Transient muscarinic calcium mobilisation in transdifferentiating as in reaggregating embryonic chick retinae

Two independent in vitro regeneration systems of the embryonic chick retina (E4-5) were used to study the mobilisation of intracellular calcium by the neurotransmitters acetylcholine (ACh) and glutamate, as measured by Fura-2 fluorescence changes. Retinal pigment epithelium (RPE) explants under the influence of basic fibroblast growth factor transdifferentiate into a retina-like tissue with normal laminar organisation, while rosetted spheres reaggregated from fully dispersed cells of the embryonic retina will achieve only an inferior tissue organisation, characterised by regions of an inverted retina [Layer et al., Neuroreport 12 (2001) A39-46]. ACh induced a pronounced Ca(2+) response in young explants, and a similar but less pronounced response in reaggregates; this response decreased almost entirely after 1 week in culture. In contrast, a Ca(2+) response to glutamate became detectable later, continuously increasing during this period. The response to ACh was strictly mediated by muscarinic ACh receptors (mAChRs), since it was inhibited by preincubation with atropine, but not tubocurarine; correspondingly, it was mimicked by muscarine, but not nicotine. Studies with mAChR blockers, preferentially acting on the m1-, m2-, or m3-receptor subtypes, suggested that the muscarine-induced Ca(2+) response is mediated by m1- and/or m3-type mAChRs, but not by the m2-type. These results show that (i) similar to the in vivo retina, in both a transdifferentiating and a reaggregating system an early muscarinic Ca(2+) response is active, which (ii) roughly parallels periods of cell proliferation, and (iii)-as evidenced by the reaggregates-does not depend on any tissue pre-organisation. In contrast, a response to glutamate becomes prominent only when tissue differentiation commences. This is the first demonstration of a physiological response in regenerating chick retinas, supporting their validity as models both of retinal development and regeneration.