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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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The mGluR2 promoter drives gene expression selectively in SACs.Whole-mount retinas from P0–P2 rats were double-labeled with HA (green) and ChAT (red) after 72 hr of transfection. Ai–Ci. Whole-mount retinas transfected with pCMV-HA-Syt I-IRES2-EGFP. Ai. Labeled with HA. Bi. Labeled with ChAT. Ci. Merged images of Ai and Bi. Scale bar, 50 µm. EGFP, enhanced green fluorescent protein. Aii–Cii. High magnification view of whole-mount retinas transfected with pCMV-HA-Syt I-IRES2-EGFP. Aii. Labeled with HA. Bii. Labeled with ChAT. Cii. Merged images of Aii and Bii. Scale bar, 25 µm. Di–Fi. Whole-mount retinas transfected with pmGluR2-HA-Syt I-IRES2-EGFP. Di. Labeled with HA. Ei. Labeled with ChAT. Fi. Merged images of Di and Ei. Scale bar, 50 µm. Dii-Fii. High magnification view of whole-mount retinas transfected with pmGluR2-HA-Syt I-IRES2-EGFP. Dii. Labeled with HA. Eii. Labeled with ChAT. Fii. Merged images of Dii and Eii. Scale bar, 25 µm. G–H. Retinal cross-sections transfected with either pCMV-HA-Syt I-IRES2-EGFP (G), or pmGluR2-HA-Syt I-IRES2-EGFP (H). The SACs and IPL were stained for ChAT (red). The transfected cells were stained for HA/GFP (green). The cell nuclei were stained with DAPI (blue). Right, Merged images (yellow). The transfected SACs were indicated by arrows. Insets showed the higher magnification of merged images. Scale bars, 25 µm.
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pone-0047465-g003: The mGluR2 promoter drives gene expression selectively in SACs.Whole-mount retinas from P0–P2 rats were double-labeled with HA (green) and ChAT (red) after 72 hr of transfection. Ai–Ci. Whole-mount retinas transfected with pCMV-HA-Syt I-IRES2-EGFP. Ai. Labeled with HA. Bi. Labeled with ChAT. Ci. Merged images of Ai and Bi. Scale bar, 50 µm. EGFP, enhanced green fluorescent protein. Aii–Cii. High magnification view of whole-mount retinas transfected with pCMV-HA-Syt I-IRES2-EGFP. Aii. Labeled with HA. Bii. Labeled with ChAT. Cii. Merged images of Aii and Bii. Scale bar, 25 µm. Di–Fi. Whole-mount retinas transfected with pmGluR2-HA-Syt I-IRES2-EGFP. Di. Labeled with HA. Ei. Labeled with ChAT. Fi. Merged images of Di and Ei. Scale bar, 50 µm. Dii-Fii. High magnification view of whole-mount retinas transfected with pmGluR2-HA-Syt I-IRES2-EGFP. Dii. Labeled with HA. Eii. Labeled with ChAT. Fii. Merged images of Dii and Eii. Scale bar, 25 µm. G–H. Retinal cross-sections transfected with either pCMV-HA-Syt I-IRES2-EGFP (G), or pmGluR2-HA-Syt I-IRES2-EGFP (H). The SACs and IPL were stained for ChAT (red). The transfected cells were stained for HA/GFP (green). The cell nuclei were stained with DAPI (blue). Right, Merged images (yellow). The transfected SACs were indicated by arrows. Insets showed the higher magnification of merged images. Scale bars, 25 µm.

Mentions: To manipulate neurotransmitter release from SACs during stage-II waves, we aimed to transfect SACs with Syt I mutants with the weakened Ca2+ binding in the C2 domains. Since the metabotropic glutamate receptor type II (mGluR2) promoter has been shown to target SACs specifically [7], [35], [36], [37], we placed our Syt constructs under the control of the mGluR2 promoter to manipulate Syt I specifically in SACs. To verify the effectiveness of our transfection strategy, we compared the specificity of the mGluR2 promoter with the ubiquitous cytomegalovirus (CMV) promoter by immnunofluorescence (Fig. 3). With the CMV promoter (Fig. 3A–C), the HA/EGFP immunoreactivity was scarcely colocalized with ChAT (the SAC marker), but mostly appeared in relatively round and large retinal neurons (∼20 µm), likely RGCs. In contrast, with the mGluR2 promoter (Fig. 3D–F), some HA/EGFP immunoreactivity was colocalized with ChAT (Fig. 3Fii, yellow), suggesting that gene expression was efficiently targeted to the SAC somata. The HA/EGFP immunoreactivity appeared in fibers originating from relatively small cells (∼5 µm), similar to SACs (Fig. 3Dii). This pattern is consistent with the expression pattern of endogenous Syt I in the IPL (Fig. 1), justifying that the mGluR2 promoter can target gene expression to SACs and the IPL.


