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Synaptotagmin I regulates patterned spontaneous activity in the developing rat retina via calcium binding to the C2AB domains.

Chiang CW, Chen YC, Lu JC, Hsiao YT, Chang CW, Huang PC, Chang YT, Chang PY, Wang CT - PLoS ONE (2012)

Bottom Line: Subsequent live Ca(2+) imaging was used to monitor the effects of these molecular perturbations on wave-associated spontaneous Ca(2+) transients.We found that targeted expression of Syt I C2A or C2B mutants in SACs significantly reduced the frequency, duration, and amplitude of wave-associated Ca(2+) transients, suggesting that both C2 domains regulate wave temporal properties.In contrast, these C2 mutants had relatively minor effects on pairwise correlations over distance for wave-associated Ca(2+) transients.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular and Cellular Biology, National Taiwan University, Taipei, Taiwan.

ABSTRACT

Background: In neonatal binocular animals, the developing retina displays patterned spontaneous activity termed retinal waves, which are initiated by a single class of interneurons (starburst amacrine cells, SACs) that release neurotransmitters. Although SACs are shown to regulate wave dynamics, little is known regarding how altering the proteins involved in neurotransmitter release may affect wave dynamics. Synaptotagmin (Syt) family harbors two Ca(2+)-binding domains (C2A and C2B) which serve as Ca(2+) sensors in neurotransmitter release. However, it remains unclear whether SACs express any specific Syt isoform mediating retinal waves. Moreover, it is unknown how Ca(2+) binding to C2A and C2B of Syt affects wave dynamics. Here, we investigated the expression of Syt I in the neonatal rat retina and examined the roles of C2A and C2B in regulating wave dynamics.

Methodology/principal findings: Immunostaining and confocal microscopy showed that Syt I was expressed in neonatal rat SACs and cholinergic synapses, consistent with its potential role as a Ca(2+) sensor mediating retinal waves. By combining a horizontal electroporation strategy with the SAC-specific promoter, we specifically expressed Syt I mutants with weakened Ca(2+)-binding ability in C2A or C2B in SACs. Subsequent live Ca(2+) imaging was used to monitor the effects of these molecular perturbations on wave-associated spontaneous Ca(2+) transients. We found that targeted expression of Syt I C2A or C2B mutants in SACs significantly reduced the frequency, duration, and amplitude of wave-associated Ca(2+) transients, suggesting that both C2 domains regulate wave temporal properties. In contrast, these C2 mutants had relatively minor effects on pairwise correlations over distance for wave-associated Ca(2+) transients.

Conclusions/significance: Through Ca(2+) binding to C2A or C2B, the Ca(2+) sensor Syt I in SACs may regulate patterned spontaneous activity to shape network activity during development. Hence, modulating the releasing machinery in presynaptic neurons (SACs) alters wave dynamics.

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Ca2+ transient frequency is reduced by weakened Ca2+ binding to the C2AB domains of Syt I.A. Left, The expression pattern of pmGluR2-IRES2EGFP in transfected whole-amount retinas. Right, The RGC layer labeled with the Ca2+ indicator fura-2 to measure the wave-associated Ca2+ transients after transfection. Scale bar for both panels, 25 µm. B. Spontaneous, correlated Ca2+ transients were monitored from randomly selected cells in the RGC layer. The example traces of fluorescent changes over time showed the Ca2+ transients in the nearby cells from one imaged region. Retinas were transfected with pmGluR2-IRES2EGFP (Control), pmGluR2-IRES2EGFP-Syt I (Syt I), pmGluR2-IRES2EGFP-Syt I-D230S (Syt I-C2A*), or pmGluR2-IRES2EGFP-Syt I-D363N (Syt I-C2B*). C. Summary of Ca2+ transient frequency after transfection. Data were from 23–39 transfected retinas and 7–15 pups (about 50% data in each group from the retinas transfected on P0 with DIV 3–4). (**p<0.01; ***p<0.001; One-Way ANOVA with post-hoc Student-Newman-Keuls test.) D. Distributions of cumulative probability for Ca2+ transient frequency from individual cells. Data were from 1196–1489 cells out of the same data sets in C. E. Summary of fraction of cells with Ca2+ transients in one imaged region after transfection. Data were from 32–58 transfected regions out of the same data sets in C. (**p<0.01; ***p<0.001; Kruskal-Wallis method followed by post-hoc Dunn test.).
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pone-0047465-g004: Ca2+ transient frequency is reduced by weakened Ca2+ binding to the C2AB domains of Syt I.A. Left, The expression pattern of pmGluR2-IRES2EGFP in transfected whole-amount retinas. Right, The RGC layer labeled with the Ca2+ indicator fura-2 to measure the wave-associated Ca2+ transients after transfection. Scale bar for both panels, 25 µm. B. Spontaneous, correlated Ca2+ transients were monitored from randomly selected cells in the RGC layer. The example traces of fluorescent changes over time showed the Ca2+ transients in the nearby cells from one imaged region. Retinas were transfected with pmGluR2-IRES2EGFP (Control), pmGluR2-IRES2EGFP-Syt I (Syt I), pmGluR2-IRES2EGFP-Syt I-D230S (Syt I-C2A*), or pmGluR2-IRES2EGFP-Syt I-D363N (Syt I-C2B*). C. Summary of Ca2+ transient frequency after transfection. Data were from 23–39 transfected retinas and 7–15 pups (about 50% data in each group from the retinas transfected on P0 with DIV 3–4). (**p<0.01; ***p<0.001; One-Way ANOVA with post-hoc Student-Newman-Keuls test.) D. Distributions of cumulative probability for Ca2+ transient frequency from individual cells. Data were from 1196–1489 cells out of the same data sets in C. E. Summary of fraction of cells with Ca2+ transients in one imaged region after transfection. Data were from 32–58 transfected regions out of the same data sets in C. (**p<0.01; ***p<0.001; Kruskal-Wallis method followed by post-hoc Dunn test.).

