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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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Related in: MedlinePlus

Gene transfer into whole-mount retinas by the homemade electroporation device.A–B. Preparation of platinum (Pt) electrodes. Ai. The arrangement of the six slides for the (+) electrode base. The colors of slides represent the slide arrangement in different layers. The numbers in the circles indicate the corresponding sequence to arrange slides. Aii. The Pt foil (15×15 mm with one extra 5×5 mm-overhanging square) was aligned to one side of the 7th slide with the overhanging square left out. Epoxy glue was applied to attach the Pt foil onto the slide and connect all the slides together. Aiii. The wire was soldered to the edge of the overhanging Pt square. Aiv. The last (8th) slide was glued onto the (+) electrode base. Bi–ii. The arrangement of the (−) electrode. A pen tube (green) with a diameter of 10 mm was used to attach the same-sized Pt foil by epoxy glue. The electric wire was inserted through the pen tube and soldered onto the Pt foil. C. The setup for electroporation in a horizontal configuration. The retinal explant was placed in a well [with dimensions 12 (length) ×12 (width) ×3 (height) mm] on the (+) electrode. The (−) electrode made contact with solution above the well and covered the retinal explant. The distance between the (+) and (−) electrodes was adjusted by a micromanipulator that held the (−) electrode. D. The P1 rat retinas were transfected with pCMV-HA (vector) or pCMV-HA-Syt I (HA-Syt I) with this electroporation device. The retinal explants were incubated for 72 hr to allow gene expression. Cellular proteins were solubilized and subjected to SDS-PAGE and Western blot analysis with antibodies indicated on the right (HA or α-tubulin). Only the retinas transfected with HA-Syt I displayed the HA signal with a size of ∼65 kD, which corresponded to the molecular weight of Syt I. Data shown were representative blots from 3 different experiments.
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pone-0047465-g002: Gene transfer into whole-mount retinas by the homemade electroporation device.A–B. Preparation of platinum (Pt) electrodes. Ai. The arrangement of the six slides for the (+) electrode base. The colors of slides represent the slide arrangement in different layers. The numbers in the circles indicate the corresponding sequence to arrange slides. Aii. The Pt foil (15×15 mm with one extra 5×5 mm-overhanging square) was aligned to one side of the 7th slide with the overhanging square left out. Epoxy glue was applied to attach the Pt foil onto the slide and connect all the slides together. Aiii. The wire was soldered to the edge of the overhanging Pt square. Aiv. The last (8th) slide was glued onto the (+) electrode base. Bi–ii. The arrangement of the (−) electrode. A pen tube (green) with a diameter of 10 mm was used to attach the same-sized Pt foil by epoxy glue. The electric wire was inserted through the pen tube and soldered onto the Pt foil. C. The setup for electroporation in a horizontal configuration. The retinal explant was placed in a well [with dimensions 12 (length) ×12 (width) ×3 (height) mm] on the (+) electrode. The (−) electrode made contact with solution above the well and covered the retinal explant. The distance between the (+) and (−) electrodes was adjusted by a micromanipulator that held the (−) electrode. D. The P1 rat retinas were transfected with pCMV-HA (vector) or pCMV-HA-Syt I (HA-Syt I) with this electroporation device. The retinal explants were incubated for 72 hr to allow gene expression. Cellular proteins were solubilized and subjected to SDS-PAGE and Western blot analysis with antibodies indicated on the right (HA or α-tubulin). Only the retinas transfected with HA-Syt I displayed the HA signal with a size of ∼65 kD, which corresponded to the molecular weight of Syt I. Data shown were representative blots from 3 different experiments.

Mentions: We constructed platinum electrodes to electroporate genes of interest into P0–P2 whole-mount retinas (Fig. 2A–B). This design achieved gene delivery within two parallel electrodes (Fig. 2C). Western blot analysis verified ectopic expression of HA-Syt I in the developing retina by this electroporation strategy (Fig. 2D). This method achieved homogenous expression across the entire retina (Fig. 3ACDF) without altering the major characteristics of retinal waves, such as the frequency, duration, and amplitude of wave-associated Ca2+ transients (Table 1). In addition, within 4 days after transfection, retinal waves were reliably blocked by the nAChR antagonist, dihydro-β-erythroidine (DHβE) (10–20 µM) [33], [34]. These results suggest that transfected retinas, which we cultured in vitro for 3–4 days after transfection, still generate stage-II waves mediated by cholinergic transmission with the same essential properties. Moreover, no significant differences in wave-associated Ca2+ transients were observed in the retinas transfected either on P0 or P1/2 (Table 1). Therefore, in the subsequent Ca2+ imaging experiments, we utilized P0–P2 transfected retinas with 3–4 day in vitro culture for studying the mechanisms mediating 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)

