THE JOURNAL OF COMPARATIVE NEUROLOGY 350:412-438 (1994) Mu, Delta, and Kappa Opioid Receptor mRNA Expression in the Rat CNS: An In Situ Hybridization Study ALFRED MANSOUR, CHARLES A. FOX, SHARON BURKE, FAN MENG, ROBERT C. THOMPSON, HUDA AKIL, AND STANLEY J. WATSON Mental Health Research Institute and Department of Psychiatry, University of Michigan, Ann Arbor, Michigan 48109-0720 ABSTRACT The p, 6, and K opioid receptors are the three main types of opioid receptors found in the central nervous system (CNS) and periphery. These receptors and the peptides with which they interact are important in a number of physiological functions, including analgesia, respiration, and hormonal regulation. This study examines the expression of p, 6, and K receptor mRNAs in the rat brain and spinal cord using in situ hybridization techniques. Tissue sections were hybridized with 35S-labeledcRNA probes to the rat IJ. (744-1,064 b), 6 (304-1,287 b), and K (1,351-2,124 b) receptors. Each mRNA demonstrates a distinct anatomical distribution that corresponds well to known receptor binding distributions. Cells expressing p receptor mRNA are localized in such regions as the olfactory bulb, caudate-putamen, nucleus accumbens, lateral and medial septum, diagonal band of Broca, bed nucleus of the stria terminalis, most thalamic nuclei, hippocampus, amygdala, medial preoptic area, superior and inferior colliculi, central gray, dorsal and median raphe, raphe magnus, locus coeruleus, parabrachial nucleus, pontine and medullary reticular nuclei, nucleus ambiguus, nucleus of the solitary tract, nucleus gracilis and cuneatus, dorsal motor nucleus of vagus, spinal cord, and dorsal root ganglia. Cellular localization of 6 receptor mRNA varied from p or K, with expression in such regions as the olfactory bulb, allo- and neocortex, caudate-putamen, nucleus accumbens, olfactory tubercle, ventromedial hypothalamus, hippocampus, amygdala, red nucleus, pontine nuclei, reticulotegmental nucleus, motor and spinal trigeminal, linear nucleus of the medulla, lateral reticular nucleus, spinal cord, and dorsal root ganglia. Cells expressing K receptor mRNA demonstrate a third pattern of expression, with cells localized in regions such as the claustrum, endopiriform nucleus, nucleus accumbens, olfactory tubercle, medial preoptic area, bed nucleus of the stria terminalis, amygdala, most hypothalamic nuclei, median eminence, infundibulum, substantia nigra, ventral tegmental area, raphe nuclei, paratrigeminal and spinal trigeminal, nucleus of the solitary tract, spinal cord, and dorsal root ganglia. These findings are discussed in relation to the physiological functions associated with the opioid receptors. D 1% Wiley-Liss, Inc. Key words: endorphins, endorphin receptors, localization, opiates, pept,ide receptors Several lines of research have demonstrated that the peptides. These early studies suggesting multiple opioid opioid receptors can be classified into three main types receptor types have been extended by numerous anatomi- referred to as mu (p), delta (61, and kappa (K; Martin et al., cal, pharmacological, biochemical, and behavioral studies 1976; Lord et al., 1977; Chang and Cuatrecasas, 1979; (Gillan and Kosterlitz, 1982; Magnun et al., 1982; Wood, Smith and Simon, 1980). The IJ. and K receptor types were 1982; Pasternak et al., 1983; Goldstein and James, 1984; initially differentiated by Martin and colleagues (1976) in James and Goldstein, 1984; Zukin and Zukin, 1984; McLean the chronic spinal dog preparation and are the sites at et al., 1986; Mansour et al., 1987; Goldstein and Naidu, which alkaloids, such as morphine, and benzomorphans, such as ethylketazocine, bind to produce their respective physiological effects. The 6 receptors, named for the mouse Accepted June 27, 1994. vas deferens preparation from which they were initially Address reprint requests to Dr. Alfred Mansour, Mental Health Research described (Lord et al., 1977),differ from the p and K binding Institute,, University of Michigan, 205 Zina Pitcher Place, Ann Arbor, MI sites and have a higher affinity for proenkephalin-derived 48109-0720. O 1994 WILEY-LISS, INC. MU, DELTA, AND KAPPA RECEPTOR mRNA IN THE RAT CNS 413 1989; Sharif and Hughes, 1989) demonstrating distinct al., 1993; Yasuda et al., 1993). Northern blot analysis opioid receptor types in the central nervous system (CNS) suggests that the opioid receptors are encoded by compar- and periphery and have led to the cloning of at least three atively large mRNAs that range from 4.5-11 kb for the rat 6 opioid receptor cDNAs that correspond to the pharmacologi- receptor (Fukada et al., 1993) to 10-16 kb for the rat p cally defined p, 6, and K receptors (Evans et al., 1992; receptor (Fukada et al., 1993; Thompson et al., 1993; Wang Kieffer et al., 1992; Chen et al., 1993; Fukada et al., 1993; et al., 1993). The mouse K receptor appears to be encoded by Meng et al., 1993; Thompson et al., 1993;Wang et al., 1993; a single mRNA transcript that is 5.2 kb in length (Yasuda et Yasuda et al., 1993). al., 1993). The relatively large size of opioid receptor The three cloned opioid receptors are