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Gβγ and the C terminus of SNAP-25 are necessary for long-term depression of transmitter release.

Zhang XL, Upreti C, Stanton PK - PLoS ONE (2011)

Bottom Line: Recent studies implicate the C terminus of the vesicle-associated protein SNAP-25 as a molecular binding target of Gβγ that transiently reduces vesicular release.Presynaptic electroporation infusion of the 14-amino acid C-terminus of SNAP-25 (Ct-SNAP-25), to scavenge Gβγ, reduced both the transient presynaptic inhibition produced by the group II metabotropic glutamate receptor stimulation, and LTD.While Gβγ binds directly to and inhibit voltage-dependent Ca(2+) channels, imaging of presynaptic [Ca(2+)] with Mg-Green revealed that low-frequency stimulation only transiently reduced presynaptic Ca(2+) influx, an effect not altered by infusion of Ct-SNAP-25.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Anatomy, New York Medical College, Valhalla, New York, United States of America.

ABSTRACT

Background: Short-term presynaptic inhibition mediated by G protein-coupled receptors involves a direct interaction between G proteins and the vesicle release machinery. Recent studies implicate the C terminus of the vesicle-associated protein SNAP-25 as a molecular binding target of Gβγ that transiently reduces vesicular release. However, it is not known whether SNAP-25 is a target for molecular modifications expressing long-term changes in transmitter release probability.

Methodology/principal findings: This study utilized two-photon laser scanning microscopy for real-time imaging of action potential-evoked [Ca(2+)] increases, in single Schaffer collateral presynaptic release sites in in vitro hippocampal slices, plus simultaneous recording of Schaffer collateral-evoked synaptic potentials. We used electroporation to infuse small peptides through CA3 cell bodies into presynaptic Schaffer collateral terminals to selectively study the presynaptic effect of scavenging the G-protein Gβγ. We demonstrate here that the C terminus of SNAP-25 is necessary for expression of LTD, but not long-term potentiation (LTP), of synaptic strength. Using type A botulinum toxin (BoNT/A) to enzymatically cleave the 9 amino acid C-terminus of SNAP-25 eliminated the ability of low frequency synaptic stimulation to induce LTD, but not LTP, even if release probability was restored to pre-BoNT/A levels by elevating extracellular [Ca(2+)]. Presynaptic electroporation infusion of the 14-amino acid C-terminus of SNAP-25 (Ct-SNAP-25), to scavenge Gβγ, reduced both the transient presynaptic inhibition produced by the group II metabotropic glutamate receptor stimulation, and LTD. Furthermore, presynaptic infusion of mSIRK, a second, structurally distinct Gβγ scavenging peptide, also blocked the induction of LTD. While Gβγ binds directly to and inhibit voltage-dependent Ca(2+) channels, imaging of presynaptic [Ca(2+)] with Mg-Green revealed that low-frequency stimulation only transiently reduced presynaptic Ca(2+) influx, an effect not altered by infusion of Ct-SNAP-25.

Conclusions/significance: The C-terminus of SNAP-25, which links synaptotagmin I to the SNARE complex, is a binding target for Gβγ necessary for both transient transmitter-mediated presynaptic inhibition, and the induction of presynaptic LTD.

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Related in: MedlinePlus

Botulinum Toxin type A pretreatment markedly reduces induction of LTD, but not LTP, at Schaffer collateral-CA1 synapses.A: Time course of reduction in field excitatory postsynaptic potential (fEPSP) slopes recorded in CA1 stratum radiatum and induced by bath application of BotoxA (200 ng/ml, 90 min; grey bar), plotting normalized fEPSP slopes (n = 9 slices). B: Time course of the effect of low frequency Schaffer collateral stimulation (2 Hz/10 min, solid bar) on fEPSP slopes in field CA1 of slices (n = 6) pretreated for 90 min with BotoxA (200 ng/ml), which eliminated LTD (P<0.01; Student’s t-test compared to untreated slice LTD). C: Time course of LTP elicited in field CA1 by four trains of theta burst Schaffer collateral stimulation (arrow; each train 10 bursts of 4 stimuli at 100 Hz frequency, 200 ms interburst interval) in slices (n = 6) pretreated for 90 min with BotoxA (200 ng/ml). D: Time course of the magnitude of depotentiation, where BotoxA (200 ng/ml, grey bar) was bath applied for 90 min prior to induction of LTP by high-frequency theta burst stimulation (arrow; 4 trains of 10 4×100 Hz bursts, 200 ms interburst interval). After LTP was established for 30 min, low frequency Schaffer collateral stimulation (2 Hz/10 min, solid bar) elicited significant depotentation reversal of LTP (n = 6). Each point mean ± SEM of n slices. Representative fEPSP waveforms insets recorded at times indicated by numbers on traces and time course.
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pone-0020500-g001: Botulinum Toxin type A pretreatment markedly reduces induction of LTD, but not LTP, at Schaffer collateral-CA1 synapses.A: Time course of reduction in field excitatory postsynaptic potential (fEPSP) slopes recorded in CA1 stratum radiatum and induced by bath application of BotoxA (200 ng/ml, 90 min; grey bar), plotting normalized fEPSP slopes (n = 9 slices). B: Time course of the effect of low frequency Schaffer collateral stimulation (2 Hz/10 min, solid bar) on fEPSP slopes in field CA1 of slices (n = 6) pretreated for 90 min with BotoxA (200 ng/ml), which eliminated LTD (P<0.01; Student’s t-test compared to untreated slice LTD). C: Time course of LTP elicited in field CA1 by four trains of theta burst Schaffer collateral stimulation (arrow; each train 10 bursts of 4 stimuli at 100 Hz frequency, 200 ms interburst interval) in slices (n = 6) pretreated for 90 min with BotoxA (200 ng/ml). D: Time course of the magnitude of depotentiation, where BotoxA (200 ng/ml, grey bar) was bath applied for 90 min prior to induction of LTP by high-frequency theta burst stimulation (arrow; 4 trains of 10 4×100 Hz bursts, 200 ms interburst interval). After LTP was established for 30 min, low frequency Schaffer collateral stimulation (2 Hz/10 min, solid bar) elicited significant depotentation reversal of LTP (n = 6). Each point mean ± SEM of n slices. Representative fEPSP waveforms insets recorded at times indicated by numbers on traces and time course.

