Structured Review

AnaSpec egta am
Decay kinetics of <t>ZnT3</t> KO mice are different from their WT littermates. A1 , Averaged sweeps of MF EPSCs evoked at various frequencies in control ACSF. Responses at 0.2 Hz (blue traces) and 1 Hz (red traces) were scaled to 0.05 Hz baseline value (black traces). A2 , Decay kinetics of evoked EPSCs recorded from WT ( n = 7) and KO ( n = 9) mice could be fitted with a single or double exponentials. A3 , <t>EGTA-AM</t> changes the decay kinetics by decreasing the occurrence of the slower component for both genotypes ( n = 7). B , Average values of the decay kinetics for WT and KO in control condition and after application of EGTA-AM. CTRL, Control.
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Images

1) Product Images from "Vesicular Zinc Regulates the Ca2+ Sensitivity of a Subpopulation of Presynaptic Vesicles at Hippocampal Mossy Fiber Terminals"

Article Title: Vesicular Zinc Regulates the Ca2+ Sensitivity of a Subpopulation of Presynaptic Vesicles at Hippocampal Mossy Fiber Terminals

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.4164-11.2011

Decay kinetics of ZnT3 KO mice are different from their WT littermates. A1 , Averaged sweeps of MF EPSCs evoked at various frequencies in control ACSF. Responses at 0.2 Hz (blue traces) and 1 Hz (red traces) were scaled to 0.05 Hz baseline value (black traces). A2 , Decay kinetics of evoked EPSCs recorded from WT ( n = 7) and KO ( n = 9) mice could be fitted with a single or double exponentials. A3 , EGTA-AM changes the decay kinetics by decreasing the occurrence of the slower component for both genotypes ( n = 7). B , Average values of the decay kinetics for WT and KO in control condition and after application of EGTA-AM. CTRL, Control.
Figure Legend Snippet: Decay kinetics of ZnT3 KO mice are different from their WT littermates. A1 , Averaged sweeps of MF EPSCs evoked at various frequencies in control ACSF. Responses at 0.2 Hz (blue traces) and 1 Hz (red traces) were scaled to 0.05 Hz baseline value (black traces). A2 , Decay kinetics of evoked EPSCs recorded from WT ( n = 7) and KO ( n = 9) mice could be fitted with a single or double exponentials. A3 , EGTA-AM changes the decay kinetics by decreasing the occurrence of the slower component for both genotypes ( n = 7). B , Average values of the decay kinetics for WT and KO in control condition and after application of EGTA-AM. CTRL, Control.

Techniques Used: Mouse Assay

2) Product Images from "The distinct roles of calcium in rapid control of neuronal glycolysis and the tricarboxylic acid cycle"

Article Title: The distinct roles of calcium in rapid control of neuronal glycolysis and the tricarboxylic acid cycle

Journal: eLife

doi: 10.7554/eLife.64821

Spontaneous oscillations in the Peredox signal may occur during the prolonged application of zero Ca 2+ -ACSF, in the presence of EGTA and LDH inhibition. Prolonged exposure to a nominal Ca 2+ -free solution, plus LDH inhibition, caused spontaneous elevations of the Peredox lifetime in 25 ± 13% of the cells in each slice (slices = 10, mice = 5), contrasting with only ~3% of cells exposed to a brief exposure as in Figure 4c . The spontaneous elevations in Peredox lifetime occurred at any time after removing Ca 2+ from the ACSF. Although these neurons were not included in the analysis, once the spontaneous transients cleared, the experiment resumed as usual. The continuous presence of 1 mM EGTA and 2 µM GSK-2837808 (LDH inhibitor) is not indicated in the figure for simplicity. In the control ACSF, the [Ca 2+ ] was adjusted accordingly to yield a free concentration of 2 mM.
Figure Legend Snippet: Spontaneous oscillations in the Peredox signal may occur during the prolonged application of zero Ca 2+ -ACSF, in the presence of EGTA and LDH inhibition. Prolonged exposure to a nominal Ca 2+ -free solution, plus LDH inhibition, caused spontaneous elevations of the Peredox lifetime in 25 ± 13% of the cells in each slice (slices = 10, mice = 5), contrasting with only ~3% of cells exposed to a brief exposure as in Figure 4c . The spontaneous elevations in Peredox lifetime occurred at any time after removing Ca 2+ from the ACSF. Although these neurons were not included in the analysis, once the spontaneous transients cleared, the experiment resumed as usual. The continuous presence of 1 mM EGTA and 2 µM GSK-2837808 (LDH inhibitor) is not indicated in the figure for simplicity. In the control ACSF, the [Ca 2+ ] was adjusted accordingly to yield a free concentration of 2 mM.

