 0

0 +–++ 0

*See Table 1 for legend. Only non-pigmented neurons. ‡ Only pigmented neurons. § Only small-sized neurons. ¶ Only medium- to large-sized neurons. †

changes for µ and ä OR binding, respectively. Given this pattern one might speculate that ä OR mediated inhibition in layer III and IV plays a important role in the processing of excitatory visual inputs. Based on binding studies, the absence of µ OR appeared to be limited to primary visual cortical areas; we did not include other primary cortical areas in our ISHH study. In rat brain, the occipital cortex did not show a similar decrease of µ OR signals.110 Expression of µ OR in rodent cerebral cortex was overall weak and signals were restricted to relatively few cells suggesting that the cortical distribution pattern in rodents is different from man. Particularly striking differences in the expression patterns in human and rodent cortex were observed for the ê OR. Areas like the frontal cortex displayed no ê OR signals in rat,111 whereas a strong signal was observed in human brain. In rat, the expression of ê OR was restricted to cortical regions like the temporal cortex, where ê OR mRNA was enriched in deeper cortical layers. The ä

OR, in contrast, was present in all cortical areas in rat with a similar laminar distribution as in man.110 Overall expression of OR mRNA in human cerebral cortex was far more prominent than in rat brain. Hippocampal formation In animals, opioids have been shown to modulate neuronal transmission in the hippocampus by OR specific mechanisms (for review see Ref. 162). It is therefore of interest to establish to what extent rodents and humans are similarly equipped in terms of OR expression. In the human hippocampal formation, we observed very weak µ OR mRNA expression in granular cell layer of the DG and low scattered signals in the CA1–4 subregions. This observation is in good agreement with binding studies demonstrating very light binding in both areas.64 Microscopic examination showed µ OR mRNA-labelled cells of variable morphology and

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Fig. 19. Higher magnification micrographs presenting examples of cellular µ (A), ê (B) and ä (C) OR hybridization signals in diverse brainstem regions. The figure shows shows a labelled neuron in the RFM (A), in the gigantocellular nucleus (B) and a group of neurons in the spinal trigeminal nerve nucleus (C). Scale bars=50 µm.

Opioid receptor mRNAs in human brain

Fig. 20. Micrographs from different brainstem sections hybridized with the ê (A) and ä (B, C) OR probes. The two first micrographs were examples of labelled neurons found in the dorsal vagal nerve nucleus. In the cuneate nucleus (C), only few cells labelled could be seen. Scale bars: (A, B)=50 µm, (C)=100 µm.

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D. Peckys and G. B. Landwehrmeyer Table 4. Distribution of opioid receptor messenger RNA in the spinal cord* µ

Region Dorsal horn Substantia gelatinosa Zona intermedia Ventral horn

ê

ä

Density of labelled neurons

Grain density per labelled neuron

Density of labelled neurons

Grain density per labelled neuron

Density of labelled neurons

Grain density per labelled neuron

+–++ ++–+++ + +

++ ++ +–++ +

+ +–++ + +

+–++ +–++ + +

0 0 0 0

0 0 0 0

*See Table 1 for legend.

size scattered throughout CA1–4. The distribution of these cells is similar to the distribution of GABAergic interneurons suggesting that µ OR may inhibit these neurons e.g., by hyperpolarization as demonstrated in the rat.103,203 In addition, pyramidal cells were weakly labelled in CA subregions. Signals generated with the ê OR probe were intense over the granular cell layer of the DG; regional signal intensities in the pyramidal cell layer of the CA1–4 subregions were low. ê binding, in contrast, seemed to be restricted to the pyramidal cell layer and did not appear in the DG.64 The absence of binding sites in the DG despite intense mRNA signals suggests an expression of ê OR in mossy fibres and agrees well with the reported presynaptic inhibition of hippocampal mossy fibre synapses by dynorphin.190 In humans therefore, modulation of long-term potentiation in hippocampus may involve ê OR as demonstrated in the rat. In addition, the presynaptic expression of ê OR in granule cells of the DG which are known to express dynorphin69 may be a particularly striking example for the hypothetical role of ê ORs as autoreceptors. At the same time the expression of ê OR mRNA and binding sites by pyramidal cells and putative interneurons suggests a postsynaptic function in these neurons. With the ä OR probe we observed moderate signals over granular cells of DG and over scattered neurons of moderate size in CA subregions in the pyramidal cell layer and the stratum oriens. ä OR binding was moderate in pyramidal cell layers and dense in the DG64 suggesting a somatodendritic local expression of ä OR in the human hippocampus. The distribution of ä and µ OR mRNA and binding sites in human hippocampus is consistent with the concept that activation of µ and ä ORs expressed in inhibitory hippocampal interneurons produces a disinhibition by decreasing GABA release.26 By this mechanism ä and µ ORs may facilitate long-term potentiation,18,197 whereas ê ORs may restrain glutamate release presynaptically at certain hippocampal synapses.126,176,186,190 In this context, it is of interest that memory impairment is not considered an important concern in the clinical application of OR agonists, although in human memory tests some minor impairments have been reported.57,85 Our data on the expression of OR mRNA in the human hippocampus match in part the OR mRNA

