Expression of AMPA/kainate receptors during development of chick embryo retina cells: in vitro versus in vivo studies.
The activity and the subunit expression of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate 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 AMPA/kainate receptors was monitored by following the changes in the intracellular free calcium concentration ([Ca(2+)](i)), in response to AMPA, kainate or to L-glutamate, and the expression of the receptor subunits GluR1, GluR2/3, GluR4 and GluR6/7 was determined in the retinospheroids and in chick retinas by immunodetection using polyclonal antibodies. The changes in [Ca(2+)](i) in response to 400 microM kainate increased from 5h in vitro to 3 days, and remained constant until day 14, whereas the [Ca(2+)](i) in response to 500 microM L-glutamate or 400 microM AMPA increased from 5h in vitro to 3 days, and thereafter decreased slightly until day 14. The [Ca(2+)](i) responses to kainate are mainly due to AMPA receptor stimulation, since the signals were abolished by LY303070, the AMPA receptor antagonist, and were not affected by MK-801, the NMDA receptor antagonist. In retinospheroids, the levels of expression of GluR1 subunit increased from 5h in vitro until day 7, then decreased until day 14. The levels of expression of GluR2/3 and GluR4 subunits increased from 5h in vitro until day 10, and remained constant until day 14. The levels of kainate receptor subunits GluR6/7 increased from 5h in vitro until day 3, and thereafter decreased slightly until day 14. In the retinas, the expression of GluR1 and GluR6/7 subunits increased from day 8 until day 15, and then decreased until day 22 (post-natal 1). The subunits GluR2/3 and GluR4 increased from day 8 until day 18, and remained constant until day 22. The results suggest that AMPA/kainate receptors are expressed at early embryonic stages, although at low levels and before synapse formation (E12). However, the AMPA receptors are not completely functional at the first stage studied since they do not respond to the agonist AMPA. Also, the patterns of AMPA/kainate receptor subunit expression in retinospheroids of chick embryo retina cells cultured in vitro and in retina cells developing in the embryo (in vivo) were similar, indicating that the AMPA/kainate receptor subunits expression in these primary cultures mimics their expression in the developing chick retina.
Cristóvão
Armando J
AJ
Department of Zoology, Centre for Neuroscience of Coimbra, University of Coimbra, 3004-517 Coimbra Codex, Portugal.
Oliveira
Catarina R
CR
Carvalho
Caetana M
CM
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D002642:Chick Embryo; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology; D018507:Gene Expression Regulation, Developmental / Q000502:physiology; D018698:Glutamic Acid / Q000378:metabolism / Q000494:pharmacology; D007150:Immunohistochemistry; D007608:Kainic Acid / Q000494:pharmacology; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D018091:Receptors, AMPA / Q000187:drug effects / Q000378:metabolism*; D018092:Kainic Acid Receptors / Q000187:drug effects / Q000378:metabolism*; D012160:Retina / Q000166:cytology / Q000254:growth & development* / Q000378:metabolism*; D018874:Spheroids, Cellular; D013569:Synapses / Q000378:metabolism* / Q000648:ultrastructure; D009435:Synaptic Transmission / Q000187:drug effects / Q000502:physiology; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid / Q000494:pharmacology; D000097806:GluK2 Kainate Receptor
D002118:Calcium; D018690:Excitatory Amino Acid Agonists; D018698:Glutamic Acid; D007608:Kainic Acid; D018091:Receptors, AMPA; D018092:Kainic Acid Receptors; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; D000097806:GluK2 Kainate Receptor; C104724:glutamate receptor ionotropic, AMPA 4; C104722:glutamate receptor ionotropic, AMPA 2