Techniques Used: Inhibition, Mouse Assay, Concentration Assay

A major Ca 2+ -dependent component of the NADH CYT also occurs under LDH inhibition. ( a ) Comparison of the ΔPeredox/ΔRCaMP values obtained in the continuous presence of the LDH inhibitor GSK-2837808A, before and after the blockade of voltage gated Ca 2+ channels with a combination of isradipine and cadmium (Isra+Cd 2+ ; N neurons = 13, N slices = 3 and N mice = 3). The slices were exposed to GSK-2837808A for at least 30 min prior to the experiment. LDH inhibition should improve the ability to detect the cytosol-only component of Ca 2+ actions on the NADH CYT transients by increasing the control responses to stimulation, as well as by preventing the potential impact of pyruvate accumulation on these transients due to lower Ca 2+ influx into the mitochondria. The effects of the manipulation on the Peredox baseline and the RCaMP spike are also included for the experiments with or without the LDH inhibitor (the sample size is reported in Figure 4 for the latter). ( b–c ) Comparisons for the application of the cell-permeable Ca 2+ chelator EGTA-AM (N neurons = 15, N slices = 3 and N mice = 3 for experiments with LDHi, sample sizes for the other group as in Figure 4 ), or the removal of Ca 2+ from the ACSF (N neurons = 136, N slices = 22 and N mice = 13 for experiments with LDHi, sample sizes for the other group as in Figure 4 ). For all panels, only neurons showing an initial ΔPeredox lifetime response ≥0.05 ns were included for analysis. A paired Student’s t test was used for comparisons between normally distributed data, or a non-parametric paired Wilcoxon test was used otherwise. LDH inhibition partially rescued the Peredox responses in Isra+Cd 2+ and EGTA-AM, but not in 0Ca 2+ +EGTA, even though some pyruvate accumulation is also expected in the last condition due to less Ca 2+ -dependent pyruvate utilization in the mitochondria during stimulation. We do not have a definitive answer for this difference. It is possible that the Ca 2+ channel blockade or Ca 2+ chelation may not be complete, especially in dendrites, triggering a component of the metabolic responses that propagates to the soma, and is better revealed during LDH inhibition.
Figure Legend Snippet: A major Ca 2+ -dependent component of the NADH CYT also occurs under LDH inhibition. ( a ) Comparison of the ΔPeredox/ΔRCaMP values obtained in the continuous presence of the LDH inhibitor GSK-2837808A, before and after the blockade of voltage gated Ca 2+ channels with a combination of isradipine and cadmium (Isra+Cd 2+ ; N neurons = 13, N slices = 3 and N mice = 3). The slices were exposed to GSK-2837808A for at least 30 min prior to the experiment. LDH inhibition should improve the ability to detect the cytosol-only component of Ca 2+ actions on the NADH CYT transients by increasing the control responses to stimulation, as well as by preventing the potential impact of pyruvate accumulation on these transients due to lower Ca 2+ influx into the mitochondria. The effects of the manipulation on the Peredox baseline and the RCaMP spike are also included for the experiments with or without the LDH inhibitor (the sample size is reported in Figure 4 for the latter). ( b–c ) Comparisons for the application of the cell-permeable Ca 2+ chelator EGTA-AM (N neurons = 15, N slices = 3 and N mice = 3 for experiments with LDHi, sample sizes for the other group as in Figure 4 ), or the removal of Ca 2+ from the ACSF (N neurons = 136, N slices = 22 and N mice = 13 for experiments with LDHi, sample sizes for the other group as in Figure 4 ). For all panels, only neurons showing an initial ΔPeredox lifetime response ≥0.05 ns were included for analysis. A paired Student’s t test was used for comparisons between normally distributed data, or a non-parametric paired Wilcoxon test was used otherwise. LDH inhibition partially rescued the Peredox responses in Isra+Cd 2+ and EGTA-AM, but not in 0Ca 2+ +EGTA, even though some pyruvate accumulation is also expected in the last condition due to less Ca 2+ -dependent pyruvate utilization in the mitochondria during stimulation. We do not have a definitive answer for this difference. It is possible that the Ca 2+ channel blockade or Ca 2+ chelation may not be complete, especially in dendrites, triggering a component of the metabolic responses that propagates to the soma, and is better revealed during LDH inhibition.