distribution known from rat and mice.33,110–112,114 Species differences were observed for µ OR mRNA expression in the CA1–4 regions: in the human CA4 region the majority of pyramidal cells displayed µ OR signals, whereas hybridization signals in rat were restricted to scattered, large cells in CA1–3. ê OR mRNA signals in the rat were weak and restricted to granular cells in the caudal parts of the ventral DG110 whereas the entire human DG was labelled. In addition, ê OR signals in the human hippocampus were seen over other cell types, among them pyramidal cells in CA4 and particularly in CA3 and smaller neurons scattered between pyramidal cells in the CA4, CA3, CA1 and in the subiculum. The entorhinal cortex and the amygdala were not adequately represented in the material studied and were therefore not included in the present analysis. The areas of the amygdala that were presently analysed suggest an intense expression of µ, ê and ä ORs in subregions of the amygdala. Basal ganglia In the striatum, mRNA expression for all three ORs was observed. However, the three ORs differed in their expression patterns. µ OR mRNA signals had macroscopically a heterogeneous distribution: the ventral areas of the anterior striatum, in particular the Acc, appeared intensely labelled. There was no preferential labelling of the shell-like or core-like divisions of the Acc. The dorsal aspects of the caudate and the putamen showed less intense mRNA expression. Binding studies in the human striatum72,185 using [3H]DAMGO in contrast demonstrate a modest dorsal to ventral high-to-low density gradient with low binding densities in the Acc: corelike and shell-like divisions of the Acc differed markedly in density of binding. Clearly the pattern of µ OR binding did not match the distribution of µ OR mRNA. Several factors may contribute to this apparent mismatch. Low µ OR binding visualized in the Acc using the µ OR agonist DAMGO may be a consequence of a preferential binding of the agonist to high affinity states of the µ OR. This assay may therefore lead to an underestimation of µ OR binding sites in the Acc. Binding studies in the primate brain using an unselective antagonist at a concentration

Fig. 21. Micrographs from cervical spinal cord sections hybridized with the µ (A, B) and ê (C, B) OR probes. Examples of labelled neurons in the dorsal horn (A, C) and in the substantia intermedia (B, D) are shown. Scale bars=50 µm.

Opioid receptor mRNAs in human brain 1121

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Fig. 22. Phosphorimager pictures of µ (A), ê (B) and ä (C) OR signals from sections of the cerebellum. Scale bars=5 µm.

thought to label prefentially µ OR binding sites, did not show low densities of binding sites in the Acc.98 Alternatively the low density of µ OR binding sites in the core of the Acc may represent the presence of a µ OR, subtype with different binding characteristics or may reflect a true low regional density of µ ORs e.g., as a consequence of transport of µ ORs into terminals projecting outside the core region or because of a low rate of translation of µ OR mRNA in this brain region. Clearly, we can not offer anything but speculation at present to explain this discrepancy. The dorsoventrally decreasing gradient in µ OR binding sites—the reverse of the gradient observed in ISHH—suggests the presence of µ ORs binding sites on extrinsic afferents to the striatum. In line with this suggestion, electrophysiological studies in striatum of the rat demonstrated a presynaptic localization of µ ORs predominantly on glutamatergic afferent terminals.79,115 Signals obtained with the µ OR probe throughout the striatum were patch-like. These patches were more prominent in the anterior striatum and appeared similar to patches in the rodent caudate– putamen.33,112 µ ORs binding in the human striatum is also heterogeneous72,143,185 but does not demonstrate patches of high density typically observed in the rat.61 Microscopically the patches of high µ OR mRNA signals represented clusters of strongly labelled medium-sized cell bodies. Their distribution and morphology is reminiscent of clusters of dynorphin mRNA expressing neurons.69 This impression is supported by double labelling in situ hybridization in the rat caudate–putamen which revealed a greater degree of co-localization of µ OR with dynorphin than of µ OR with enkephalin.53 This observation in rat does not rule out, however, that µ ORs may be preferentially expressed in enkephalin-containing neurons in the human brain. We are currently addressing this issue using a double labelling technique. Macroscopically, signals obtained with the ê OR probe were more widely distributed in the striatum than those seen with the µ OR probe. High signal intensities were seen in medioventral areas, including the Acc. A heterogeneous patchy ê OR probe hy-

