Muscarinic acetylcholine responses in the early embryonic chick retina

The action of acetylcholine on cytoplasmic Ca2+ concentration ([Ca2+]i) was studied in early embryonic chick retinae. Whole neural retinae were isolated from embryonic day 3 (E3) chicks and loaded with a Ca(2+)-sensitive fluorescent dye (Fura-2). Increases in [Ca2+]i were evoked by the puff application of acetylcholine at concentrations higher than 0.1 microM. The Ca2+ response became larger in a dose-dependent manner up to 10 microM of acetylcholine applied. The rise in [Ca2+]i was not due to the influx of Ca2+ through calcium channels, but to the release of Ca2+ from internal stores. A calcium channel antagonist, nifedipine, which completely blocks the Ca2+ rise caused by depolarization with 100 mM K+, had no effects on the acetylcholine response and the Ca2+ response to acetylcholine occurred even in a Ca(2+)-free medium. The Ca2+ response to acetylcholine was mediated by muscarinic receptors. Atropine of 1 microM abolished the response to 10 microM acetylcholine, whereas d-tubocurarine of 100 microM had no effects. Two muscarinic agonists, muscarine and carbamylcholine (100 microM each), evoked comparable responses with that to 10 microM acetylcholine. The developmental change of the muscarinic response was examined from E3 to E13. The Ca2+ response to 100 microM carbamylcholine was intense at E3-E5, then rapidly declined until E8. The muscarinic Ca2+ mobilization we found in the early embryonic chick retina may be regarded as a part of the "embryonic muscarinic system" proposed by Drew's group, which appears transiently and ubiquitously at early embryonic stages in relation to organogenesis.