title: Fluorescent visualisation of the hypothalamic oxytocin neurones activated by cholecystokinin-8 in rats expressing c-fos-enhanced green fluorescent protein and oxytocin-monomeric red fluorescent protein 1 fusion transgenes.
abstract: The up-regulation of c-fos gene expression is widely used as a marker of neuronal activation elicited by various stimuli. Anatomically precise observation of c-fos gene products can be achieved at the RNA level by in situ hybridisation or at the protein level by immunocytochemistry. Both of these methods are time and labour intensive. We have developed a novel transgenic rat system that enables the trivial visualisation of c-fos expression using an enhanced green fluorescent protein (eGFP) tag. These rats express a transgene consisting of c-fos gene regulatory sequences that drive the expression of a c-fos-eGFP fusion protein. In c-fos-eGFP transgenic rats, robust nuclear eGFP fluorescence was observed in osmosensitive brain regions 90 min after i.p. administration of hypertonic saline. Nuclear eGFP fluorescence was also observed in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) 90 min after i.p. administration of cholecystokinin (CCK)-8, which selectively activates oxytocin (OXT)-secreting neurones in the hypothalamus. In double transgenic rats that express c-fos-eGFP and an OXT-monomeric red fluorescent protein 1 (mRFP1) fusion gene, almost all mRFP1-positive neurones in the SON and PVN expressed nuclear eGFP fluorescence 90 min after i.p. administration of CCK-8. It is possible that not only a plane image, but also three-dimensional reconstruction image may identify cytoplasmic vesicles in an activated neurone at the same time.
authors.affiliation: Department of Physiology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan; Otorhynolaryngology-Head and Neck Surgery, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan; Department of Otorhynolaryngology, National Kyushu Medical Center, Fukuoka, Japan.
authors.forename: A
authors.initials: A
authors.lastname: Katoh
authors.forename: K
authors.initials: K
authors.lastname: Shoguchi
authors.forename: H
authors.initials: H
authors.lastname: Matsuoka
authors.forename: M
authors.initials: M
authors.lastname: Yoshimura
authors.forename: J-I
authors.initials: JI
authors.lastname: Ohkubo
authors.forename: T
authors.initials: T
authors.lastname: Matsuura
authors.forename: T
authors.initials: T
authors.lastname: Maruyama
authors.forename: T
authors.initials: T
authors.lastname: Ishikura
authors.forename: T
authors.initials: T
authors.lastname: Aritomi
authors.forename: H
authors.initials: H
authors.lastname: Fujihara
authors.forename: H
authors.initials: H
authors.lastname: Hashimoto
authors.forename: H
authors.initials: H
authors.lastname: Suzuki
authors.forename: D
authors.initials: D
authors.lastname: Murphy
authors.forename: Y
authors.initials: Y
authors.lastname: Ueta
mesh_terms: D000818:Animals; D002766:Cholecystokinin / Q000494:pharmacology*; D005455:Fluorescent Antibody Technique; D049452:Green Fluorescent Proteins / Q000096:biosynthesis; D007031:Hypothalamus / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D008164:Luminescent Proteins / Q000096:biosynthesis; D009474:Neurons / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure*; D015514:Oncogene Proteins, Fusion / Q000235:genetics; D010121:Oxytocin / Q000502:physiology*; D010446:Peptide Fragments / Q000494:pharmacology*; D016760:Proto-Oncogene Proteins c-fos / Q000096:biosynthesis*; D051381:Rats; D055647:Rats, Transgenic; D017208:Rats, Wistar; D019076:Transgenes / Q000235:genetics*; D000097573:Red Fluorescent Protein
chemical_list: D002766:Cholecystokinin; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D015514:Oncogene Proteins, Fusion; D010121:Oxytocin; D010446:Peptide Fragments; D016760:Proto-Oncogene Proteins c-fos; D000097573:Red Fluorescent Protein; C410885:cholecystokinin 8; C485184:enhanced green fluorescent protein
keywords: c-fos; green fluorescent protein; oxytocin; rat; red fluorescent protein