Techniques Used: Inhibition, Mouse Assay

3) Product Images from "Autocrine activation of neuronal NMDA receptors by aspartate mediates dopamine- and cAMP-induced CREB-dependent gene transcription"

Article Title: Autocrine activation of neuronal NMDA receptors by aspartate mediates dopamine- and cAMP-induced CREB-dependent gene transcription

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

doi: 10.1523/JNEUROSCI.1166-09.2009

Stimulation of CREB-dependent transcription by forskolin requires an increase in [Ca 2+ ] within a microdomain. One day after transfecting with 4x CRE reporter plasmid, cultures were preincubated with Bapta-AM or EGTA-AM (10 μM) for 1 hr and then
Figure Legend Snippet: Stimulation of CREB-dependent transcription by forskolin requires an increase in [Ca 2+ ] within a microdomain. One day after transfecting with 4x CRE reporter plasmid, cultures were preincubated with Bapta-AM or EGTA-AM (10 μM) for 1 hr and then

Techniques Used: Plasmid Preparation

4) Product Images from "Autocrine activation of neuronal NMDA receptors by aspartate mediates dopamine- and cAMP-induced CREB-dependent gene transcription"

Article Title: Autocrine activation of neuronal NMDA receptors by aspartate mediates dopamine- and cAMP-induced CREB-dependent gene transcription

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

doi: 10.1523/JNEUROSCI.1166-09.2009

Stimulation of CREB-dependent transcription by forskolin requires an increase in [Ca 2+ ] within a microdomain. One day after transfecting with 4x CRE reporter plasmid, cultures were preincubated with Bapta-AM or EGTA-AM (10 μM) for 1 hr and then
Figure Legend Snippet: Stimulation of CREB-dependent transcription by forskolin requires an increase in [Ca 2+ ] within a microdomain. One day after transfecting with 4x CRE reporter plasmid, cultures were preincubated with Bapta-AM or EGTA-AM (10 μM) for 1 hr and then

Techniques Used: Plasmid Preparation

5) Product Images from "The distinct roles of calcium in rapid control of neuronal glycolysis and the tricarboxylic acid cycle"

Article Title: The distinct roles of calcium in rapid control of neuronal glycolysis and the tricarboxylic acid cycle

Journal: eLife

doi: 10.7554/eLife.64821

Spontaneous oscillations in the Peredox signal may occur during the prolonged application of zero Ca 2+ -ACSF, in the presence of EGTA and LDH inhibition. Prolonged exposure to a nominal Ca 2+ -free solution, plus LDH inhibition, caused spontaneous elevations of the Peredox lifetime in 25 ± 13% of the cells in each slice (slices = 10, mice = 5), contrasting with only ~3% of cells exposed to a brief exposure as in Figure 4c . The spontaneous elevations in Peredox lifetime occurred at any time after removing Ca 2+ from the ACSF. Although these neurons were not included in the analysis, once the spontaneous transients cleared, the experiment resumed as usual. The continuous presence of 1 mM EGTA and 2 µM GSK-2837808 (LDH inhibitor) is not indicated in the figure for simplicity. In the control ACSF, the [Ca 2+ ] was adjusted accordingly to yield a free concentration of 2 mM.
Figure Legend Snippet: Spontaneous oscillations in the Peredox signal may occur during the prolonged application of zero Ca 2+ -ACSF, in the presence of EGTA and LDH inhibition. Prolonged exposure to a nominal Ca 2+ -free solution, plus LDH inhibition, caused spontaneous elevations of the Peredox lifetime in 25 ± 13% of the cells in each slice (slices = 10, mice = 5), contrasting with only ~3% of cells exposed to a brief exposure as in Figure 4c . The spontaneous elevations in Peredox lifetime occurred at any time after removing Ca 2+ from the ACSF. Although these neurons were not included in the analysis, once the spontaneous transients cleared, the experiment resumed as usual. The continuous presence of 1 mM EGTA and 2 µM GSK-2837808 (LDH inhibitor) is not indicated in the figure for simplicity. In the control ACSF, the [Ca 2+ ] was adjusted accordingly to yield a free concentration of 2 mM.

