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This paper reviews some of the findings that have led to this conclusion and the anatomical distributions of these sites in the rat brain. Their relation to the opioid peptides and some of the proposed functions mediated by these receptor systems are also discussed.

The mu, delta, and kappa 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 mu, delta, and kappa receptor mRNAs in the rat brain and spinal cord using in situ hybridization techniques. Tissue sections were hybridized with 35S-labeled cRNA probes to the rat mu (744-1,064 b), delta (304-1,287 b), and kappa (1,351-2,124 b) receptors. Each mRNA demonstrates a distinct anatomical distribution that corresponds well to known receptor binding distributions. Cells expressing mu 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 delta receptor mRNA varied from mu or kappa, 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 kappa 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.

Several lines of evidence suggest the existence of multiple dopamine receptor subtypes, referred to as D1 and D2. The present study examines the distribution of these dopamine binding sites in the rat brain and pituitary in relation to the distribution of D2 receptor mRNA using a combination of in vitro receptor autoradiographic and in situ hybridization techniques. 3H-Raclopride and 3H-SCH23390 (in the presence of 1 microM ketanserin) were used to label D2 and D1 receptor binding sites, respectively, while a 495 bp cRNA probe synthesized from the Sac I-Bgl II fragment of a rat D2 receptor cDNA was used to visualize the D2 receptor mRNA. Analysis of adjacent tissue sections in which receptor autoradiography and in situ hybridization had been performed revealed several brain regions where the D2 binding site and corresponding mRNA appear to be similarly distributed, including the caudate-putamen, nucleus accumbens, olfactory tubercle, globus pallidus, substantia nigra, and ventral tegmental area. In the pituitary gland, D2 binding sites and mRNA appear to be codistributed with very dense levels in the intermediate lobe and individually labeled cells in the anterior lobe. Brain regions demonstrating a lack of correspondence between the distribution of the D2 binding site and D2 receptor mRNA include the olfactory bulb, neocortex, paleocortex, hippocampus, and zona incerta. Several hypotheses are discussed to explain the lack of correspondence in certain brain regions; these include the localization of receptor binding sites on both fibers and cell bodies and receptor transport. These studies provide a better understanding of the anatomical distribution of the D2 receptor and serve as a framework for future regulatory and anatomical mapping studies. By focusing on specific brain regions, such as the nigrostriatal system, hippocampus, and olfactory bulb, they provide insights into D2 receptor synthesis, transport, and insertion into cell membranes.

We have isolated a rat cDNA clone that displays 75% amino acid homology with the mouse delta and rat kappa opioid receptors. The cDNA (designated pRMuR-12) encodes a protein of 398 amino acids comprising, in part, seven hydrophobic domains similar to those described for other G protein-linked receptors. Data from binding assays conducted with COS-1 cells transiently transfected with a CMV mammalian expression vector containing the full coding region of pRMuR-12 demonstrated mu receptor selectivity. In situ hybridization mRNA analysis revealed an mRNA distribution in rat brain that corresponds well to the distribution of binding sites labeled with mu-selective ligands. Based upon these observations, we conclude that pRMuR-12 encodes a mu opioid receptor.

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A full-length cDNA was isolated from a rat striatal library by using low-stringency screening with two PCR fragments, one spanning transmembrane domains 3-6 of the mouse 6 opioid receptor and the other unidentified but homologous to the mouse 6 receptor from rat brain. The novel cDNA had a long open reading frame encoding a protein of 380 residues with 59% identity to the mouse 6 receptor and topography consistent with a seven-helix guanine nucleotidebinding protein-coupled receptor. COS-1 cells transfected with the coding region of this clone showed high-affinity binding to ic opioid receptor-selective ligands such as dynorphin A and U-50,488 and also nonselective opioid ligands such as bremazocine, ethylketocyclazocine, and naloxone. Not bound at all (or bound with low affimity) were dynorphin A-(2-13), enantiomers of naloxone and levophanol [i.e., (+)-naloxone and dextrorphan], and selective ,u and 8 opioid receptor ligands. Activation of the expressed receptor by X receptor agonists led to inhibition of cAMP. Finally, in situ hybridization revealed a mRNA distribution in rat brain that corresponded well to the distribution of binding sites labeled with i-selective ligands. These observations indicate that we have cloned a cDNA encoding a rat ic receptor of the wc subtype.

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