GABAA-receptor-mediated increase in intracellular Ca2+ concentration in the regenerating retina of adult newt.

We used optical recording with the Ca(2+)-sensitive dye, fura-2, in living slice preparations from the newt retina at different stages of regeneration. gamma-Aminobutyric acid (GABA) induced pronounced [Ca(2+)](i) rise in progenitor cells and differentiating ganglion cells in the 'intermediate' stage of retinal regeneration. This [Ca(2+)](i) rise became less pronounced at the beginning of synapse formation in the late regenerating retina. At the late period of the late regenerating retina with the IPL thickness comparable to that of the control retina, GABA-induced [Ca(2+)](i) rise became undetectable or sometimes a small decrease in [Ca(2+)](i) was observed in regenerated ganglion cells. In contrast, N-methyl-d-aspartate (NMDA)-induced [Ca(2+)](i) rise appeared in premature ganglion cells and became prominent gradually as the regeneration proceeded. The [Ca(2+)](i) rise to GABA was mediated by GABA(A) receptors. This was shown by inhibition of GABA-induced Ca(2+) response with the preincubation of the GABA(A) receptor antagonist, bicuculline. The [Ca(2+)](i) rise due to GABA was suppressed in the absence of extracellular Ca(2+) or in the presence of the L-type voltage-gated Ca(2+) channel blocker, verapamil, suggesting that Ca(2+) may be entered through L-type Ca(2+) channels. Transient appearance of [Ca(2+)](i) rise to GABA during regeneration and origin of GABA-induced [Ca(2+)](i) rise were similar to those in the developing retina [J. Neurobiol. 24 (1993) 1600]. These similarities may suggest that common mechanisms may control neurogenesis and/or synaptogenesis during development and regeneration.

Muscarinic calcium mobilization in the regenerating retina of adult newt.

We used optical recording with a Ca(2+)-sensitive dye, fura2, in living slice preparations from the newt retina at different stages of regeneration. ACh produced the most pronounced [Ca2+]i rise in progenitor cells and premature ganglion cells of the earlier stage of retinal regeneration, but less pronounced Ca2+ response in ganglion cells just before, or at the beginning of, synaptogenesis. The [Ca2+]i rise to ACh was mediated by mAChRs. This was shown by inhibition of the ACh-induced Ca2+ response with a preincubation of the mAChR antagonist atropine as well as with direct stimulation of the [Ca2+]i rise by the mAChR agonist muscarine. This muscarine-induced [Ca2+]i rise was more greatly suppressed by the M1 and/or M3 preferring mAChR antagonists than by the M2 preferring mAChR antagonist. The [Ca2+]i rise due to muscarine was not suppressed in the absence of extracellular Ca2+, but suppressed in part in the presence of the L-type voltage-gated Ca2+ channel blockers, verapamil or nicardipine. Furthermore, thapsigargin (TG), a Ca-ATPase inhibitor, abolished the muscarine-induced [Ca2+]i rise in the absence of extracellular Ca2+. These results suggest that the mAChR-mediated [Ca2+]i rise is mainly a result of a release of Ca2+ from intracellular stores. TG produced a slow rise in the resting level of [Ca2+]i. This [Ca2+]i raise was suppressed as extracellular Ca2+ was omitted, whereas a rapid rise in [Ca2+]i occurred when extracellular Ca2+ was reintroduced, suggesting the occurrence of the capacitative Ca2+ influx in the progenitor cells and premature ganglion cells of the regenerating newt retina.

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.