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Boosting of synaptic potentials and spine Ca transients by the peptide toxin SNX-482 requires alpha-1E-encoded voltage-gated Ca channels.

Giessel AJ, Sabatini BL - PLoS ONE (2011)

Bottom Line: The majority of glutamatergic synapses formed onto principal neurons of the mammalian central nervous system are associated with dendritic spines.Spines are tiny protuberances that house the proteins that mediate the response of the postsynaptic cell to the presynaptic release of glutamate.In order to specifically test this conclusion, we examined the effects of SNX-482 on synaptic signals in acute hippocampal slices from knock-out mice lacking the Alpha-1E gene.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
The majority of glutamatergic synapses formed onto principal neurons of the mammalian central nervous system are associated with dendritic spines. Spines are tiny protuberances that house the proteins that mediate the response of the postsynaptic cell to the presynaptic release of glutamate. Postsynaptic signals are regulated by an ion channel signaling cascade that is active in individual dendritic spines and involves voltage-gated calcium (Ca) channels, small conductance (SK)-type Ca-activated potassium channels, and NMDA-type glutamate receptors. Pharmacological studies using the toxin SNX-482 indicated that the voltage-gated Ca channels that signal within spines to open SK channels belong to the class Ca(V)2.3, which is encoded by the Alpha-1E pore-forming subunit. In order to specifically test this conclusion, we examined the effects of SNX-482 on synaptic signals in acute hippocampal slices from knock-out mice lacking the Alpha-1E gene. We find that in these mice, application of SNX-482 has no effect on glutamate-uncaging evoked synaptic potentials and Ca influx, indicating that that SNX-482 indeed acts via the Alpha-1E-encoded Ca(V)2.3 channel.

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Uncaging-evoked synaptic responses in Alpha-1E knock-out mice are unaffected by SNX-482.(A) High-magnification image of a spiny dendrite of a CA1 hippocampal pyramidal cell formed from the red fluorescence of Alexa Fluor-594. The pyramidal neuron is in an acute slice cut from of an Alpha-1E knock-out mouse. (B) Example of fluorescence collected during a line scan, shown in the yellow line in (A), which intersects the dendrite (den) and spine head (sp) during glutamate uncaging at the spine head. The increase in green signal indicates a rise in intracellular [Ca]. The inset traces show the simultaneously recorded uEPSP (top, amplitude 1.2 mV) and the quantification of the green fluorescence in the spine head (bottom, amplitude 10% ΔG/Gsat). (C) Average uEPSPs (top) and Ca-dependent changes in green fluorescence in the spine head (bottom) evoked by uncaging of glutamate in control conditions (black) and in the presence of SNX-482 (red). Data are shown as the mean (line) ± SEM (shaded region). (D) Summary of amplitudes of uEPSPs (left) and Δ[Ca]spine (right) in control conditions (black) and in the presence of SNX-482 (red).
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pone-0020939-g001: Uncaging-evoked synaptic responses in Alpha-1E knock-out mice are unaffected by SNX-482.(A) High-magnification image of a spiny dendrite of a CA1 hippocampal pyramidal cell formed from the red fluorescence of Alexa Fluor-594. The pyramidal neuron is in an acute slice cut from of an Alpha-1E knock-out mouse. (B) Example of fluorescence collected during a line scan, shown in the yellow line in (A), which intersects the dendrite (den) and spine head (sp) during glutamate uncaging at the spine head. The increase in green signal indicates a rise in intracellular [Ca]. The inset traces show the simultaneously recorded uEPSP (top, amplitude 1.2 mV) and the quantification of the green fluorescence in the spine head (bottom, amplitude 10% ΔG/Gsat). (C) Average uEPSPs (top) and Ca-dependent changes in green fluorescence in the spine head (bottom) evoked by uncaging of glutamate in control conditions (black) and in the presence of SNX-482 (red). Data are shown as the mean (line) ± SEM (shaded region). (D) Summary of amplitudes of uEPSPs (left) and Δ[Ca]spine (right) in control conditions (black) and in the presence of SNX-482 (red).

Mentions: In order to determine if the effects of SNX-482 on uncaging-evoked potentials and Ca transients were mediated through Alpha-1E encoded VGCCs, we examined responses evoked by 2-photon laser uncaging of glutamate onto apical spines of CA1 pyramidal neurons from animals which were homozygous for the Alpha1E subunit (Figure 1) [11]. Whole-cell current-clamp recordings were obtained from neurons in acute slices of the hippocampus (post-natal day 15–18) and 2-photon laser-scanning microscopy was used to image cellular and dendritic morphology as well as to monitor intracellular Ca transients (Figure 1A–B). Neurons were filled with a green-fluorescing Ca indicator (300 µM Fluo-5F) and a Ca-insensitive red fluorophore (10 µM Alexa Fluor-594) through a whole-cell recording electrode. Individual dendritic spines were stimulated by uncaging glutamate using 500 µs pulses of 725 nm laser light directed at a spot near the spine head (see methods). This resulted in an uncaging-evoked postsynaptic potential (uEPSP) detectable at the soma and increases in green fluorescence (ΔG/Gsat, see methods) in the spine head, indicative of elevated [Ca] (Δ[Ca]spine, Figure 1B). Analysis was limited to spines with clearly defined heads that were well separated from the parent dendrite and located less than 150 µm from the soma on radial oblique dendrites in order to use identical experimental conditions as in previous studies [2], [3], [5].


