Visualization of glucocorticoid receptor in the brain of green fluorescent protein-glucocorticoid receptor knockin mice.
Glucocorticoids exert various neuroendocrinological effects, including stress response, in the central nervous system via glucocorticoid receptor (GR). GRs are transported from the cytoplasm to the nucleus upon ligand binding, and then exert the transcriptional activity. Although it is important for unraveling the actual property of the GR in vivo, subcellular dynamics of the GR are still unclear within the brain tissue in which the neuronal circuitry is maintained. To address this issue, we generated green fluorescent protein (GFP)-GR knockin mice, whose GR has been replaced by a GFP-GR fusion protein that is functionally indistinguishable from endogenous GR. In fixed brain sections of the GFP-GR knockin mice, the distribution of the green fluorescence was similar to that of GR immunoreactivity. By subtracting autofluorescence using fluorescent emission fingerprinting method with confocal laser scanning microscope, nuclear localization of GFP-GR was identifiable in the hippocampal CA3 subregion, where subcellular localization of the GR has been unsolved compared with other areas. To examine the subcellular trafficking of GFP-GR in vivo, we performed adrenalectomy on the GFP-GR knockin mice. GFP-GR was translocated from the nucleus to the cytoplasm and neurites two days after adrenalectomy. Furthermore, laser scanning cytometry by which fluorescence intensity in situ can be quantitatively measured revealed the entire GFP-GR expression level was increased. We then examined the dynamic changes in the subcellular localization of GFP-GR in living hippocampal neurons both in dissociated culture and in tissue slices. GFP-GR was localized in not only the perikarya but also neurites in the absence of ligand, and nuclear translocation following ligand treatment was observed. This is the first report visualizing subcellular trafficking of the GR in the mouse brain in more physiological condition. The present results propose new avenues for the research of the GR dynamics both in vitro and in vivo.
Usuku
T
T
Department of Anatomy and Neurobiology, Kyoto Prefectural University of Medicine, Kawaramachi Hirokoji, Kamigyo-Ku, Kyoto, Japan.
Nishi
M
M
Morimoto
M
M
Brewer
J A
JA
Muglia
L J
LJ
Sugimoto
T
T
Kawata
M
M
D000315:Adrenalectomy; D000818:Animals; D001921:Brain / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D051379:Mice; D008817:Mice, Mutant Strains; D018613:Microscopy, Confocal; D009474:Neurons / Q000378:metabolism*; D009924:Organ Culture Techniques; D021381:Protein Transport / Q000502:physiology; D011965:Receptors, Glucocorticoid / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*
D011965:Receptors, Glucocorticoid; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins