A novel neuronal cell line derived from the ventrolateral region of the suprachiasmatic nucleus.
The suprachiasmatic nucleus of the anterior hypothalamus is the center of an internal biological clock in mammals. Glutamate is the neurotransmitter of retino-hypothalamic tract responsible for mediating the circadian actions of light in rodents. N-methyl-d-aspartate receptors, particularly NR2B subunit are reported to be principally involved in photic resetting of the biological clock in vivo and in slice culture. But, the precise cellular mechanisms of the resetting are not elucidated, because no adequate neuronal cell lines derived from the suprachiasmatic nucleus have been established. We established a neuronal cell line, N14.5, derived from the suprachiasmatic nucleus of a transgenic rat harboring the temperature-sensitive simian virus 40 large T-antigen gene. When the cells were cultured at 39 degrees C, the morphological features were turned fibroblastic into neuronal round cell body with neurite extensions. These cells showed immunoreactivities for neuronal markers (betaIII-tubulin, microtubule-associated protein 2 and TAU2) and as well as for vasoactive intestinal peptide which is expressed in the ventrolateral region of the suprachiasmatic nucleus. The cells expressed N-methyl-d-aspartate receptors, particularly NR1 and NR2B subunits as revealed by quantitative PCR. N-methyl-d-aspartate activated phosphorylation of p44/42 mitogen-activated protein kinase and increased expression level of Per1 and Per2 mRNA. These results suggest that the N14.5 is a novel neuronal cell line derived from the ventrolateral region of the suprachiasmatic nucleus, and that N-methyl-d-aspartate receptors expressed in the cells are a functional receptor. The N14.5 cells may be a useful tool to elucidate numerous chronobiological processes, especially resetting mechanism induced by an external light signal.
Matsushita
T
T
Molecular Medicine Laboratories, Institute for Drug Discovery Research, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba-shi, Ibaraki 305-8585 Japan.
Amagai
Y
Y
Terai
K
K
Kojima
T
T
Obinata
M
M
Hashimoto
S
S
D000818:Animals; D030801:Animals, Genetically Modified; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics; D001683:Biological Clocks / Q000235:genetics* / Q000528:radiation effects; D015415:Biomarkers / Q000378:metabolism; D018797:Cell Cycle Proteins; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D002940:Circadian Rhythm / Q000235:genetics* / Q000528:radiation effects; D005786:Gene Expression Regulation / Q000502:physiology; D005822:Genetic Vectors / Q000235:genetics; D008027:Light; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000378:metabolism* / Q000528:radiation effects; D009687:Nuclear Proteins / Q000235:genetics; D056950:Period Circadian Proteins; D010775:Photic Stimulation; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000235:genetics*; D013493:Suprachiasmatic Nucleus / Q000166:cytology / Q000378:metabolism* / Q000528:radiation effects; D014157:Transcription Factors / Q000235:genetics; D014162:Transfection / Q000379:methods; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism
D000952:Antigens, Polyomavirus Transforming; D015415:Biomarkers; D018797:Cell Cycle Proteins; C409466:NR1 NMDA receptor; C121001:NR2B NMDA receptor; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; C539023:Per1 protein, mouse; C539024:Per1 protein, rat; C513312:Per2 protein, mouse; C513311:Per2 protein, rat; D056950:Period Circadian Proteins; D016194:Receptors, N-Methyl-D-Aspartate; D014157:Transcription Factors; D014660:Vasoactive Intestinal Peptide; D048052:Mitogen-Activated Protein Kinase 3