bridization pattern, already visible in the anterior striatum, but less patchy than the µ OR hybridization pattern, became more apparent in posterior parts of the putamen. In the rostrocaudal direction, a decrease in signal intensity was visible; mRNA expression in the ventromedial parts was slightly stronger than in the dorsal region. ê OR binding in the human striatum using both unselective143,183 and selective agonist ligands144 matched the distribution of ê OR mRNA demonstrating a similar gradient and patches of high density binding. A localization of ê OR on somatodendritic domains of striatal neurons is therefore likely. On microscopic examination, ê OR mRNA was expressed by numerous medium-sized cells suggesting an expression in striatal projection neurons. Cells showing moderate to high ê OR mRNA levels were aggregated into clusters, raising the question to what extent these clusters represent clusters of dynorphin-expressing striatal neurons. An expression of ê OR by dynorphin-expressing striatal neurons might imply a function of ê OR as an autoreceptor; to our knowledge peptide autoreceptors have not yet been demonstrated in functional assays. There is indirect evidence for a possible function of ê OR as autoreceptors: ê OR agonists inhibit the release of antidiuretic hormone151 which is know to co-localize with dynorphin in neurosecretory vesicles of the pituitary.193 Consistent with a possible function of ê OR as autoreceptors is the observation that ê OR binding sites are present in the SN32,144 which might reflect in part binding sites on striatonigral afferents. To what extent there is a preferential expression of ê and µ OR in the direct (dynorphin/ substance P [SP]-positive) and in the indirect (enkephalin-positive) striatofugal pathways and to what extent ê and µ OR co-localize to the same neuronal populations is an important question which will be addressed in our ongoing double label ISHH study. The macroscopic hybridization signal produced by the ä OR probe was punctate and uniform throughout all parts of the striatum. ä OR binding, in contrast, is dense and homogeneous in the human striatum.16,143 Cellular hybridization signals for the ä

Opioid receptor mRNAs in human brain

Fig. 23. Micrographs demonstrating cellular µ (A), ê (B) and ä (C) OR mRNA expression signals in the cerebellar cortex. Labelled Golgi and granular cells can be seen in sections hybridized with the µ and ê OR probe. In sections hybridized with the ä OR probe, only Golgi cells displayed hybridization signals. Scale bars=50 µm.

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OR mRNA were seen over large, polygonal cells, scattered throughout the entire striatum, probably representing cholinergic striatal interneurons. In rat, an expression of ä OR by cholinergic interneurons has been demonstrated.96 Signals from the ä OR probe in the striatum were clearly restricted to this distinct cell population, which displayed no labelling with the µ or ê OR probes. No other striatal cell population was labelled with the ä OR probe. Cholinergic interneurons that contain both SP and ä ORs, at least in rat,49,96 might be a site of convergence of the two striatal projection neuron populations differentially expressing SP and enkephalin. Cholinergic interneurons in turn may differentially influence the two striatal projection neuron populations by muscarinic receptors selectively expressed by striatal projection neurons.12 We observed very few neurons in the GPe unequivocally labelled by the ä OR probe. In addition, µ OR mRNAs were observed in both GPe and GPi, i.e. in both output nuclei of the basal ganglia. However, ISHH signals were not detectable in all pallidal neurons. The low number of pallidal neurons exhibiting clear hybridization signals may explain the low density of µ and ê OR binding sites in both segments of the GP.183,185 Considering the dense enkephalinergic and dynorphinergic projections to the GPe and GPi, respectively, there appears to be at least a quantitative mismatch between the abundance of the putative neurotransmitters and their respective receptors.60 It is possible that the OR binding techniques previously used are not able to detect all forms of ê and µ OR binding sites. Immunohistochemical studies are needed to establish the predicted somatodendritic localization of µ ORs in the human pallidal neurons. Since striatal OR binding distributions in rodents111,112,161,179 and in rhesus monkeys98 are different from each other and from those in humans, species differences in the striatal compartmental OR architecture are likely. The most obvious difference between man and rat were the more widely distributed expression of ê OR mRNA in the human striatum and the presence of µ OR mRNA in the human Acc core-like portion. On the other hand, there were similarities between the distribution of human and rodent striatal OR mRNA, e.g., the prominent expression of ä OR mRNA in large cholinergic interneurons, the organization of µ and ê OR-labelled neurons into clusters and the rostrocaudal decreasing gradient in µ and ê OR signal intensities. Thalamus Neurons expressing µ OR were present in all ‘‘specific’’ thalamic nuclei studied with the exception of the ventroposterior lateral nucleus. The largest ‘‘non-specific’’ thalamic nucleus, the centromedian nucleus, contained no clearly µ OR-labelled cells, whereas the neurons of the intralaminar nuclei

