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Serotonin 5‐HT4 receptors are widely distributed in the periphery and in brain, where they modulate the release of various neurotransmitters and have been implicated in learning and memory. Nine C‐terminal splice variants of this receptor have been cloned in mammalian species. In the rat, three such variants have been described: 5‐HT4(a), 5‐HT4(b), and 5‐HT4(e). In the present study, we have examined several aspects of the distribution of these receptors in brain. First, we provide, in rat and guinea pig, a detailed comparison of the distribution of 5‐HT4 receptors labeled by the antagonist [125I]‐SB 207710 with the distribution of their encoding mRNA visualized by in situ hybridization histochemistry (ISHH). The results suggest that, in several projection systems (striato‐nigral and striato‐pallidal pathways, projection from dentate granule cells to field CA3, habenulo‐interpeduncular pathway), 5‐HT4 receptors are located both somatodendritically and axonally. Second, we have analyzed the distribution of mRNA for the three known rat splice variants by reverse transcription‐polymerase chain reaction (RT‐PCR) and by ISHH. RT‐PCR indicates that all three variants are widely distributed, with 5‐HT4(b) mRNA being present in all regions examined (olfactory tubercle, striatum, hippocampus, inferior colliculus, substantia nigra, parietal cortex) and 5‐HT4(a) and 5‐HT4(e) showing a somewhat more restricted distribution. In other regions (periaqueductal gray, reticular formation, medial septum, diagonal band), faint ISHH signals are observed for 5‐HT4(a)+4(e) mRNAs, whereas 5‐HT4(b) mRNA signals are almost undetectable. Finally, neurotoxic lesions of basal ganglia components in guinea pig also indicate a location of these receptors on terminals of striatal projection neurons. J. Comp. Neurol. 484:418–439, 2005. © 2005 Wiley‐Liss, Inc.

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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...

In recent years the family of mammalian serotonin receptors has grown to 14 different subtypes, characterized by pharmacological or molecular biological techniques. In parallel, new ligand molecules have been developed for their study. However, selective ligands are not yet available to study every one of them. In addition the degree of selectivity of ligands, hitherto regarded as specific for a particular receptor subtype has been called in question by their affinities for newly discovered receptors. Consequently, a re-evaluation of past ligand receptor autoradiography work is necessary in view of the redefined receptor profiles of these ligands, and the introduction of newly developed ligands. A further difficulty for the characterization of these receptors is the absence of selective antagonist ligands which, for some of the subtypes, have become available only recently. In an attempt to overcome these difficulties we have combined in situ hybridization histochemistry and receptor ligand autoradiography to study the regional and cellular localization of several serotonin receptors in the rodent brain. In addition, for some receptors, we have expanded these studies to primates, including humans. We have found that the distribution of 5-HT1A receptors in monkey brain, labelled with the agonist 3H-8-OH-DPAT and the antagonist 3H-WAY 100635 was very similar at the levels examined, and corresponded well with that observed for the cells containing mRNA coding for this receptor, confirming the somatodendritic localization of 5-HT1A receptors in monkey brain. The labelling conditions to visualize 5-HT1F receptors in guinea pig brain, namely 3H-sumatriptan in the presence of 10(-8) M 5-CT to block 5-HT1D receptors, are suitable for visualizing this receptor, since the results agreed with those observed by in situ hybridization. By using 3H-ketanserin and 3H-mesulergine in parallel with in situ hybridization using the corresponding oligonucleotides, we were able to show that these ligands label respectively 5-HT2A and 5-HT2C binding sites in monkey brain. 5-HT4 receptors were localized in the brain of several species including humans by using 125I-SB 207710. In situ hybridization experiments performed in guinea pig confirmed that 5-HT4 receptors are localized on the terminals of the striatopallidal and striatonigral projections. 5-HT7 binding sites were labelled in rat and guinea pig brains by incubating with 3H-5-CT in the presence of 100 microM WAY 100135 and 250 microM GR 127935; the distribution obtained in both species agreed, in general, with that of the corresponding mRNA coding for them. These results are an illustration of the understanding of our current knowledge of the chemical neuroanatomy of the mammalian 5-HT system.

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