Mentions: Botulinum toxin type A (BoNT/A) partially suppresses vesicular transmitter release [29], [32] by cleaving 9 amino acids from the C-terminus of SNAP-25 [33], [34], a t-SNARE protein that modulates vesicular release at excitatory synapses. To determine whether the C-terminus of SNAP-25 might contain a regulatory site necessary for long-term plasticity of transmitter release, we applied BoNT/A (200 ng/ml) to hippocampal slices, which produced a slow reduction in evoked fEPSP slope and amplitude which took ∼1 hr to plateau at ∼50% of baseline fEPSP values (Fig. 1A). To test the importance of the C-terminus of SNAP25 to LTD, we preincubated hippocampal slices for 90 minutes with BoNT/A (200 ng/ml) and then applied a 2 Hz/10 minute low frequency Schaffer collateral stimulus (LFS) train previously shown to induce robust LTD [35], [36]. Pretreatment with BoNT/A completely inhibited the induction of stimulus evoked LTD (Fig. 1B; P<0.05, Student's t-test compared to control LTD 30 min post-LFS), while not altering transient LFS-evoked depression in these slices. The occlusion of LTD by BoNT/A pretreatment indicates that the C-terminus of SNAP-25 containing residues 198–206 is critical for the induction of stimulus evoked LTD in acute hippocampal slices.


Gβγ and the C terminus of SNAP-25 are necessary for long-term depression of transmitter release.

Zhang XL, Upreti C, Stanton PK - PLoS ONE (2011)

Botulinum Toxin type A pretreatment markedly reduces induction of LTD, but not LTP, at Schaffer collateral-CA1 synapses.A: Time course of reduction in field excitatory postsynaptic potential (fEPSP) slopes recorded in CA1 stratum radiatum and induced by bath application of BotoxA (200 ng/ml, 90 min; grey bar), plotting normalized fEPSP slopes (n = 9 slices). B: Time course of the effect of low frequency Schaffer collateral stimulation (2 Hz/10 min, solid bar) on fEPSP slopes in field CA1 of slices (n = 6) pretreated for 90 min with BotoxA (200 ng/ml), which eliminated LTD (P<0.01; Student’s t-test compared to untreated slice LTD). C: Time course of LTP elicited in field CA1 by four trains of theta burst Schaffer collateral stimulation (arrow; each train 10 bursts of 4 stimuli at 100 Hz frequency, 200 ms interburst interval) in slices (n = 6) pretreated for 90 min with BotoxA (200 ng/ml). D: Time course of the magnitude of depotentiation, where BotoxA (200 ng/ml, grey bar) was bath applied for 90 min prior to induction of LTP by high-frequency theta burst stimulation (arrow; 4 trains of 10 4×100 Hz bursts, 200 ms interburst interval). After LTP was established for 30 min, low frequency Schaffer collateral stimulation (2 Hz/10 min, solid bar) elicited significant depotentation reversal of LTP (n = 6). Each point mean ± SEM of n slices. Representative fEPSP waveforms insets recorded at times indicated by numbers on traces and time course.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3102109&req=5