produced relatively intense signals. µ OR binding showed an identical distribution pattern143,185 suggesting a local expression of the µ OR mRNA. Neurons in the reticular nucleus were intensely labelled with the µ OR probe. Since this nucleus consist of GABAergic neurons which project to other thalamic nuclei, µ ORs may exert, through these sites, an indirect disinhibitory effect on the entire thalamus. ê OR mRNA-expressing cells were also detected in all ‘‘specific’’ thalamic nuclei. In the rat, in contrast, the homologous ventrolateral thalamic nucleus seems to lack ê OR mRNA.111 No ê OR-positive neurons were seen in human the centromedian or reticular nuclei. Similar to the µ OR, the binding sites for ê OR matched the distribution of mRNA.143,183 ä OR mRNA as well as binding sites16,143 were absent from the human thalamus. The finding that within the labelled thalamic nuclei µ and ê OR mRNA signals were present in almost all neurons suggests a coexpression of µ and ê OR mRNA in at least some thalamic neuronal populations. The source of endogenous ligands for ORs in thalamus is unknown. Endogenous opioids are not expressed by intrinsic thalamic neurons.69,172 Therefore OR-activating ligands may be released from terminals afferent to the thalamus. It has been shown for instance in the rhesus monkey that hypothalamic efferents containing proopiomelanocorticotropin-derived peptides, like â-endorphin, form synaptic contacts within thalamic nuclei.87 Hypothalamus It is well established in both man and rodents that opioid agonists have neuroendocrine effects.13,39,52 It was not surprising therefore to discover a prominent expression of OR mRNA in human hypothalamus. Interestingly, signals were obtained with µ and ê OR probes, but not with the ä probe at the levels studied, the tuberal region and the mammillary region, suggesting (combined with observations in binding studies16) the absence of ä OR in human hypothalamus. µ OR mRNA was expressed in cells in the periventricular, ventromedial, dorsomedial and lateral hypohalamic areas and was particularly prominent in the area of the PVN. The mammillary nucleus did not contain labelled cells. ê OR mRNA had a similar widespread expression in the human hypothalamus with strong signals in the area of the PVN and no signals in the mammillary nuclei. However, ê OR mRNA expression was more prominent in the ventro- and dorsomedial and the lateral hypothalamic area compared to the µ OR mRNA. Since there are no detailed studies on µ and ê OR binding in the human hypothalamus, it is difficult to know, to what extent the distribution of OR mRNA is mirrored by the distribution of OR binding. There is evidence that OR agonists modulate the release of hormones from the anterior (adrenocorticotropin, thyrotropin, growth hormone, luteinizing hormone), intermediate

Opioid receptor mRNAs in human brain

and posterior lobe (arginine vasopressin [AVP], oxytocin) of the pituitary. In animals, AVP97,151 and oxytocin release14 have been shown to be restrained by OR agonists. Effects of naloxone on insulininduced hypoglycemic stimulation of AVP and oxytocin release suggested an inhibitory role of endogenous opioids in humans as well.24,160 Neurons expressing AVP and oxytocin and sending their axons to the posterior pituitary lobe are located in the PVN and SON; they are of medium or large size. In the PVN, we found hybridization signals with µ and ê OR probes over magnocellular neurons; the percentage of OR labelled large cells was low. In the SON, we detected a high number of cells labelled with the µ OR probe but no neurons with clear signals from the ê OR probe. Together these results suggest an expression of both µ and ê OR by AVP and oxytocin neurons. This interpretation is in agreement with a recent receptor binding study of the human pituitary, in which ê and µ OR binding were detected in the neural lobe82 arguing for an expression of ê and µ OR on AVP and oxytocin terminals in the posterior lobe. In rodents, ORs have a different expression: in rat pituitary, ê ORs in the posterior lobe were the only OR binding site detected.62 Correspondingly, ê OR mRNA expression predominates in the rat PVN.113 Double-labelled ISHH studies are required to establish the extent of co-expression of the ê and µ OR mRNA with AVP and oxytocin, respectively. However, the induction of diuresis is one of the characteristic effects of ê OR agonist in both humans and rodents and has been linked to a decrease in AVP release.37,166 Since application of morphine (a µ OR agonist) has no effect on urine output in human and primates, one may predict that ê OR expression is likely to be more prominent in human AVP neurons (although mechanisms other than inhibition of AVP release may contribute to the diuretic effect of ê OR agonists9). Aside from the direct modulation of hormones secreted by the neuronal lobe, the release of hormones from the anterior pituitary is modulated by opioids as well, presumably by interactions with ORs in hypothalamus, i.e. by an effect on releasing hormones, since there are no OR binding sites in the anterior pituitary lobe.82 For instance, in humans, OR agonists inhibit and OR antagonists stimulate the hypothalamus–pituitary–adrenal axis.27,132 The corticotropin-releasing hormone (CRH) is synthesized by a population of small neurons in the PVN; hybridization signals with µ and ê OR probes were observed in about 40% of parvocellular neurons in PVN. We do not know whether µ or ê ORs (or both) are co-expressed with CRH, but the morphineinduced reduction of serum concentrations of cortisol in humans would be consistent with an expression of inhibitory µ OR in CRH neurons. ê OR activation on the other hand has been shown to stimulate CRH release in rat21 and cortisol release in humans.180 In addition, some parvocellular neurons synthesize the

