title: Expression of functional N-methyl-D-aspartate receptors during development of chick embryo retina cells: in vitro versus in vivo studies.
abstract: The N-methyl-D-aspartate (NMDA) ionotropic glutamate receptors were studied in retina cells developing in chick embryos and in retina cells cultured as retinospheroids, at the same stages of development. In the retinospheroids, the activity of the NMDA receptors was followed by monitoring the changes in the intracellular free calcium concentration ([Ca2+](i)), in response to NMDA or to L-glutamate. The expression of the subunits NMDAR1, NMDAR2A/B and NMDAR2C in the retinospheroids and in chick retinas were determined by Western blot analyses. The changes in [Ca2+](i) in response to 400 microM NMDA increased from 5 h in vitro to 3 days in vitro (DIV) and remained constant until 14 DIV, whereas the [Ca2+](i) response to 500 microM L-glutamate increased from 5 h in vitro to 3 DIV and decreased slightly until 14 DIV. In the retinospheroids, the expression of the NMDAR1 and NMDAR2A/B subunits increased from 5 h in vitro until 14 DIV, whereas the NMDAR2C subunit increased from 5 h in vitro until 10 DIV and remained constant until 14 DIV. In the retinas, the expression of NMDAR1 increased from embryonic day 8 (E8) until E15, decreased until E18, and increased again until day 22 (post-hatched 1, PH1). The NMDAR2A/B increased from E8 until E18 and decreased slightly until PH1, whereas the NMDAR2C subunit increased from E8 until E15, remained constant until E18, and increased again until PH1. The results suggest that NMDA receptors are expressed and functionally active at early embryonic stages in the retina and in retinospheroids, before synapse formation (E12). However, the calcium responses to NMDA were relatively constant from 3 DIV until 14 DIV, showing no correlation with the increase in the expression of the studied NMDA receptor subunit during the same period. Also, the patterns of NMDA receptor subunits expressed in chick embryo retina cells cultured in vitro and in retina cells developing in vivo were similar.
authors.affiliation: Center for Neuroscience of Coimbra, Department of Zoology, Faculty of Science and Technology, University of Coimbra, 3004-517, Portugal.
authors.forename: Armando J
authors.initials: AJ
authors.lastname: Cristóvão
authors.forename: Catarina R
authors.initials: CR
authors.lastname: Oliveira
authors.forename: Caetana M
authors.initials: CM
authors.lastname: Carvalho
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D002642:Chick Embryo; D018507:Gene Expression Regulation, Developmental / Q000502:physiology*; D018698:Glutamic Acid / Q000378:metabolism* / Q000494:pharmacology; D016202:N-Methylaspartate / Q000494:pharmacology; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D016194:Receptors, N-Methyl-D-Aspartate / Q000187:drug effects / Q000378:metabolism*; D012160:Retina / Q000166:cytology / Q000196:embryology* / Q000378:metabolism; D009435:Synaptic Transmission / Q000187:drug effects / Q000502:physiology*; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology
chemical_list: D002118:Calcium; D018698:Glutamic Acid; D016202:N-Methylaspartate; D016194:Receptors, N-Methyl-D-Aspartate; C121001:NR2B NMDA receptor