Gene transfer into whole-mount retinas by the homemade electroporation device.A–B. Preparation of platinum (Pt) electrodes. Ai. The arrangement of the six slides for the (+) electrode base. The colors of slides represent the slide arrangement in different layers. The numbers in the circles indicate the corresponding sequence to arrange slides. Aii. The Pt foil (15×15 mm with one extra 5×5 mm-overhanging square) was aligned to one side of the 7th slide with the overhanging square left out. Epoxy glue was applied to attach the Pt foil onto the slide and connect all the slides together. Aiii. The wire was soldered to the edge of the overhanging Pt square. Aiv. The last (8th) slide was glued onto the (+) electrode base. Bi–ii. The arrangement of the (−) electrode. A pen tube (green) with a diameter of 10 mm was used to attach the same-sized Pt foil by epoxy glue. The electric wire was inserted through the pen tube and soldered onto the Pt foil. C. The setup for electroporation in a horizontal configuration. The retinal explant was placed in a well [with dimensions 12 (length) ×12 (width) ×3 (height) mm] on the (+) electrode. The (−) electrode made contact with solution above the well and covered the retinal explant. The distance between the (+) and (−) electrodes was adjusted by a micromanipulator that held the (−) electrode. D. The P1 rat retinas were transfected with pCMV-HA (vector) or pCMV-HA-Syt I (HA-Syt I) with this electroporation device. The retinal explants were incubated for 72 hr to allow gene expression. Cellular proteins were solubilized and subjected to SDS-PAGE and Western blot analysis with antibodies indicated on the right (HA or α-tubulin). Only the retinas transfected with HA-Syt I displayed the HA signal with a size of ∼65 kD, which corresponded to the molecular weight of Syt I. Data shown were representative blots from 3 different experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0047465-g002: Gene transfer into whole-mount retinas by the homemade electroporation device.A–B. Preparation of platinum (Pt) electrodes. Ai. The arrangement of the six slides for the (+) electrode base. The colors of slides represent the slide arrangement in different layers. The numbers in the circles indicate the corresponding sequence to arrange slides. Aii. The Pt foil (15×15 mm with one extra 5×5 mm-overhanging square) was aligned to one side of the 7th slide with the overhanging square left out. Epoxy glue was applied to attach the Pt foil onto the slide and connect all the slides together. Aiii. The wire was soldered to the edge of the overhanging Pt square. Aiv. The last (8th) slide was glued onto the (+) electrode base. Bi–ii. The arrangement of the (−) electrode. A pen tube (green) with a diameter of 10 mm was used to attach the same-sized Pt foil by epoxy glue. The electric wire was inserted through the pen tube and soldered onto the Pt foil. C. The setup for electroporation in a horizontal configuration. The retinal explant was placed in a well [with dimensions 12 (length) ×12 (width) ×3 (height) mm] on the (+) electrode. The (−) electrode made contact with solution above the well and covered the retinal explant. The distance between the (+) and (−) electrodes was adjusted by a micromanipulator that held the (−) electrode. D. The P1 rat retinas were transfected with pCMV-HA (vector) or pCMV-HA-Syt I (HA-Syt I) with this electroporation device. The retinal explants were incubated for 72 hr to allow gene expression. Cellular proteins were solubilized and subjected to SDS-PAGE and Western blot analysis with antibodies indicated on the right (HA or α-tubulin). Only the retinas transfected with HA-Syt I displayed the HA signal with a size of ∼65 kD, which corresponded to the molecular weight of Syt I. Data shown were representative blots from 3 different experiments.
Mentions: We constructed platinum electrodes to electroporate genes of interest into P0–P2 whole-mount retinas (Fig. 2A–B). This design achieved gene delivery within two parallel electrodes (Fig. 2C). Western blot analysis verified ectopic expression of HA-Syt I in the developing retina by this electroporation strategy (Fig. 2D). This method achieved homogenous expression across the entire retina (Fig. 3ACDF) without altering the major characteristics of retinal waves, such as the frequency, duration, and amplitude of wave-associated Ca2+ transients (Table 1). In addition, within 4 days after transfection, retinal waves were reliably blocked by the nAChR antagonist, dihydro-β-erythroidine (DHβE) (10–20 µM) [33], [34]. These results suggest that transfected retinas, which we cultured in vitro for 3–4 days after transfection, still generate stage-II waves mediated by cholinergic transmission with the same essential properties. Moreover, no significant differences in wave-associated Ca2+ transients were observed in the retinas transfected either on P0 or P1/2 (Table 1). Therefore, in the subsequent Ca2+ imaging experiments, we utilized P0–P2 transfected retinas with 3–4 day in vitro culture for studying the mechanisms mediating 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