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thyrotropin-releasing hormone (TRH). We do not know whether these TRH-positive cells express OR mRNA, but a stimulatory effect of µ OR agonists52 and a suppressive effect of ê OR agonists140 on thyrotropin secretion has been observed in humans. A crucial role in the regulation of fertility and sexual behaviour is played by the gonadotropinreleasing hormone (GnRH), synthesized in neurons scattered throughout the preoptic and the ventromedial area of the hypothalamus. µ OR-mediated inhibition of GnRH gene expression99 and GnRH secretion has been documented in animals36,86 and in humans.78,202 In the rat, a double labelling ISHH study failed to detect any of the three OR mRNAs in GnRH neurons, but µ, ê and ä OR mRNA-expressing cells were seen in close proximity to GnRH-labelled cells.156 Double labelling studies in human hypothalamus are required to address this question. However, ê OR mRNAexpressing cells (and to a lesser extent µ OR mRNAcontaining neurons) are found in the ventromedial area in agreement with the therapeutic efficacy of OR antagonist treatment in hypothalamic amenorrhoea.47 The periventricular and preoptic areas and the PVN are implicated in thermoregulation.17,148 µ and ê OR agonists, respectively, are known to influence the regulation of body temperature in animals and humans.75,84,123 The expression of µ and ê OR mRNA in the PVN and in the periventricular nucleus is consistent with a direct effect of opioids at these sites. Behaviours ensuring homeostasis and preserving the species, like feeding and sexual behaviour, are thought to be regulated by centres in the medial and lateral hypothalamus.25,43,104 In humans, both behaviours can be modulated by OR activation;177,196 there is even evidence for a role of endogenous opioids in the pathophysiology of eating disorders like anorexia nervosa and bulemia.31,81 Our findings of strong signals for µ and ê OR mRNAs in these hypothalamic areas provide an anatomical basis for these behavioural effects of opioids. It has to be remembered, however, that the expression of ORs on neuroendocrine cells is only one of several mechanisms, by which ORs may modulate hypothalamic circuits and that indirect effects by modulation of interneurons or by a modulation of dopaminergic and noradrenergic afferents are likely to play an important role. Subcortical projection systems The SNpc and the medial continuation of this nucleus, the paranigral nucleus (or ventral tegmental area in the rat), is the source of the dopaminergic innervation of basal ganglia, neocortex and the hippocampal formation. Our study confirmed the recently reported expression of ê OR by dopaminergic neurons in the human SN200 and demonstrated that hybridization with µ or ä OR do not label

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pigmented neurons in SN suggesting a low or absent expression of µ and ä OR in dopaminergic neurons. In the rat brain, ê OR mRNAs are in a similar way prominently expressed in putative dopaminergic cells in SN.33,110 ê OR binding is low in the rat SN112 suggesting an expression of ê OR on nigrostriatal terminals. Release studies in rat indeed show that ê OR agonists inhibit striatal dopamine release.191 In the human brain, an activation of nigrostriatal ê OR may have a similar inhibitory effect on striatal dopamine release and may contribute to the dysphoric effects of ê OR agonists. The euphoric effect of morphine in contrast may be related to an indirect stimulation of dopamine release by a µ OR-mediated inhibition of a population of striatal GABAergic neurons projecting to dopaminergic cells.50 Consistent with this hypothesis µ OR binding is high in SN and can be significantly reduced by striatal lesions.131 In addition, moderate to strong µ OR mRNA signals were seen over many unpigmented cells in SNpr, adjacent to or mixed with unlabelled putative dopaminergic neurons. µ OR activation on these presumably GABAergic projection neurons (which at least in rat and guinea-pig form inhibitory synaptic contacts with dopaminergic neurons56,174) may lead to a disinhibition of the dopaminergic cells.67 In the rat brain, activation of µ and ê ORs mediate opposed effects on dopamine release and metabolism also in mesolimbic regions.89,168 The LC is the source of an extensive noradrenergic innervation of the telencephalon and is thought to play a role in attention and vigilance4 as well as in emotions like fear and alarm.129 From animal studies it is known that opioids inhibit neuronal activity in the LC1 and decrease noradrenaline release,55,83,117,150 most likely by µ ORs which are highly expressed by neurons of the LC.33,112,181 In the rat brain, a more limited expression of ê OR mRNA in the LC has been reported.111 In the human brain, the large, pigmented noradrenergic neurons of the LC displayed no OR mRNA at the levels studied. µ and ê OR signals were restricted to adjacent non-pigmented cells. µ OR signals were observed over neurons of small size, whereas ê OR signals were associated with larger neurons double in size. Clearly, the distribution of OR in the LC is different in the human and rat. The intense fear and panic, seen in opioid addicts during withdrawal, has been linked to an overshooting activity of noradrenergic neurons following the termination of long-standing opioid receptor stimulation. The apparent absence of ORs in human noradrenergic neurons suggests that the effects of opioids on noradrenalin release are indirect and may be mediated by ORs on neighbouring cells. The dorsal raphe nucleus is the main source of ascending serotonergic fibres to the telencephalon. In addition, there are important descending serotonergic projections from the raphe magnus to the spinal cord. Signals for µ and ê OR mRNAs were found

