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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

Selective filling of presynaptic CA3 pyramidal neurons using Electroporation.Schematic of a hippocampal slice illustrating in red the region where peptides were infused by electroporation into somata of multiple CA3 pyramidal neurons, and in pink the region where Schaffer collateral presynaptic terminals were imaged to confirm successful fill. Blunt patch electrodes were filled with 1 mM AlexaFluoro-594, and trains of 10 square current pulses (30 V/200 ms, 0.5 Hz) were applied at 50, 100, and 150 µm depth, after which the electrode was removed from the slice, shift laterally 20 µM, and the depth electroporations were repeated. One hour after electroporation, two-photon laser scanning microscopy (63x objective) was used to acquire the attached images of CA3 pyramidal neuron cell bodies (lower left) and Schaffer collateral terminals (upper left) to confirm successful presynaptic infusion.
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pone-0020500-g005: Selective filling of presynaptic CA3 pyramidal neurons using Electroporation.Schematic of a hippocampal slice illustrating in red the region where peptides were infused by electroporation into somata of multiple CA3 pyramidal neurons, and in pink the region where Schaffer collateral presynaptic terminals were imaged to confirm successful fill. Blunt patch electrodes were filled with 1 mM AlexaFluoro-594, and trains of 10 square current pulses (30 V/200 ms, 0.5 Hz) were applied at 50, 100, and 150 µm depth, after which the electrode was removed from the slice, shift laterally 20 µM, and the depth electroporations were repeated. One hour after electroporation, two-photon laser scanning microscopy (63x objective) was used to acquire the attached images of CA3 pyramidal neuron cell bodies (lower left) and Schaffer collateral terminals (upper left) to confirm successful presynaptic infusion.

Mentions: To directly test the potential role of Gβγ in presynaptic LTD, we used the multiple electroporation method of Haas et al. [46] to selectively load many presynaptic CA3 pyramidal neurons (Fig. 5) with the 14 amino acid cleaved product of the C-terminus of SNAP-25 (rat Ct-SNAP-25, residues 193–206, DEANQRATKMLGSG) previously show to be a potent scavenger of free Gβγ [29]. Alexa-Fluoro-594 (1 mM) was included in the patch pipette along with the Ct-SNAP-25 peptide (1 mM) and injected into multiple regions (depicted in red, Fig. 5) of field CA3 in the hippocampal slice using multiple trains of square current pulses [46]. Successful presynaptic infusion of the peptide was verified using two photon imaging of field CA1 (Fig. 5, pink region), one hour after electroporation. During the two hours allowed for presynaptic infusion prior to attempting to induce LTD, the time course of evoked fEPSPs confirmed that Ct-SNAP-25 infusion into CA3 pyramidal neuron terminals had no long-term effects alone on evoked synaptic potentials (data not shown).


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)

Selective filling of presynaptic CA3 pyramidal neurons using Electroporation.Schematic of a hippocampal slice illustrating in red the region where peptides were infused by electroporation into somata of multiple CA3 pyramidal neurons, and in pink the region where Schaffer collateral presynaptic terminals were imaged to confirm successful fill. Blunt patch electrodes were filled with 1 mM AlexaFluoro-594, and trains of 10 square current pulses (30 V/200 ms, 0.5 Hz) were applied at 50, 100, and 150 µm depth, after which the electrode was removed from the slice, shift laterally 20 µM, and the depth electroporations were repeated. One hour after electroporation, two-photon laser scanning microscopy (63x objective) was used to acquire the attached images of CA3 pyramidal neuron cell bodies (lower left) and Schaffer collateral terminals (upper left) to confirm successful presynaptic infusion.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3102109&req=5

pone-0020500-g005: Selective filling of presynaptic CA3 pyramidal neurons using Electroporation.Schematic of a hippocampal slice illustrating in red the region where peptides were infused by electroporation into somata of multiple CA3 pyramidal neurons, and in pink the region where Schaffer collateral presynaptic terminals were imaged to confirm successful fill. Blunt patch electrodes were filled with 1 mM AlexaFluoro-594, and trains of 10 square current pulses (30 V/200 ms, 0.5 Hz) were applied at 50, 100, and 150 µm depth, after which the electrode was removed from the slice, shift laterally 20 µM, and the depth electroporations were repeated. One hour after electroporation, two-photon laser scanning microscopy (63x objective) was used to acquire the attached images of CA3 pyramidal neuron cell bodies (lower left) and Schaffer collateral terminals (upper left) to confirm successful presynaptic infusion.
Mentions: To directly test the potential role of Gβγ in presynaptic LTD, we used the multiple electroporation method of Haas et al. [46] to selectively load many presynaptic CA3 pyramidal neurons (Fig. 5) with the 14 amino acid cleaved product of the C-terminus of SNAP-25 (rat Ct-SNAP-25, residues 193–206, DEANQRATKMLGSG) previously show to be a potent scavenger of free Gβγ [29]. Alexa-Fluoro-594 (1 mM) was included in the patch pipette along with the Ct-SNAP-25 peptide (1 mM) and injected into multiple regions (depicted in red, Fig. 5) of field CA3 in the hippocampal slice using multiple trains of square current pulses [46]. Successful presynaptic infusion of the peptide was verified using two photon imaging of field CA1 (Fig. 5, pink region), one hour after electroporation. During the two hours allowed for presynaptic infusion prior to attempting to induce LTD, the time course of evoked fEPSPs confirmed that Ct-SNAP-25 infusion into CA3 pyramidal neuron terminals had no long-term effects alone on evoked synaptic potentials (data not shown).

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