Mentions: To study the roles of Syt I C2 domains in retinal waves, we used this established electroporation strategy to introduce Syt I mutants with weakened Ca2+-binding C2 domains. With the aid of the mGluR2 promoter, transfected retinal explants reliably demonstrated EGFP fluorescence in small cells (∼5 µm), compared to relatively large RGCs (∼20 µm) stained by the Ca2+ indicator fura-2 (Fig. 4A). Moreover, the mGluR2 promoter-driven EGFP was localized to the deeper region underneath the RGC layer. The EGFP expression pattern was essentially the same among all transfection groups, suggesting that transfection efficiency was comparable in all groups.


Synaptotagmin I regulates patterned spontaneous activity in the developing rat retina via calcium binding to the C2AB domains.

Chiang CW, Chen YC, Lu JC, Hsiao YT, Chang CW, Huang PC, Chang YT, Chang PY, Wang CT - PLoS ONE (2012)

Ca2+ transient frequency is reduced by weakened Ca2+ binding to the C2AB domains of Syt I.A. Left, The expression pattern of pmGluR2-IRES2EGFP in transfected whole-amount retinas. Right, The RGC layer labeled with the Ca2+ indicator fura-2 to measure the wave-associated Ca2+ transients after transfection. Scale bar for both panels, 25 µm. B. Spontaneous, correlated Ca2+ transients were monitored from randomly selected cells in the RGC layer. The example traces of fluorescent changes over time showed the Ca2+ transients in the nearby cells from one imaged region. Retinas were transfected with pmGluR2-IRES2EGFP (Control), pmGluR2-IRES2EGFP-Syt I (Syt I), pmGluR2-IRES2EGFP-Syt I-D230S (Syt I-C2A*), or pmGluR2-IRES2EGFP-Syt I-D363N (Syt I-C2B*). C. Summary of Ca2+ transient frequency after transfection. Data were from 23–39 transfected retinas and 7–15 pups (about 50% data in each group from the retinas transfected on P0 with DIV 3–4). (**p<0.01; ***p<0.001; One-Way ANOVA with post-hoc Student-Newman-Keuls test.) D. Distributions of cumulative probability for Ca2+ transient frequency from individual cells. Data were from 1196–1489 cells out of the same data sets in C. E. Summary of fraction of cells with Ca2+ transients in one imaged region after transfection. Data were from 32–58 transfected regions out of the same data sets in C. (**p<0.01; ***p<0.001; Kruskal-Wallis method followed by post-hoc Dunn test.).
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pone-0047465-g004: Ca2+ transient frequency is reduced by weakened Ca2+ binding to the C2AB domains of Syt I.A. Left, The expression pattern of pmGluR2-IRES2EGFP in transfected whole-amount retinas. Right, The RGC layer labeled with the Ca2+ indicator fura-2 to measure the wave-associated Ca2+ transients after transfection. Scale bar for both panels, 25 µm. B. Spontaneous, correlated Ca2+ transients were monitored from randomly selected cells in the RGC layer. The example traces of fluorescent changes over time showed the Ca2+ transients in the nearby cells from one imaged region. Retinas were transfected with pmGluR2-IRES2EGFP (Control), pmGluR2-IRES2EGFP-Syt I (Syt I), pmGluR2-IRES2EGFP-Syt I-D230S (Syt I-C2A*), or pmGluR2-IRES2EGFP-Syt I-D363N (Syt I-C2B*). C. Summary of Ca2+ transient frequency after transfection. Data were from 23–39 transfected retinas and 7–15 pups (about 50% data in each group from the retinas transfected on P0 with DIV 3–4). (**p<0.01; ***p<0.001; One-Way ANOVA with post-hoc Student-Newman-Keuls test.) D. Distributions of cumulative probability for Ca2+ transient frequency from individual cells. Data were from 1196–1489 cells out of the same data sets in C. E. Summary of fraction of cells with Ca2+ transients in one imaged region after transfection. Data were from 32–58 transfected regions out of the same data sets in C. (**p<0.01; ***p<0.001; Kruskal-Wallis method followed by post-hoc Dunn test.).
Mentions: To study the roles of Syt I C2 domains in retinal waves, we used this established electroporation strategy to introduce Syt I mutants with weakened Ca2+-binding C2 domains. With the aid of the mGluR2 promoter, transfected retinal explants reliably demonstrated EGFP fluorescence in small cells (∼5 µm), compared to relatively large RGCs (∼20 µm) stained by the Ca2+ indicator fura-2 (Fig. 4A). Moreover, the mGluR2 promoter-driven EGFP was localized to the deeper region underneath the RGC layer. The EGFP expression pattern was essentially the same among all transfection groups, suggesting that transfection efficiency was comparable in all groups.

