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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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Pairwise correlation is not altered by weakened Ca2+ binding to the C2AB domain of Syt I.A. Representative pairwise cross-correlograms of 49 cells in one imaged region from the transfected retinas. Each event was a Ca2+ transient in an individual cell. Delay times were the time differences between Ca2+ transient peaks in cell pairs. Note that all correlograms for control, Syt I, Syt I-C2A* and Syt I-C2B* were very similar with sharp peaks at t = 0 sec (see Table 2 for statistics). The oscillatory behavior of the correlograms beyond / t / > ∼7.5 sec might reflect the bursting behavior of the distant cells. Bin widths for all correlograms were 1 sec. B. Pairwise C.I. values as a function of intercellular distance for control, Syt I, Syt I-C2A* and Syt I-C2B*. Data points were averages of medians within the 50 µm bins from 17–23 transfected retinas (about 50% data in each group from the retinas transfected on P0 with DIV 3–4). The error bars were the standard errors of the C.I. values in each bin.
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pone-0047465-g006: Pairwise correlation is not altered by weakened Ca2+ binding to the C2AB domain of Syt I.A. Representative pairwise cross-correlograms of 49 cells in one imaged region from the transfected retinas. Each event was a Ca2+ transient in an individual cell. Delay times were the time differences between Ca2+ transient peaks in cell pairs. Note that all correlograms for control, Syt I, Syt I-C2A* and Syt I-C2B* were very similar with sharp peaks at t = 0 sec (see Table 2 for statistics). The oscillatory behavior of the correlograms beyond / t / > ∼7.5 sec might reflect the bursting behavior of the distant cells. Bin widths for all correlograms were 1 sec. B. Pairwise C.I. values as a function of intercellular distance for control, Syt I, Syt I-C2A* and Syt I-C2B*. Data points were averages of medians within the 50 µm bins from 17–23 transfected retinas (about 50% data in each group from the retinas transfected on P0 with DIV 3–4). The error bars were the standard errors of the C.I. values in each bin.

Mentions: Both Syt I-C2A* and Syt I-C2B* dampened wave-associated Ca2+ transients, but it is unknown if these mutants also affect the spatial properties of stage-II waves (i.e. the correlated propagation over distance). To assess wave correlation structure across cells, we computed cross-correlograms over cell pairs [38], [39] within imaged regions (340×460 µm). The representative cross-correlograms for control, Syt I, Syt I-C2A* and Syt I-C2B* were shown in Fig. 6A. The sharp peaks at t = 0 sec in all the correlograms for cell pairs (Fig. 6A; Data summarized in Table 2) indicated that the waves were well correlated in the imaged region. Syt I-C2B* had more oscillatory behavior outside of t = 0 sec compared to all other groups. We thus suspected that waves in distant cell pairs lose their synchrony in the Syt I-C2B* transfected retina.


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)

Pairwise correlation is not altered by weakened Ca2+ binding to the C2AB domain of Syt I.A. Representative pairwise cross-correlograms of 49 cells in one imaged region from the transfected retinas. Each event was a Ca2+ transient in an individual cell. Delay times were the time differences between Ca2+ transient peaks in cell pairs. Note that all correlograms for control, Syt I, Syt I-C2A* and Syt I-C2B* were very similar with sharp peaks at t = 0 sec (see Table 2 for statistics). The oscillatory behavior of the correlograms beyond / t / > ∼7.5 sec might reflect the bursting behavior of the distant cells. Bin widths for all correlograms were 1 sec. B. Pairwise C.I. values as a function of intercellular distance for control, Syt I, Syt I-C2A* and Syt I-C2B*. Data points were averages of medians within the 50 µm bins from 17–23 transfected retinas (about 50% data in each group from the retinas transfected on P0 with DIV 3–4). The error bars were the standard errors of the C.I. values in each bin.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3472990&req=5

pone-0047465-g006: Pairwise correlation is not altered by weakened Ca2+ binding to the C2AB domain of Syt I.A. Representative pairwise cross-correlograms of 49 cells in one imaged region from the transfected retinas. Each event was a Ca2+ transient in an individual cell. Delay times were the time differences between Ca2+ transient peaks in cell pairs. Note that all correlograms for control, Syt I, Syt I-C2A* and Syt I-C2B* were very similar with sharp peaks at t = 0 sec (see Table 2 for statistics). The oscillatory behavior of the correlograms beyond / t / > ∼7.5 sec might reflect the bursting behavior of the distant cells. Bin widths for all correlograms were 1 sec. B. Pairwise C.I. values as a function of intercellular distance for control, Syt I, Syt I-C2A* and Syt I-C2B*. Data points were averages of medians within the 50 µm bins from 17–23 transfected retinas (about 50% data in each group from the retinas transfected on P0 with DIV 3–4). The error bars were the standard errors of the C.I. values in each bin.
Mentions: Both Syt I-C2A* and Syt I-C2B* dampened wave-associated Ca2+ transients, but it is unknown if these mutants also affect the spatial properties of stage-II waves (i.e. the correlated propagation over distance). To assess wave correlation structure across cells, we computed cross-correlograms over cell pairs [38], [39] within imaged regions (340×460 µm). The representative cross-correlograms for control, Syt I, Syt I-C2A* and Syt I-C2B* were shown in Fig. 6A. The sharp peaks at t = 0 sec in all the correlograms for cell pairs (Fig. 6A; Data summarized in Table 2) indicated that the waves were well correlated in the imaged region. Syt I-C2B* had more oscillatory behavior outside of t = 0 sec compared to all other groups. We thus suspected that waves in distant cell pairs lose their synchrony in the Syt I-C2B* transfected retina.

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