over neurons in the human dorsal raphe. In the rat, the raphe nuclei show high ê OR, moderate µ OR and very low ä OR signals. The serotonergic system is thought to play a role in the regulation of sleep and food intake and has been implicated in the pathophysiology of depression, obsessive-compulsive disorders and panic attacks.10,35,80 The function of ORs on 5-hydroxytryptamine (5-HT) neurons involves modulation of 5-HT release;29 in the rat, an increase of extracellular 5-HT levels in the diencephalon has been found following the administration of morphine.173 There are no published data on the influence of opioids on the human serotonergic system. Cholinergic cell bodies in the NBM are the main source of a cortical cholinergic innervation in humans135,136 which is associated with cognitive functions. Similar to the rat33 moderate signals with the µ OR probe are observed in a major population of large NBM cells. µ OR binding has been observed in the region of the NBM143 suggesting a local expression of the µ OR mRNA. We were unable to detect unequivocal labelling of large neurons within the NBM by ê or ä OR probes. However, we observed large neurons in the white matter tracts lining the GP including the ventral internal capsula labelled with the ê OR probe. The nature of these neurons is not well defined at present, but they might represent a subpopulation of cholinergic neurons as suggested by Saper.157 ê OR binding sites have been described in the NBM using several ligands.143,144 Although these binding sites may be expressed on afferent fibres to the NBM (e.g., from the amygdala, cerebral cortex, hypothalamus or brainstem nuclei expressing ê ORs153), they may indicate an expression of ê OR mRNA on cholinergic neurons. In addition, human cortical acetylcholine release studies suggest a presynaptic localization of ê ORs on cholinergic terminals.42 However, at the levels of the NBM studied, we did not observe neurons labelled with the ê OR probe within the region of NBM. It is possible, that the expression level of the ê OR mRNA in the NBM neurons is below our detection limit or that expression of ê OR is limited to a subpopulation of cholinergic neurons missed in our random samples. Functional neuronal systems Somatosensory system. The localization of OR mRNAs within well-known circuits in the human brain can help to identify the neuronal populations that are potential targets of opioid drugs. For example, in the present study we demonstrate the presence of specific OR mRNAs in several relay stations of somatosensory pathways. Cells expressing OR mRNA were observed in the dorsal horn of the spinal cord, nucleus gracilis and cuneatus, sensory and spinal trigeminal nerve nucleus and the somatosensory thalamic nuclei. Dorsal root ganglia and

Opioid receptor mRNAs in human brain

primary sensory cortex were not included in the present study. In dorsal root ganglia, the rat mRNA coding for all three ORs has been described108 and OR binding sites have been detected on primary afferents in the periphery as well as on terminals in the dorsal root entry zone170 where ê and ä OR inhibit the release of SP and calcitonin gene-related peptide from small primary afferents.198,199 We do not know whether ORs in human dorsal root ganglia have a similar expression. The afferent fibres from the dorsal root ganglia synapse by axon collaterals with second order neurons in the dorsal horn, giving rise to the spinothalamic tract, transmit pain and temperature information. We observed strong signals for µ and ê OR mRNAs in these neurons suggesting that µ and ê opioid agonist may hyperpolarize these neurons as demonstrated in the rat (for review see Ref. 198). Unlike the rat, we did not observe ä OR mRNA in spinal cord. Interestingly, intrathecal application of a ä OR agonist produced analgesia in man,130 raising the possibility that ä OR may be present on afferent fibres from dorsal root ganglia. Other afferents from dorsal root ganglia forming the posterior columns and mediating information about tactile sensation and limb proprioception have their second order neurons in the nucleus gracilis and cuneatus, where weak to moderate hybridization signals with the ä and ê OR probes were seen. In contrast to the rat brain, where µ OR signals predominated, we could not detect µ OR signals in these nuclei. In the sensory and spinal nuclei of the trigeminal nerve nucleus we also observed ä in addition to µ and ê OR mRNA signals. Forming the medial lemniscus somatosensory fibres synapse on thalamic third order neurons. Signals for the ê OR mRNA were observed in the ventral posterior nuclei of the lateral nuclear group of the thalamus. In these thalamic nuclei no, or very few, neurons were found to be labelled with the µ OR probe, a finding in good agreement with binding studies and the observation that thalamic microinjection of morphine does not result in analgesia.137 In addition to the thalamus, the claustrum is thought to serve as a relay nucleus conveying somatosensory and nociceptive information from the brain stem and the spinal cord to the cerebral cortex by-passing the thalamus.125,167 This brain region displayed very high signal intensities for ê OR mRNA; neurons expressing µ OR mRNA were also detected at low levels. We have no data on the OR mRNA expression within the primary sensory cortex, however, binding studies would suggest that the primary sensory cortex, unlike the occipital cortex, does not differ substantially in terms of OR distribution from other cortical areas.143 Nociception is influenced by descending pathways in the brain stem which inhibit spinal nociceptive reflexes and reduce spinal neuronal activity evoked by noxious stimuli. An important supraspinal site in

