Sheep 5HT2A receptors: partial cloning of the coding sequence and mRNA localization by in situ hybridization in the ewe hypothalamus

Serotonin and serotonin receptors of class II (5HT2-R) are thought to be in volved in the neural mecha nisms which regulate the LH release associated w ith photoperiodic changes in sheep. A specific premammillary hypothalamic a rea displaying a significant binding of H-3-ketanserin, a potent 5HT2-R ant agonist, was previously identified. The aim of the present study was to asc ertain by in situ hybridization (ISH) that 5HT2-R mRNA-containing cells wer e also present in this specific hypothalamic area. Total RNA was prepared f rom sheep pars tuberalis/median eminence, and a cDNA fragment of 546 bp was amplified by reverse transcriptase polymerase chain reaction (RT-PCR) usin g degenerated primers deduced from the human and rat 5HT2A-R sequences. Aft er cloning and sequencing, the sheep nucleotide sequence had the highest ho mology (85.1-92.3%) with the other known mammalian 5HT2-R or 5HT2A-R sequen ces. Homology with other 5HT-R subtypes or other monoamine receptors was mu ch lower, 60% at maximum. After ISH using sense and antisense S-35-riboprob es, specific labelling was found in different parts of the hypothalamus, es pecially in the mammillary bodies where the binding was higher. Within the hypothalamus, the density of labelled cells, mainly neurons, varied conside rably. It was maximal in the mammillary bodies and also in a restricted ven tral region of the premammillary hypothalamus located from about 500/700 mu m to 1200/1400 mu m in front of the mammillary recess, where 3H-ketanserin binding was previously reported. In conclusion: (1) the structural study o f the sequence indicated that the new cloned cDNA corresponds to the sheep 5HT2-R class and, probably, to the 5HT2A-R subtype and (2) the ISH studies revealed that a restricted area of the premammillary hypothalamus shows a l arge number of 5HT2-R mRNA-containing neurons.