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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 size is reduced by weakened Ca2+ binding to the C2AB domains of Syt I.A. Top, Definitions of Ca2+ transient duration and amplitude. The peaks of Ca2+ transients were automatically picked by Igor Pro according to the criteria in Materials and Methods. The RMS noise (Noise) was measured from the trace between 30 sec prior to the peak and 50 sec following the peak. The starting point (x0, y0) (indicated by solid arrows) was defined by the latest time point where the first derivative was zero (i.e. dy/dx  = 0, where y was the fluorescence changes in % ΔF/F and x was the recording time in sec) before the peak. To define the end point (x′, y′) (indicated by open arrows), a line was drawn to connect the time points where the trace of Ca2+ transients returned to within the RMS noise of the baseline, with the minimal fluctuation of fluorescence between the starting and end points (i.e. y′−y0 = minimum). Bottom, The Ca2+ transient duration was the interval between the starting and end points. The Ca2+ transient amplitude was the fluorescence change from the baseline to peak. B. Representative individual Ca2+ transients from retinas transfected with Control (black), Syt I (blue), Syt I-C2A* (green), or Syt I-C2B* (red). C. Summary of Ca2+ transient duration for each group. Data were from 22–39 transfected retinas and 7–15 pups (about 50% data in each group from the retinas transfected on P0 with DIV 3–4). (*p<0.05; **p<0.01; ***p<0.001; Kruskal-Wallis method followed by post-hoc Dunn test.) D. Distributions of cumulative probability for Ca2+ transient duration from individual cells. Data were from 860–1106 cells out of the same data sets in C. E. Summary of Ca2+ transient amplitude after transfection. Data sets were the same as in C. (**p<0.01; Kruskal-Wallis method followed by post-hoc Dunn test.) F. Distributions of cumulative probability for Ca2+ transient amplitude from individual cells. Data sets were the same as in D.
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pone-0047465-g005: Ca2+ transient size is reduced by weakened Ca2+ binding to the C2AB domains of Syt I.A. Top, Definitions of Ca2+ transient duration and amplitude. The peaks of Ca2+ transients were automatically picked by Igor Pro according to the criteria in Materials and Methods. The RMS noise (Noise) was measured from the trace between 30 sec prior to the peak and 50 sec following the peak. The starting point (x0, y0) (indicated by solid arrows) was defined by the latest time point where the first derivative was zero (i.e. dy/dx  = 0, where y was the fluorescence changes in % ΔF/F and x was the recording time in sec) before the peak. To define the end point (x′, y′) (indicated by open arrows), a line was drawn to connect the time points where the trace of Ca2+ transients returned to within the RMS noise of the baseline, with the minimal fluctuation of fluorescence between the starting and end points (i.e. y′−y0 = minimum). Bottom, The Ca2+ transient duration was the interval between the starting and end points. The Ca2+ transient amplitude was the fluorescence change from the baseline to peak. B. Representative individual Ca2+ transients from retinas transfected with Control (black), Syt I (blue), Syt I-C2A* (green), or Syt I-C2B* (red). C. Summary of Ca2+ transient duration for each group. Data were from 22–39 transfected retinas and 7–15 pups (about 50% data in each group from the retinas transfected on P0 with DIV 3–4). (*p<0.05; **p<0.01; ***p<0.001; Kruskal-Wallis method followed by post-hoc Dunn test.) D. Distributions of cumulative probability for Ca2+ transient duration from individual cells. Data were from 860–1106 cells out of the same data sets in C. E. Summary of Ca2+ transient amplitude after transfection. Data sets were the same as in C. (**p<0.01; Kruskal-Wallis method followed by post-hoc Dunn test.) F. Distributions of cumulative probability for Ca2+ transient amplitude from individual cells. Data sets were the same as in D.

Mentions: Retinal waves occur with a periodicity on the order of minutes. Spontaneous Ca2+ transients associated with waves can last for tens of seconds following relatively brief spontaneous depolarizations [3]. To examine if the wave patterns are changed by Syt I mutants, we measured the duration and amplitude of single Ca2+ transients in an unbiased way (Fig. 5A, see Materials and Methods for details). The representative Ca2+ transients were shown in Fig. 5B. Both Syt I-C2A* and Syt I-C2B* reduced Ca2+ transient size compared to wild-type Syt I and control, including a decrease in Ca2+ transient duration and amplitude, and this impression was confirmed by quantitative analysis.