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antinociception is the PAG, where local microinjection of opioid agonists results in a marked reduction in pain behaviour in several species.198 In animals, GABAergic neurons in the PAG have been shown to exert a tonic inhibitory modulatory control over bulbospinal pathways thought to inhibit spinal nociceptive activity.38 µ OR agonists inhibit these GABAergic interneurons147 permitting a disinhibition of bulbospinal pathways. Using µ and ê OR probes we observed signals over scattered neurons in the human PAG consistent with an expression of µ and ê OR on GABAergic interneurons in this region. In addition, there is evidence that activation of serotonergic and noradrenergic brainstem neurons play a major role in descending antinociceptive pathways.128,146 Aside from these descending pathways numerous rostral projections arise from the LC and raphe nuclei that influence forebrain systems involved in motivational and affective components of pain behaviour such as the Acc and the amygdala. In the region of the LC we found signals indicating µ and ê OR mRNA expression on putative interneurons. µ and ê OR signals are visible in subpopulations of the nucleus raphe neurons. These observations suggest that in the human brain ORs may play a role in the modulation of these components of supraspinal pain control as they do in animals. There is evidence that ORs participate in sensory processing outside the somatosensory system. Neurons expressing OR mRNA can be found in regions regarded as visual or auditory relay stations. The differential expression of ORs in the primary visual cortex (where µ ORs were absent) suggests that specific ORs have distinct functional roles. In humans, the ê OR agonist ketocyclazocine has been reported to alter visual and auditory perception;95 µ OR agonists on the other hand had no effect95 or appeared to improve auditory sensitivity.154 Motor system. Neurons expressing OR mRNA were present in several brainstem motor nuclei, basal ganglia, motor nuclei of the thalamus, frontal cortex, cerebellum and pontine nuclei suggesting that ORs are involved in motor control. In the rat, µ and ä OR agonists produce an increase in motor activity at low doses; high doses of µ OR agonist result in catalepsy and muscular rigidity.194 ê OR agonist have no effects on locomotion,76 but may reduce muscle tone at least in mice22 and rhesus monkey.37 These effects have been linked to the dopaminergic system.34,127 In humans, no µ OR agonist-induced motor effects have been reported. However, µ OR agonists can induce muscular rigidity in humans.91,171 The minor motor effects of OR activation come as a surprise given that the basal ganglia, which have a central modulatory role in locomotor circuits, contains one of the highest levels of endogenous opioids in the brain. Since the antinociceptive effects of opioids are not readily apparent in subjects at rest it might well be that the

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functional role of opioids in basal ganglia is apparent only under certain conditions which are presently ill defined. Interestingly, marked changes in endogenous opioid expression189 as well as alterations in opioid receptor binding142 has been shown in Parkinson’s disease patients experiencing L-DOPA-induced dyskinesias. A therapeutic use of ê OR ligands for the treatment of dyskinesias in Parkinson’s disease patients has been proposed.59,63,106 The function of ORs in cerebellum and pontine nuclei is unknown. The expression of µ and ä ORs in subpopulations of Golgi cells suggest that these receptors may serve a role as autoreceptors on the subpopulations of Golgi cells expressing enkephalin. Double labelling studies are required to further substantiate this hypothesis. Vegetative nervous system. The clinical use and dosage of opioids is limited by their influence on vegetative functions. Morphine is known to cause respiratory depression which represents one of its major side effects in clinical practice. Other unwanted consequences of opioid receptor activation are nausea and constipation; effects on blood pressure and heart rate are less marked. Hence it was not surprising to find, in particular, the µ OR mRNA widely expressed in brain regions associated with vegetative functions. Among the regions serving the central regulation of respiration and cardiovascular functions are the nucleus parabrachialis, nucleus ambiguus, nucleus of the solitary tract and the dorsal nucleus of the vagus. In all these nuclei neurons expressing µ and/or ê OR mRNAs were found. Generally µ OR mRNA was more widely expressed than ê OR mRNA, with the exception of the nucleus ambiguus, where both OR mRNAs were expressed in a similar number of neurons. The function of ê OR in these nuclei is unclear since ê OR agonists in contrast to morphine did not have major respiratory or cardiovascular effects in primates and humans.51,68 The physiological role of endogenous opioids within the autonomic system is unknown. Opioids, while relatively quiescent in the resting state, are thought to modify circulatory homeostatic mechanisms only during intense stimulation.120 Effects on vegetative functions can also arise from opioid-evoked modulation of the neuroendocrine system. Endogenous opioids appear to be present in neurons connecting autonomic centres in the formatio reticularis and the hypothalamus.8,159 These bidirectional connections are well suited for co-ordinating neuroendocrine and autonomic responses. Limbic system. The limbic system is closely related to the hypothalamus and involved in behaviour and emotions. The most prominent emotional effects produced by µ OR agonists like morphine in healthy subjects, are pleasant feelings or at higher doses even euphoria. In contrast, ê OR agonists produce dysphoria and sensations like derealization, depersonalization and sometimes pseudohallucinations

