Serotonin receptors in brain revisited.

UNLABELLED: In the early 1980's, the dispute on the existence of a multiplicity of receptors for neurotransmitter was at its height. Several subtypes of serotonin (5-HT) receptors were proposed on the basis of radioligand binding assays. In order to provide further support to the existence of these receptors we performed quantitative autoradiographic mapping of the binding of several ligands for the 5-HT1 receptor labeling the subtypes 5-HT1A, 5-HT1B and 5-HT1C, and characterized pharmacologically these different receptors. The results demonstrated differential localization of the subtypes of 5-HT1 receptors indicating that they were expressed by different cell populations, probably neurons, in the brain and further supporting their reality. Shortly afterwards, the cloning of the genes coding for these 5-HT receptors, and many others, ended the dispute by demonstrating that they were different proteins. The advent of Molecular Biology provided new methodologies for the study of the chemical and molecular anatomy of 5-HT receptors in brain, by visualizing cells expressing their mRNA by in situ hybridization and showed that the family of mammalian 5-HT receptors has 14 members, a figure much larger than ever suspected at that time. ORIGINAL ARTICLE ABSTRACT: QUANTITATIVE AUTORADIOGRAPHIC MAPPING OF SEROTONIN RECEPTORS IN THE RAT BRAIN. I. SEROTONIN-1 RECEPTORS: The distribution of serotonin-1 (5-HT1) receptors in the rat brain was studied by light microscopic quantitative autoradiography. Receptors were labeled with [(3)H]serotonin (5-[(3)H]HT), 8-hydroxy-2-[H-dipropylamino-(3)H]tetralin (8-OH-[(3)H]DPAT), [(3)H]LSD and [(3)H]mesulergine, and the densities quantified by microdensitometry with the aid of a computer-assisted image-analysis system. Competition experiments for 5-[(3)H]HT binding by several serotonin-1 agonizts led to the identification of brain areas enriched in each one of the three subtypes of 5-HT1 recognition sites already described (5-HT1A, 5-HT1B, 5-HT1C). The existence of these׳selective׳ areas allowed a detailed pharmacological characterization of these sites to be made in a more precise manner than has been attained in membrane-binding studies. While 5-[(3)H]HT labeled with nanomolar affinity all the 5-HT1 subtypes, the other (3)H-labeled ligands labeled selectively 5-HT1A (8-OH-[(3)H]DPAT), 5-HT1C ([(3)H]mesulergine) and both of them ([(3)H]LSD). Very high concentrations of 5-HT1 receptors were localized in the choroid plexus, lateroseptal nucleus, globus pallidus and ventral pallidum, dentate gyrus, dorsal subiculum, olivary pretectal nucleus, substantia nigra, reticular and external layer of the entorhinal cortex. The different fields of the hippocampus (CA1-CA4), some nuclei of the amygdaloid complex, the hypothalamic nuclei and the dorsal raphé, among others, also presented high concentrations of sites. Areas containing intermediate densities of 5-HT1 receptors included the claustrum, olfactory tubercle, accumbens, central gray and lateral cerebellar nucleus. The nucleus caudate-putamen and the cortex, at the different levels studied, presented receptor densities ranging from intermediate to low. Finally, in other brain areas-pons, medulla, and spinal cord-only low or very low concentrations of 5-HT1 receptors were found. From the areas strongly enriched in 5-HT1 sites, dentate gyrus and septal nucleus contained 5-HT1A sites, while globus pallidus, dorsal subiculum, substantia nigra and olivary pretectal nucleus were enriched in 5-HT1B. The sites in the choroid plexus, which presented the highest density of receptors in the rat brain, were of the 5-HT1C subtype. The distribution of 5-HT1 receptors reported here is discussed in correlation with the distribution of serotoninergic neurons and fibers, the related anatomical pathways and the effects which appear to be mediated by these sites. © 1985.This article is part of a Special Issue entitled SI:50th Anniversary Issue. This article is part of a Special Issue entitled SI:50th Anniversary Issue.

Effective microPET imaging of brain 5-HT(1A) receptors in rats with [(18) F]MeFWAY by suppression of radioligand defluorination.

INTRODUCTION: [(18) F]MeFWAY has been developed for imaging the serotonin 1A receptors in the brain. The purpose of this study were to verify the metabolic stability of [(18) F]MeFWAY, to measure the degree of defluorination of [(18) F]MeFWAY in vivo, to investigate methods of inhibition of defluorination of [(18) F]MeFWAY, and to assess the efficacy of [(18) F]MeFWAY in rat brains in vivo. METHODS: MicroPET experiments in rats were conducted to confirm the distribution of radioactivity in the brain. Nondisplaceable binding potential (BP(ND) ) in the hippocampus and frontal cortex were also analyzed. Miconazole and fluconazole were tested for the ability to suppress defluorination of [(18) F]MeFWAY. We conducted a blockade and displacement experiment by treating with WAY-100635. RESULTS: In vitro stability tests showed that MeFWAY was very stable in serum for 6 h, but PET revealed that authentic [(18) F]MeFWAY underwent significant defluorination in vivo. In vitro inhibition study against decreasing parent activity in liver microsomes, miconazole and fluconazole suppressed metabolic elimination of MeFWAY. However, in the PET study, fluconazole showed more potent inhibitory activity than miconazole. In the suppression of metabolizing enzymes using fluconazole, radioactivity in skull was dramatically decreased by 81% (compared with 69% with miconazole) and it was coupled with an increase in brain uptake. Moreover, BP(ND) in hippocampus was 5.53 and 2.66 in frontal cortex. The blockade and displacement study showed the specificity of [(18) F]MeFWAY to 5-HT(1A) receptors. CONCLUSION: In the rat brain, [(18) F]MeFWAY microPET showed skull uptake due to defluorination in vivo. We can effectively overcome this drawback with fluconazole.

Molecular cloning and functional expression of the Penaeus monodon 5-HT receptor.

Serotonin (5-HT) mediates a number of diverse physiological functions in crustaceans by interacting with various 5-HT receptor subtypes. A putative 5-HT receptor cloned from the ovary of the black tiger prawn (Penaeus monodon) consisted of 2291 nucleotides, encoding a putative 5-HT(1Pem) receptor protein of 591 amino acids. Transient expression of 5-HT(1Pem) in HEK293 cells demonstrated a saturable [3H]-5-HT binding with a Kd of 10.43+/-1.13 nM and Bmax of 1.53+/-0.06 pmol/mg. The putative 5-HT(1Pem) receptor is expressed in all tissues examined and is constitutively expressed in the ovary during ovarian maturation and spent phase. Polyclonal antibodies against the third intracellular loop (i3 loop) of the 5-HT receptor showed that the 5-HT(1Pem) receptor protein was expressed in the trabeculae of ovarian stages 1 and 2 but on the cortical rod and surrounding the oocyte membrane of stages 3 and 4, suggesting that receptor localization plays a critical role in regulating ovarian maturation and spawning in penaeus shrimp.