Bottom Line: Subsequent live Ca(2+) imaging was used to monitor the effects of these molecular perturbations on wave-associated spontaneous Ca(2+) transients.We found that targeted expression of Syt I C2A or C2B mutants in SACs significantly reduced the frequency, duration, and amplitude of wave-associated Ca(2+) transients, suggesting that both C2 domains regulate wave temporal properties.In contrast, these C2 mutants had relatively minor effects on pairwise correlations over distance for wave-associated Ca(2+) transients.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular and Cellular Biology, National Taiwan University, Taipei, Taiwan.

ABSTRACT

Background: In neonatal binocular animals, the developing retina displays patterned spontaneous activity termed retinal waves, which are initiated by a single class of interneurons (starburst amacrine cells, SACs) that release neurotransmitters. Although SACs are shown to regulate wave dynamics, little is known regarding how altering the proteins involved in neurotransmitter release may affect wave dynamics. Synaptotagmin (Syt) family harbors two Ca(2+)-binding domains (C2A and C2B) which serve as Ca(2+) sensors in neurotransmitter release. However, it remains unclear whether SACs express any specific Syt isoform mediating retinal waves. Moreover, it is unknown how Ca(2+) binding to C2A and C2B of Syt affects wave dynamics. Here, we investigated the expression of Syt I in the neonatal rat retina and examined the roles of C2A and C2B in regulating wave dynamics.

Methodology/principal findings: Immunostaining and confocal microscopy showed that Syt I was expressed in neonatal rat SACs and cholinergic synapses, consistent with its potential role as a Ca(2+) sensor mediating retinal waves. By combining a horizontal electroporation strategy with the SAC-specific promoter, we specifically expressed Syt I mutants with weakened Ca(2+)-binding ability in C2A or C2B in SACs. Subsequent live Ca(2+) imaging was used to monitor the effects of these molecular perturbations on wave-associated spontaneous Ca(2+) transients. We found that targeted expression of Syt I C2A or C2B mutants in SACs significantly reduced the frequency, duration, and amplitude of wave-associated Ca(2+) transients, suggesting that both C2 domains regulate wave temporal properties. In contrast, these C2 mutants had relatively minor effects on pairwise correlations over distance for wave-associated Ca(2+) transients.

Conclusions/significance: Through Ca(2+) binding to C2A or C2B, the Ca(2+) sensor Syt I in SACs may regulate patterned spontaneous activity to shape network activity during development. Hence, modulating the releasing machinery in presynaptic neurons (SACs) alters wave dynamics.

Show MeSH
Related in: MedlinePlus