members of the mRNAs is primarily due to their long 3’-untranslated seven transmembrane superfamily of G-protein coupled regions; the protein-coding and 5‘-untranslated regions receptors and are homologous to one another at both the range from 1.2-1.5 kb. amino acid and the nucleic acid levels, particularly in Single genes have been suggested for the p, 6, and K regions spanning the putative transmembrane domains receptors (Evans et al., 1992; Thompson et al., 1993; and cytosolic loops. The most divergent regions of these Yasuda et al., 1993). These genes contain multiple introns receptors are the N- and C-termini, extracellular loops 2 that may result in the transcription of alternatively spliced and 3, and transmembrane 4 (Chen et al., 1993; Fukada et mRNA forms of each receptor type. The function of these al., 1993; Meng et al., 1993; Thompson et al., 1993; Wang et long 3’-untranslated regions and the multiple mRNA tran- Abbreuiations 3 oculomotor nucleus LO lateral orbital cortex 3v third ventricle LRt lateral reticular nucleus 10 dorsal motor nucleus of vagus LS lateral septum 12 hypoglossal nucleus Md medullary reticular nucleus ac anterior commissure ME median eminence Acb nucleus accumbens Me medial amygdala AHPM amygdalohippocampalarea, posteromedial MG medial geniculate, thalamus A1 agranular insular cortex MH medial habenula Amb ambiguus nucleus Mi mitral cell layer olfadory bulb AOD anterior olfactory nucleus, dorsal Mn median raphe AOL anterior olfactory nucleus, lateral MnR median raphe AOM anterior olfactory nucleus, medial MPA medial preoptic area AOV anterior olfactory nucleus, ventral MS medial septum Arc arcuate hypothalamic nucleus oc occipital cortex ATg anterior tegmental nucleus Or oriens layer, hippocampus BL basolateral amygdala ox optic chiasm BM basomedial amygdala Pa paraventricular hypothalamus CA1 field CA1 of Ammon’s horn Pa5 paratrigeminal nucleus CA2 field CA2 of Ammon’s horn Par parietal cortex CA3 field CA3 of Ammon’s horn PB parabrachial nucleus cc central canal Pir piriform cortex Ce central amygdala PMCo posteromedial cortical amygdala CG central gray Pn pontine nuclei cg cingulate cortex PnRt pontine reticular c1 claustrum PrH prepositus hypoglossal nucleus CLi caudal linear raphe PrS presubiculum CM centromedial thalamus PS parastrial nucleus CPU caudate-putamen PV paraventricular thalamus cu nucleus cuneatus PY pyramidal cell layer, hippocampus DB diagonal band of Broca Rad stratum radiatum, hippocampus DG dentate gyrus Re reuniens DMH dorsomedial hypothalamus RMC red nucleus, magnocellular DPGi dorsal paragigantocellular nucleus RMg raphe magnus DpMe deep mesencephalic nucleus Rob raphe obscurus DR dorsal raphe RtTg reticulotegmental nucleus, pons DRG dorsal root ganglia S subiculum ECu external cuneate nucleus sc superior colliculus En endopiriform nucleus SCh suprachiasmatic nucleus Ent entorhinal cortex SHY septohypothalamic nucleus Fr frontal cortex SN substantia nigra Gi gigantocellular reticular nucleus SNC substantia nigra, pars compacta GI glomerular layer olfactory bulb SNR substantia nigra, pars reticulata Gr nucleus gracilis so supraoptic nucleus HPC dorsal hippocampus Sol nucleus solitary tract IC inferior colliculus SOlC nucleus solitary tract, conimissural IGr internal granular layer of olfactory bulb SolR nucleus solitary tract, rostral In intercalated nuclei, amygdala SP5 spinal trigeminal nucleus InfS infundibular stem SpVe spinal vestibular nucleus IPR interpeduncular nucleus, rostral Te temporal cortex IRt intermediate reticular nucleus Tu olfactory tubercle LC locus coeruleus VMH ventromedial hypothalamus LD laterodorsal thalamus VP ventral pallidurn LH lateral hypothalamus WL ventral posterolateral thalamic nucleus Li linear nucleus, medulla VTA ventral tegmental area LL nucleus of the lateral lemniscus ZI zona incerta 414 A. MANSOUR ET AL. scripts that are possible from a single receptor gene are then transferred to sealed humidifying chambers contain- presently unknown. ing 50% formamide and hybridized overnight at 55°C. The focus of this study is to compare the anatomical The next day, the slides were rinsed in 2x SSC (5 localization of the p, 6, and K receptor mRNAs in the rat minutes) and treated with RNase A (200 pg/ml in 100 mM CNS using in situ hybridization techniques. Previous stud- Tris, pH 8.0, and 0.5 M NaCl) for 60 minutes at 37°C. ies examining the distribution of p and K receptor mRNAs Subsequently, the sections were rinsed in 2~ SSC for 5 in the rat (Mansour et al., 1994a,b) and 6 receptor mRNA in minutes (22°C) and 0.1~SSC for 60 minutes (65°C). the mouse (Mansour et al., 1993) have compared opioid Following the low-salt wash, sections were rinsed in water, receptor mRNA and receptor binding. The emphasis here is dehydrated through graded alcohols, and air dried. Sections comparison of all three receptor mRNAs simultaneously in were apposed to Kodak XAR-5 X-ray film for 7 (S), 10 (p), or the rat CNS to gain further insights into their specific 11 (K) days and dipped in NTBB film emulsion. Brain receptor distributions and their diverse functions. This sections used to visualize IJ.