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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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Syt I is strongly expressed in neonatal SACs and IPL.Ai–Aiii. Merged images of Syt I (green) and ChAT (red) staining in retinal cross-sections from P0–P2 rats. The colocalization of Syt I and ChAT labeling was found in the IPL (yellow). Scale bars, 25 µm. ChAT, choline acetyltransferase. NBL, neuroblast layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Bi–Biii. Immunofluorescence labeling of Syt I (green) in the IPL of retinal cross-sections from P0–P2 rats. Ci–Ciii. Immunofluorescence staining of ChAT (red) labeling SACs and the IPL in retinal cross-sections from P0–P2 rats. Di–Diii. Merged images of Syt I and ChAT staining in the same retinal cross-sections. The Syt I and ChAT labeling was colocalized to the SAC somata (yellow) as indicated by arrows. Scale bars for B-D, 50 µm. Ei–Eiii. The high magnification for merged images of Syt I and ChAT staining in the boxes of Di–Diii. Scale bars for E, 5 µm.
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pone-0047465-g001: Syt I is strongly expressed in neonatal SACs and IPL.Ai–Aiii. Merged images of Syt I (green) and ChAT (red) staining in retinal cross-sections from P0–P2 rats. The colocalization of Syt I and ChAT labeling was found in the IPL (yellow). Scale bars, 25 µm. ChAT, choline acetyltransferase. NBL, neuroblast layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Bi–Biii. Immunofluorescence labeling of Syt I (green) in the IPL of retinal cross-sections from P0–P2 rats. Ci–Ciii. Immunofluorescence staining of ChAT (red) labeling SACs and the IPL in retinal cross-sections from P0–P2 rats. Di–Diii. Merged images of Syt I and ChAT staining in the same retinal cross-sections. The Syt I and ChAT labeling was colocalized to the SAC somata (yellow) as indicated by arrows. Scale bars for B-D, 50 µm. Ei–Eiii. The high magnification for merged images of Syt I and ChAT staining in the boxes of Di–Diii. Scale bars for E, 5 µm.

Mentions: Syt I is the major Syt isoform found in many parts of the nervous system [15]. However, the expression of Syt I in the developing SACs, which release neurotransmitters to initiate stage-II waves, remains to be determined. We thus examined if Syt I is expressed in SACs during stage-II waves. In P0–P2 rat retinal cross-sections, double immunolabeling for Syt I and choline acetyltransferase (ChAT, the SAC marker) showed that Syt I was mainly localized to the inner plexiform layer (IPL) surrounding the SACs (Fig. 1). The high-magnification image from the IPL indicated that Syt I was also localized to the somata of SACs (Fig. 1Ei–iii, yellow), suggesting that Syt I is expressed in the cholinergic synapses originating from SACs. Thus, Syt I is located where it can serve as a Ca2+ sensor in triggering neurotransmitter release during stage-II waves.


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)

Syt I is strongly expressed in neonatal SACs and IPL.Ai–Aiii. Merged images of Syt I (green) and ChAT (red) staining in retinal cross-sections from P0–P2 rats. The colocalization of Syt I and ChAT labeling was found in the IPL (yellow). Scale bars, 25 µm. ChAT, choline acetyltransferase. NBL, neuroblast layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Bi–Biii. Immunofluorescence labeling of Syt I (green) in the IPL of retinal cross-sections from P0–P2 rats. Ci–Ciii. Immunofluorescence staining of ChAT (red) labeling SACs and the IPL in retinal cross-sections from P0–P2 rats. Di–Diii. Merged images of Syt I and ChAT staining in the same retinal cross-sections. The Syt I and ChAT labeling was colocalized to the SAC somata (yellow) as indicated by arrows. Scale bars for B-D, 50 µm. Ei–Eiii. The high magnification for merged images of Syt I and ChAT staining in the boxes of Di–Diii. Scale bars for E, 5 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0047465-g001: Syt I is strongly expressed in neonatal SACs and IPL.Ai–Aiii. Merged images of Syt I (green) and ChAT (red) staining in retinal cross-sections from P0–P2 rats. The colocalization of Syt I and ChAT labeling was found in the IPL (yellow). Scale bars, 25 µm. ChAT, choline acetyltransferase. NBL, neuroblast layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Bi–Biii. Immunofluorescence labeling of Syt I (green) in the IPL of retinal cross-sections from P0–P2 rats. Ci–Ciii. Immunofluorescence staining of ChAT (red) labeling SACs and the IPL in retinal cross-sections from P0–P2 rats. Di–Diii. Merged images of Syt I and ChAT staining in the same retinal cross-sections. The Syt I and ChAT labeling was colocalized to the SAC somata (yellow) as indicated by arrows. Scale bars for B-D, 50 µm. Ei–Eiii. The high magnification for merged images of Syt I and ChAT staining in the boxes of Di–Diii. Scale bars for E, 5 µm.
Mentions: Syt I is the major Syt isoform found in many parts of the nervous system [15]. However, the expression of Syt I in the developing SACs, which release neurotransmitters to initiate stage-II waves, remains to be determined. We thus examined if Syt I is expressed in SACs during stage-II waves. In P0–P2 rat retinal cross-sections, double immunolabeling for Syt I and choline acetyltransferase (ChAT, the SAC marker) showed that Syt I was mainly localized to the inner plexiform layer (IPL) surrounding the SACs (Fig. 1). The high-magnification image from the IPL indicated that Syt I was also localized to the somata of SACs (Fig. 1Ei–iii, yellow), suggesting that Syt I is expressed in the cholinergic synapses originating from SACs. Thus, Syt I is located where it can serve as a Ca2+ sensor in triggering neurotransmitter release during stage-II waves.

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