Localization of the mRNA for the dopamine D2 receptor in the rat brain by in situ hybridization histochemistry.

Abstract

ABSTRACT32P-labeled oligonucleotides derived from the coding region of rat dopamine D2 receptor cDNA were used as probes to localize cells in the rat brain that contain the mRNA coding for this receptor by using in situ hybridization histochemistry. The highest level of hybridization was found in the intermediate lobe of the pituitary gland. High mRNA content was observed in the anterior lobe of the pituitary gland, the nuclei caudate-putamen and accumbens, and the olfactory tubercle. Lower levels were seen in the substantia nigra pars compacta and the ventral tegmental area, as well as in the lateral mammillary body. In these areas the distribution was comparable to that of the dopamine D2 receptor binding sites as visualized by autoradiography using ligand. However, in some areas such as the olfactory bulb, neocortex, hippocampus, superior colliculus, and cerebellum, D2 receptors have been visualized but no significant hybridization signal could be detected. The mRNA coding for these receptors in these areas could be contained in cells outside those brain regions, be different from the one recognized by our probes, or be present at levels below the detection limits of our procedure. The possibility of visualizing and quantifying the mRNA coding for dopamine D2 receptor at the microscopic level will yield more information about the in vivo regulation of the synthesis of these receptors and their alteration following selective lesions or drug treatments.Two types of dopamine receptors mediate the effects of this neurotransmitter in the central nervous system and in the periphery, the so-called D1 and D2 receptor subtypes (1). Dopamine D2 receptors are the target for drugs used in the treatment of schizophrenia and Parkinson disease and for regulation of prolactin secretion (2). Their distribution in the mammalian brain, including human, has been extensively examined by using receptor binding autoradiography (3). Dopamine D2 receptors have been directly visualized in living humans by positron-emission tomography (PET) (4). Binding and PET studies have suggested that dopamine D2 receptor levels may be elevated in schizophrenics (refs. 5, 6; see, however, ref. 7). Although dopamine receptors can be visualized at the light microscopic level by means of receptor binding autoradiography, the cellular localization of these receptors cannot be determined with this technique. The use of neurotoxin or mechanical brain lesions combined with binding studies has resulted in contradictory results (8-10). In order to establish the exact cellular localization of dopamine receptors, tools with higher anatomical resolution are necessary. The cloning and sequencing of the gene coding for the rat dopamine D2 receptor has been reported recently (11), thus opening the way to the development of these tools. On the basis of this sequence we have synthesized oligonucleotide probes and used them to visualize the regional distribution of the cells containing mRNA coding for the dopamine D2 receptor in the rat brain and pituitary gland b...

Pharmacology for the Treatment of Premature Ejaculation

“…Among all types of dopamine receptors, only D 2 and D 3 subtypes can be presynaptic autoreceptors regulating dopamine release and metabolism (Mercuri et al, 1997). High density of D 1 receptor has been found in limbic system, hypothalamus, thalamus, and PVN (Mengod et al, 1989;Czyrak et al, 2000). D 2 -like receptors have also been detected in limbic areas, hypothalamus, and thalamus.…”

Dopamine Receptor Gene Expression in the Human Medial Temporal Lobe

“…Although the determination of both mRNA and binding sites in some ways provide complemen tary information, the determination of dopamine recep tor mRNA distributions allows the identifIcation of specific dopaminoceptive cells. The consensus of a number of studies is that the only region that expresses all fIve of these receptor mRNAs is the hippocam pal formation; 02, 03, 04, and Os are all expressed throughout the hippocampal formation of the rat, pri marily in the pyramidal cell layer of the hippocampus and in the granular cell layer of the dentate gyrus (Tiberi et al 1991;Meador-Woodruff et al 1989Sokoloff et al 1992;O'Malley et al 1992;Najlerahim et al 1989;Weiner and Brann 1989;Mengod et al 1989;Bouthe net et al 1991;Landwehrmeyer et al 1993). 0 1 recep tor mRNA was found to be more limited in its distribu tion, being located predominantly in the ventral portion of the dentate gyrus (Mansour et al 1991;Meador Woodruff et al 1991;Fremeau et al 1991;Mengod et al 1991;Weiner et al 1991).…”

Distribution and cellular localization of mRNA coding for 5-HT1A receptor in the rat brain: correlation with receptor binding

“…The probes were synthesized on a 380B Applied Biosystems DNA synthesizer (Foster City Biosystems, Foster City, CA) and purified on a 20% polyacrylamide/8 M urea preparative sequencing gel. The oligonucleotides were labeled at their 3' end with terminal deoxynucleotidyltransferase (Boehringer Mannheim) and GP-dATP (3000 Ci/ mmol; DuPont New England Nuclear, Boston, MA) following the protocol described previously (Mengod et al, 1989).…”

“…PolyA+ RNA isolated from different dissected rat brain regions was blotted onto nylon membranes, and blots were then hybridized with the corresponding oligonucleotide as in Mengod et al (1989).…”

“…The probes were synthesized on a 380B Applied Biosystems DNA synthesizer (Foster City Biosystems, Foster City, CA) and purified on a 20% polyacrylamide/8 M urea preparative sequencing gel. The oligonucleotides were labeled at their 3' end with terminal deoxynucleotidyltransferase (Boehringer Mannheim) and GP-dATP (3000 Ci/ mmol; DuPont New England Nuclear, Boston, MA) following the protocol described previously (Mengod et al, 1989).PolyA+ RNA isolated from different dissected rat brain regions was blotted onto nylon membranes, and blots were then hybridized with the corresponding oligonucleotide as in Mengod et al (1989).In situ hybridization histochemistry. Frozen tissue sections were fixed and then hybridized with the corresponding labeled oligonucleotide probe as described in Vilar6 et al (199 1 b) except that the hybridization buffer contained 4OW formamide and in addition 10% dextran sulfate and that the washing temperature was 60°C.…”