Neurones in the supraoptic nucleus (SON) of the hypothalamus possess intrinsic osmosensing mechanisms, which are lost in transient receptor potential vanilloid 1 (Trpv1)-knock-out mice. The molecular nature of the osmosensory mechanism in SON neurones is believed to be associated with the N-terminal splice variant of Trpv1, although their entire molecular structures have not been hitherto identified. In this study, we sought for TRPV1-related molecules and their function in the rat SON. We performed RT-PCR and immunohistochemistry to detect TRPV1-related molecules in the SON, and patch-clamp and imaging of the cytosolic Ca(2+) concentration ([Ca(2+)]i) to measure responses to osmolality changes and TRPV-related drugs in acutely dissociated SON neurones of rats. RT-PCR analysis revealed full-length Trpv1 and a new N-terminal splice variant, Trpv1_SON (LC008303) in the SON. Positive immunostaining was observed using an antibody against the N-terminal portion of TRPV1 in arginine vasopressin (AVP)-immunoreactive neurones, but not in oxytocin (OT)-immunoreactive neurones. Approximately 20% of SON neurones responded to mannitol (50 mM) with increased action potential firing, inward currents, and [Ca(2+)]i mobilization. Mannitol-induced responses were observed in AVP neurones isolated from AVP-eGFP transgenic rats and identified by GFP fluorescence, but not in OT neurones isolated from OT-mRFP transgenic rats and identified by RFP fluorescence. The mannitol-induced [Ca(2+)]i responses were reversibly blocked by the non-selective TRPV antagonist, ruthenium red (10 μM) and the TRPV1 antagonists, capsazepine (10 μM) and BCTC (10 μM). Although the TRPV1 agonist, capsaicin (100 nM) evoked no response at room temperature, it triggered cationic currents and [Ca(2+)]i elevation when the temperature was increased to 36°C. These results suggest that AVP neurones in the rat SON possess functional full-length TRPV1. Moreover, differences between the responses to capsaicin or hyperosmolality obtained in rat SON neurones and those obtained from dorsal root ganglion neurones or TRPV1-expressing cells indicate that the osmoreceptor expressed in the SON may be a heteromultimer in which TRPV1 is co-assembled with some other, yet unidentified, molecules.

Department of Molecular Neurophysiology Institute of Experimental Medicine Academy of Sciences of the Czech Republic Prague Czech Republic; Department of Neuroscience Institute of Experimental Medicine Academy of Sciences of the Czech Republic Prague Czech Republic

Department of Molecular Neurophysiology Institute of Experimental Medicine Academy of Sciences of the Czech Republic Prague Czech Republic; Université Montpellier 2 Montpellier France; Institut National de la Santé et de la Recherche Médicale U710 Montpellier France; Ecole Pratique des Hautes Etudes Paris France

Laboratory of Veterinary Anatomy Joint Department of Veterinary Medicine Faculty of Agriculture Tottori University Tottori Japan; The United Graduate School of Veterinary Science Yamaguchi University Yamaguchi Japan

Laboratory of Veterinary Biochemistry Joint Department of Veterinary Medicine Faculty of Agriculture Tottori University Tottori Japan; The United Graduate School of Veterinary Science Yamaguchi University Yamaguchi Japan

Laboratory of Veterinary Physiology Joint Department of Veterinary Medicine Faculty of Agriculture Tottori University Tottori Japan

Laboratory of Veterinary Physiology Joint Department of Veterinary Medicine Faculty of Agriculture Tottori University Tottori Japan; The United Graduate School of Veterinary Science Yamaguchi University Yamaguchi Japan

Laboratory of Veterinary Physiology Joint Department of Veterinary Medicine Faculty of Agriculture Tottori University Tottori Japan; The United Graduate School of Veterinary Science Yamaguchi University Yamaguchi Japan; Department of Molecular Neurophysiology Institute of Experimental Medicine Academy of Sciences of the Czech Republic Prague Czech Republic

University of Manchester School of Biological Sciences Oxford Road Manchester UK; Achucarro Center for Neuroscience IKERBASQUE Basque Foundation for Science 48011 Bilbao Spain; Department of Neurosciences University of the Basque Country UPV EHU and CIBERNED Leioa Spain

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