Boosting of synaptic potentials and spine Ca transients by the peptide toxin SNX-482 requires alpha-1E-encoded voltage-gated Ca channels.

Giessel AJ, Sabatini BL - PLoS ONE (2011)

Uncaging-evoked synaptic responses in Alpha-1E knock-out mice are unaffected by SNX-482.(A) High-magnification image of a spiny dendrite of a CA1 hippocampal pyramidal cell formed from the red fluorescence of Alexa Fluor-594. The pyramidal neuron is in an acute slice cut from of an Alpha-1E knock-out mouse. (B) Example of fluorescence collected during a line scan, shown in the yellow line in (A), which intersects the dendrite (den) and spine head (sp) during glutamate uncaging at the spine head. The increase in green signal indicates a rise in intracellular [Ca]. The inset traces show the simultaneously recorded uEPSP (top, amplitude 1.2 mV) and the quantification of the green fluorescence in the spine head (bottom, amplitude 10% ΔG/Gsat). (C) Average uEPSPs (top) and Ca-dependent changes in green fluorescence in the spine head (bottom) evoked by uncaging of glutamate in control conditions (black) and in the presence of SNX-482 (red). Data are shown as the mean (line) ± SEM (shaded region). (D) Summary of amplitudes of uEPSPs (left) and Δ[Ca]spine (right) in control conditions (black) and in the presence of SNX-482 (red).
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Related In: Results  -  Collection

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pone-0020939-g001: Uncaging-evoked synaptic responses in Alpha-1E knock-out mice are unaffected by SNX-482.(A) High-magnification image of a spiny dendrite of a CA1 hippocampal pyramidal cell formed from the red fluorescence of Alexa Fluor-594. The pyramidal neuron is in an acute slice cut from of an Alpha-1E knock-out mouse. (B) Example of fluorescence collected during a line scan, shown in the yellow line in (A), which intersects the dendrite (den) and spine head (sp) during glutamate uncaging at the spine head. The increase in green signal indicates a rise in intracellular [Ca]. The inset traces show the simultaneously recorded uEPSP (top, amplitude 1.2 mV) and the quantification of the green fluorescence in the spine head (bottom, amplitude 10% ΔG/Gsat). (C) Average uEPSPs (top) and Ca-dependent changes in green fluorescence in the spine head (bottom) evoked by uncaging of glutamate in control conditions (black) and in the presence of SNX-482 (red). Data are shown as the mean (line) ± SEM (shaded region). (D) Summary of amplitudes of uEPSPs (left) and Δ[Ca]spine (right) in control conditions (black) and in the presence of SNX-482 (red).
Mentions: In order to determine if the effects of SNX-482 on uncaging-evoked potentials and Ca transients were mediated through Alpha-1E encoded VGCCs, we examined responses evoked by 2-photon laser uncaging of glutamate onto apical spines of CA1 pyramidal neurons from animals which were homozygous for the Alpha1E subunit (Figure 1) [11]. Whole-cell current-clamp recordings were obtained from neurons in acute slices of the hippocampus (post-natal day 15–18) and 2-photon laser-scanning microscopy was used to image cellular and dendritic morphology as well as to monitor intracellular Ca transients (Figure 1A–B). Neurons were filled with a green-fluorescing Ca indicator (300 µM Fluo-5F) and a Ca-insensitive red fluorophore (10 µM Alexa Fluor-594) through a whole-cell recording electrode. Individual dendritic spines were stimulated by uncaging glutamate using 500 µs pulses of 725 nm laser light directed at a spot near the spine head (see methods). This resulted in an uncaging-evoked postsynaptic potential (uEPSP) detectable at the soma and increases in green fluorescence (ΔG/Gsat, see methods) in the spine head, indicative of elevated [Ca] (Δ[Ca]spine, Figure 1B). Analysis was limited to spines with clearly defined heads that were well separated from the parent dendrite and located less than 150 µm from the soma on radial oblique dendrites in order to use identical experimental conditions as in previous studies [2], [3], [5].

Bottom Line: The majority of glutamatergic synapses formed onto principal neurons of the mammalian central nervous system are associated with dendritic spines.Spines are tiny protuberances that house the proteins that mediate the response of the postsynaptic cell to the presynaptic release of glutamate.In order to specifically test this conclusion, we examined the effects of SNX-482 on synaptic signals in acute hippocampal slices from knock-out mice lacking the Alpha-1E gene.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
The majority of glutamatergic synapses formed onto principal neurons of the mammalian central nervous system are associated with dendritic spines. Spines are tiny protuberances that house the proteins that mediate the response of the postsynaptic cell to the presynaptic release of glutamate. Postsynaptic signals are regulated by an ion channel signaling cascade that is active in individual dendritic spines and involves voltage-gated calcium (Ca) channels, small conductance (SK)-type Ca-activated potassium channels, and NMDA-type glutamate receptors. Pharmacological studies using the toxin SNX-482 indicated that the voltage-gated Ca channels that signal within spines to open SK channels belong to the class Ca(V)2.3, which is encoded by the Alpha-1E pore-forming subunit. In order to specifically test this conclusion, we examined the effects of SNX-482 on synaptic signals in acute hippocampal slices from knock-out mice lacking the Alpha-1E gene. We find that in these mice, application of SNX-482 has no effect on glutamate-uncaging evoked synaptic potentials and Ca influx, indicating that that SNX-482 indeed acts via the Alpha-1E-encoded Ca(V)2.3 channel.

Show MeSH
Related in: MedlinePlus