pone-0020500-g001: Botulinum Toxin type A pretreatment markedly reduces induction of LTD, but not LTP, at Schaffer collateral-CA1 synapses.A: Time course of reduction in field excitatory postsynaptic potential (fEPSP) slopes recorded in CA1 stratum radiatum and induced by bath application of BotoxA (200 ng/ml, 90 min; grey bar), plotting normalized fEPSP slopes (n = 9 slices). B: Time course of the effect of low frequency Schaffer collateral stimulation (2 Hz/10 min, solid bar) on fEPSP slopes in field CA1 of slices (n = 6) pretreated for 90 min with BotoxA (200 ng/ml), which eliminated LTD (P<0.01; Student’s t-test compared to untreated slice LTD). C: Time course of LTP elicited in field CA1 by four trains of theta burst Schaffer collateral stimulation (arrow; each train 10 bursts of 4 stimuli at 100 Hz frequency, 200 ms interburst interval) in slices (n = 6) pretreated for 90 min with BotoxA (200 ng/ml). D: Time course of the magnitude of depotentiation, where BotoxA (200 ng/ml, grey bar) was bath applied for 90 min prior to induction of LTP by high-frequency theta burst stimulation (arrow; 4 trains of 10 4×100 Hz bursts, 200 ms interburst interval). After LTP was established for 30 min, low frequency Schaffer collateral stimulation (2 Hz/10 min, solid bar) elicited significant depotentation reversal of LTP (n = 6). Each point mean ± SEM of n slices. Representative fEPSP waveforms insets recorded at times indicated by numbers on traces and time course.
Mentions: Botulinum toxin type A (BoNT/A) partially suppresses vesicular transmitter release [29], [32] by cleaving 9 amino acids from the C-terminus of SNAP-25 [33], [34], a t-SNARE protein that modulates vesicular release at excitatory synapses. To determine whether the C-terminus of SNAP-25 might contain a regulatory site necessary for long-term plasticity of transmitter release, we applied BoNT/A (200 ng/ml) to hippocampal slices, which produced a slow reduction in evoked fEPSP slope and amplitude which took ∼1 hr to plateau at ∼50% of baseline fEPSP values (Fig. 1A). To test the importance of the C-terminus of SNAP25 to LTD, we preincubated hippocampal slices for 90 minutes with BoNT/A (200 ng/ml) and then applied a 2 Hz/10 minute low frequency Schaffer collateral stimulus (LFS) train previously shown to induce robust LTD [35], [36]. Pretreatment with BoNT/A completely inhibited the induction of stimulus evoked LTD (Fig. 1B; P<0.05, Student's t-test compared to control LTD 30 min post-LFS), while not altering transient LFS-evoked depression in these slices. The occlusion of LTD by BoNT/A pretreatment indicates that the C-terminus of SNAP-25 containing residues 198–206 is critical for the induction of stimulus evoked LTD in acute hippocampal slices.

Bottom Line: Recent studies implicate the C terminus of the vesicle-associated protein SNAP-25 as a molecular binding target of Gβγ that transiently reduces vesicular release.Presynaptic electroporation infusion of the 14-amino acid C-terminus of SNAP-25 (Ct-SNAP-25), to scavenge Gβγ, reduced both the transient presynaptic inhibition produced by the group II metabotropic glutamate receptor stimulation, and LTD.While Gβγ binds directly to and inhibit voltage-dependent Ca(2+) channels, imaging of presynaptic [Ca(2+)] with Mg-Green revealed that low-frequency stimulation only transiently reduced presynaptic Ca(2+) influx, an effect not altered by infusion of Ct-SNAP-25.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Anatomy, New York Medical College, Valhalla, New York, United States of America.

ABSTRACT

Background: Short-term presynaptic inhibition mediated by G protein-coupled receptors involves a direct interaction between G proteins and the vesicle release machinery. Recent studies implicate the C terminus of the vesicle-associated protein SNAP-25 as a molecular binding target of Gβγ that transiently reduces vesicular release. However, it is not known whether SNAP-25 is a target for molecular modifications expressing long-term changes in transmitter release probability.

Methodology/principal findings: This study utilized two-photon laser scanning microscopy for real-time imaging of action potential-evoked [Ca(2+)] increases, in single Schaffer collateral presynaptic release sites in in vitro hippocampal slices, plus simultaneous recording of Schaffer collateral-evoked synaptic potentials. We used electroporation to infuse small peptides through CA3 cell bodies into presynaptic Schaffer collateral terminals to selectively study the presynaptic effect of scavenging the G-protein Gβγ. We demonstrate here that the C terminus of SNAP-25 is necessary for expression of LTD, but not long-term potentiation (LTP), of synaptic strength. Using type A botulinum toxin (BoNT/A) to enzymatically cleave the 9 amino acid C-terminus of SNAP-25 eliminated the ability of low frequency synaptic stimulation to induce LTD, but not LTP, even if release probability was restored to pre-BoNT/A levels by elevating extracellular [Ca(2+)]. Presynaptic electroporation infusion of the 14-amino acid C-terminus of SNAP-25 (Ct-SNAP-25), to scavenge Gβγ, reduced both the transient presynaptic inhibition produced by the group II metabotropic glutamate receptor stimulation, and LTD. Furthermore, presynaptic infusion of mSIRK, a second, structurally distinct Gβγ scavenging peptide, also blocked the induction of LTD. While Gβγ binds directly to and inhibit voltage-dependent Ca(2+) channels, imaging of presynaptic [Ca(2+)] with Mg-Green revealed that low-frequency stimulation only transiently reduced presynaptic Ca(2+) influx, an effect not altered by infusion of Ct-SNAP-25.

Conclusions/significance: The C-terminus of SNAP-25, which links synaptotagmin I to the SNARE complex, is a binding target for Gβγ necessary for both transient transmitter-mediated presynaptic inhibition, and the induction of presynaptic LTD.

Show MeSH
Related in: MedlinePlus