(‘‘psychotomimesis’’; for review see Refs 95 and 139). The neurobiological basis of these effects is not understood. The powerful reinforcing effects of µ OR agonists are mainly attributed to activation of the mesolimbic dopaminergic reward system (for review see Ref. 94). By means of dopamine D2 receptor and µ OR knock-out mice it has been demonstrated that both, D2-receptors and µ ORs, are necessary for the mediation of reward effects of opiates.105,118 Many studies have shown that drug abuse alters levels of mRNAs encoding endogenous opioid peptides or their receptors44,71,169 even after a single application.70 Further studies of OR mRNA expression in psychiatric patients will provide clues to an understanding of the adaptive mechanisms associated with drug dependence and the role of the OR system in posttraumatic stress disorders,182 where opioid antagonist have been shown to reduce flash backs.15 General species differences One striking difference between rodents and man was the low intensity of hybridization signals for all three OR probes in the human brain tissue. Many factors may influence the abundance of mRNA in post mortem human brain tissue, among others the agonal state (e.g., perimortal hypoxia19,58), and account for some variability of signals among subjects. However, in all brains studied, we consistently observed relatively low in situ hybridization signals for the ORs despite the fact that probes targeting other neurotransmitter receptor mRNAs resulted in intense signals in the same specimen, often in close-by sections. A particularly high sensitivity of human µ, ê and ä OR mRNA to perimortal RNA degradation can not be excluded. Alternatively, the low apparent mRNA levels measured may reflect low steady state levels of OR mRNAs in the human brain. Another striking species difference between human and rat was the more extensive expression of ê OR mRNA in many human brain regions almost devoid of ê OR signals in rat brain. ä OR mRNA on the other hand appeared to have a more limited expression in the human brain. For instance, no ä OR signals were detectable in human spinal cord; in rat spinal cord, in contrast, ä OR expression was evident in numerous neurons. It is conceivable, however, that the apparently more limited expression of ä OR mRNA reported in this study reflects in part the somewhat lower specific activity of the ä OR probe used. µ OR mRNA had a similar expression pattern in both the rat and human brain. Specific species differences are apparent in telencephalic regions: µ and ê OR signals were found in far more neurons in the human cortex and hippocampus than in the rat. This was particularly true for the ê OR, which had a very restricted expression in the

Opioid receptor mRNAs in human brain

rat cortex and caudate–putamen and was almost absent from rat hippocampus. In the diencephalon, ê OR signals in the human thalamus were more widespread than in rat, whereas µ OR signals in the human hypothalamus were more prominent than in rat. In the human LC, no OR mRNA signals were observed over noradrenergic neurons, whereas in the rat µ and ê OR mRNA are expressed in neurons of the LC. Some brainstem nuclei, like the pontine, gigantocellular, gracile and cuneate nuclei, displayed no µ OR mRNA signals in human, but strong µ OR mRNA signals were detected in the rat brain. The trigeminal nerve nucleus in contrast displayed very intense µ OR mRNA signals in the human and weak signals in the rat. Finally, in the human cerebellar cortex, strong µ and ê OR signals were found, whereas in the rat only ä OR signals were observed. In the human cerebellar nuclei on the other hand, no OR expression was detectable; in the rat the µ and ê OR mRNAs are present. In view of these species differences one has to exert some caution when data from animal studies are consulted to explain phenomena observed in humans.

1129 CONCLUSIONS

In summary, this study presents the first comparative data on the distribution of µ, ê and ä OR mRNAs in the human brain. Overall, OR mRNA distribution in the human brain shows many similarities to that reported in rodent brains. However, we also noted species differences in a number of regions, in particular a more widespread occurrence of ê OR mRNA in the human brain. Further studies are required to clarify the expression of µ, ê and ä OR mRNAs in neurochemically-identified neurons to extend our understanding of the role of opioids in neuronal circuits involved in pain perception, neuroendocrine regulation, motor control and reward in humans. Acknowledgements—This work was supported by the Deutsche Forschungsgemeinschaft (SFB 505). We thank Dr B. Volk and Dr K. Mueller from the Department of Neuropathology, Albert-Ludwigs-University Freiburg, for providing us with human brain tissue and Dr B. Kieffer and Dr F. Simonin for human OR cDNAs. We would like to thank Dr T. J. Feuerstein for stimulating discussions and Dr R. Jackisch for critically reading the manuscript.

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