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 size is reduced by weakened Ca2+ binding to the C2AB domains of Syt I.A. Top, Definitions of Ca2+ transient duration and amplitude. The peaks of Ca2+ transients were automatically picked by Igor Pro according to the criteria in Materials and Methods. The RMS noise (Noise) was measured from the trace between 30 sec prior to the peak and 50 sec following the peak. The starting point (x0, y0) (indicated by solid arrows) was defined by the latest time point where the first derivative was zero (i.e. dy/dx  = 0, where y was the fluorescence changes in % ΔF/F and x was the recording time in sec) before the peak. To define the end point (x′, y′) (indicated by open arrows), a line was drawn to connect the time points where the trace of Ca2+ transients returned to within the RMS noise of the baseline, with the minimal fluctuation of fluorescence between the starting and end points (i.e. y′−y0 = minimum). Bottom, The Ca2+ transient duration was the interval between the starting and end points. The Ca2+ transient amplitude was the fluorescence change from the baseline to peak. B. Representative individual Ca2+ transients from retinas transfected with Control (black), Syt I (blue), Syt I-C2A* (green), or Syt I-C2B* (red). C. Summary of Ca2+ transient duration for each group. Data were from 22–39 transfected retinas and 7–15 pups (about 50% data in each group from the retinas transfected on P0 with DIV 3–4). (*p<0.05; **p<0.01; ***p<0.001; Kruskal-Wallis method followed by post-hoc Dunn test.) D. Distributions of cumulative probability for Ca2+ transient duration from individual cells. Data were from 860–1106 cells out of the same data sets in C. E. Summary of Ca2+ transient amplitude after transfection. Data sets were the same as in C. (**p<0.01; Kruskal-Wallis method followed by post-hoc Dunn test.) F. Distributions of cumulative probability for Ca2+ transient amplitude from individual cells. Data sets were the same as in D.
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Related In: Results  -  Collection

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

pone-0047465-g005: Ca2+ transient size is reduced by weakened Ca2+ binding to the C2AB domains of Syt I.A. Top, Definitions of Ca2+ transient duration and amplitude. The peaks of Ca2+ transients were automatically picked by Igor Pro according to the criteria in Materials and Methods. The RMS noise (Noise) was measured from the trace between 30 sec prior to the peak and 50 sec following the peak. The starting point (x0, y0) (indicated by solid arrows) was defined by the latest time point where the first derivative was zero (i.e. dy/dx  = 0, where y was the fluorescence changes in % ΔF/F and x was the recording time in sec) before the peak. To define the end point (x′, y′) (indicated by open arrows), a line was drawn to connect the time points where the trace of Ca2+ transients returned to within the RMS noise of the baseline, with the minimal fluctuation of fluorescence between the starting and end points (i.e. y′−y0 = minimum). Bottom, The Ca2+ transient duration was the interval between the starting and end points. The Ca2+ transient amplitude was the fluorescence change from the baseline to peak. B. Representative individual Ca2+ transients from retinas transfected with Control (black), Syt I (blue), Syt I-C2A* (green), or Syt I-C2B* (red). C. Summary of Ca2+ transient duration for each group. Data were from 22–39 transfected retinas and 7–15 pups (about 50% data in each group from the retinas transfected on P0 with DIV 3–4). (*p<0.05; **p<0.01; ***p<0.001; Kruskal-Wallis method followed by post-hoc Dunn test.) D. Distributions of cumulative probability for Ca2+ transient duration from individual cells. Data were from 860–1106 cells out of the same data sets in C. E. Summary of Ca2+ transient amplitude after transfection. Data sets were the same as in C. (**p<0.01; Kruskal-Wallis method followed by post-hoc Dunn test.) F. Distributions of cumulative probability for Ca2+ transient amplitude from individual cells. Data sets were the same as in D.
Mentions: Retinal waves occur with a periodicity on the order of minutes. Spontaneous Ca2+ transients associated with waves can last for tens of seconds following relatively brief spontaneous depolarizations [3]. To examine if the wave patterns are changed by Syt I mutants, we measured the duration and amplitude of single Ca2+ transients in an unbiased way (Fig. 5A, see Materials and Methods for details). The representative Ca2+ transients were shown in Fig. 5B. Both Syt I-C2A* and Syt I-C2B* reduced Ca2+ transient size compared to wild-type Syt I and control, including a decrease in Ca2+ transient duration and amplitude, and this impression was confirmed by quantitative analysis.

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