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)

The mGluR2 promoter drives gene expression selectively in SACs.Whole-mount retinas from P0–P2 rats were double-labeled with HA (green) and ChAT (red) after 72 hr of transfection. Ai–Ci. Whole-mount retinas transfected with pCMV-HA-Syt I-IRES2-EGFP. Ai. Labeled with HA. Bi. Labeled with ChAT. Ci. Merged images of Ai and Bi. Scale bar, 50 µm. EGFP, enhanced green fluorescent protein. Aii–Cii. High magnification view of whole-mount retinas transfected with pCMV-HA-Syt I-IRES2-EGFP. Aii. Labeled with HA. Bii. Labeled with ChAT. Cii. Merged images of Aii and Bii. Scale bar, 25 µm. Di–Fi. Whole-mount retinas transfected with pmGluR2-HA-Syt I-IRES2-EGFP. Di. Labeled with HA. Ei. Labeled with ChAT. Fi. Merged images of Di and Ei. Scale bar, 50 µm. Dii-Fii. High magnification view of whole-mount retinas transfected with pmGluR2-HA-Syt I-IRES2-EGFP. Dii. Labeled with HA. Eii. Labeled with ChAT. Fii. Merged images of Dii and Eii. Scale bar, 25 µm. G–H. Retinal cross-sections transfected with either pCMV-HA-Syt I-IRES2-EGFP (G), or pmGluR2-HA-Syt I-IRES2-EGFP (H). The SACs and IPL were stained for ChAT (red). The transfected cells were stained for HA/GFP (green). The cell nuclei were stained with DAPI (blue). Right, Merged images (yellow). The transfected SACs were indicated by arrows. Insets showed the higher magnification of merged images. Scale bars, 25 µm.
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getmorefigures.php?uid=PMC3472990&req=5

pone-0047465-g003: The mGluR2 promoter drives gene expression selectively in SACs.Whole-mount retinas from P0–P2 rats were double-labeled with HA (green) and ChAT (red) after 72 hr of transfection. Ai–Ci. Whole-mount retinas transfected with pCMV-HA-Syt I-IRES2-EGFP. Ai. Labeled with HA. Bi. Labeled with ChAT. Ci. Merged images of Ai and Bi. Scale bar, 50 µm. EGFP, enhanced green fluorescent protein. Aii–Cii. High magnification view of whole-mount retinas transfected with pCMV-HA-Syt I-IRES2-EGFP. Aii. Labeled with HA. Bii. Labeled with ChAT. Cii. Merged images of Aii and Bii. Scale bar, 25 µm. Di–Fi. Whole-mount retinas transfected with pmGluR2-HA-Syt I-IRES2-EGFP. Di. Labeled with HA. Ei. Labeled with ChAT. Fi. Merged images of Di and Ei. Scale bar, 50 µm. Dii-Fii. High magnification view of whole-mount retinas transfected with pmGluR2-HA-Syt I-IRES2-EGFP. Dii. Labeled with HA. Eii. Labeled with ChAT. Fii. Merged images of Dii and Eii. Scale bar, 25 µm. G–H. Retinal cross-sections transfected with either pCMV-HA-Syt I-IRES2-EGFP (G), or pmGluR2-HA-Syt I-IRES2-EGFP (H). The SACs and IPL were stained for ChAT (red). The transfected cells were stained for HA/GFP (green). The cell nuclei were stained with DAPI (blue). Right, Merged images (yellow). The transfected SACs were indicated by arrows. Insets showed the higher magnification of merged images. Scale bars, 25 µm.
Mentions: To manipulate neurotransmitter release from SACs during stage-II waves, we aimed to transfect SACs with Syt I mutants with the weakened Ca2+ binding in the C2 domains. Since the metabotropic glutamate receptor type II (mGluR2) promoter has been shown to target SACs specifically [7], [35], [36], [37], we placed our Syt constructs under the control of the mGluR2 promoter to manipulate Syt I specifically in SACs. To verify the effectiveness of our transfection strategy, we compared the specificity of the mGluR2 promoter with the ubiquitous cytomegalovirus (CMV) promoter by immnunofluorescence (Fig. 3). With the CMV promoter (Fig. 3A–C), the HA/EGFP immunoreactivity was scarcely colocalized with ChAT (the SAC marker), but mostly appeared in relatively round and large retinal neurons (∼20 µm), likely RGCs. In contrast, with the mGluR2 promoter (Fig. 3D–F), some HA/EGFP immunoreactivity was colocalized with ChAT (Fig. 3Fii, yellow), suggesting that gene expression was efficiently targeted to the SAC somata. The HA/EGFP immunoreactivity appeared in fibers originating from relatively small cells (∼5 µm), similar to SACs (Fig. 3Dii). This pattern is consistent with the expression pattern of endogenous Syt I in the IPL (Fig. 1), justifying that the mGluR2 promoter can target gene expression to SACs and the IPL.

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