Techniques Used: Inhibition, Mouse Assay, Concentration Assay

A major Ca 2+ -dependent component of the NADH CYT also occurs under LDH inhibition. ( a ) Comparison of the ΔPeredox/ΔRCaMP values obtained in the continuous presence of the LDH inhibitor GSK-2837808A, before and after the blockade of voltage gated Ca 2+ channels with a combination of isradipine and cadmium (Isra+Cd 2+ ; N neurons = 13, N slices = 3 and N mice = 3). The slices were exposed to GSK-2837808A for at least 30 min prior to the experiment. LDH inhibition should improve the ability to detect the cytosol-only component of Ca 2+ actions on the NADH CYT transients by increasing the control responses to stimulation, as well as by preventing the potential impact of pyruvate accumulation on these transients due to lower Ca 2+ influx into the mitochondria. The effects of the manipulation on the Peredox baseline and the RCaMP spike are also included for the experiments with or without the LDH inhibitor (the sample size is reported in Figure 4 for the latter). ( b–c ) Comparisons for the application of the cell-permeable Ca 2+ chelator EGTA-AM (N neurons = 15, N slices = 3 and N mice = 3 for experiments with LDHi, sample sizes for the other group as in Figure 4 ), or the removal of Ca 2+ from the ACSF (N neurons = 136, N slices = 22 and N mice = 13 for experiments with LDHi, sample sizes for the other group as in Figure 4 ). For all panels, only neurons showing an initial ΔPeredox lifetime response ≥0.05 ns were included for analysis. A paired Student’s t test was used for comparisons between normally distributed data, or a non-parametric paired Wilcoxon test was used otherwise. LDH inhibition partially rescued the Peredox responses in Isra+Cd 2+ and EGTA-AM, but not in 0Ca 2+ +EGTA, even though some pyruvate accumulation is also expected in the last condition due to less Ca 2+ -dependent pyruvate utilization in the mitochondria during stimulation. We do not have a definitive answer for this difference. It is possible that the Ca 2+ channel blockade or Ca 2+ chelation may not be complete, especially in dendrites, triggering a component of the metabolic responses that propagates to the soma, and is better revealed during LDH inhibition.
Figure Legend Snippet: A major Ca 2+ -dependent component of the NADH CYT also occurs under LDH inhibition. ( a ) Comparison of the ΔPeredox/ΔRCaMP values obtained in the continuous presence of the LDH inhibitor GSK-2837808A, before and after the blockade of voltage gated Ca 2+ channels with a combination of isradipine and cadmium (Isra+Cd 2+ ; N neurons = 13, N slices = 3 and N mice = 3). The slices were exposed to GSK-2837808A for at least 30 min prior to the experiment. LDH inhibition should improve the ability to detect the cytosol-only component of Ca 2+ actions on the NADH CYT transients by increasing the control responses to stimulation, as well as by preventing the potential impact of pyruvate accumulation on these transients due to lower Ca 2+ influx into the mitochondria. The effects of the manipulation on the Peredox baseline and the RCaMP spike are also included for the experiments with or without the LDH inhibitor (the sample size is reported in Figure 4 for the latter). ( b–c ) Comparisons for the application of the cell-permeable Ca 2+ chelator EGTA-AM (N neurons = 15, N slices = 3 and N mice = 3 for experiments with LDHi, sample sizes for the other group as in Figure 4 ), or the removal of Ca 2+ from the ACSF (N neurons = 136, N slices = 22 and N mice = 13 for experiments with LDHi, sample sizes for the other group as in Figure 4 ). For all panels, only neurons showing an initial ΔPeredox lifetime response ≥0.05 ns were included for analysis. A paired Student’s t test was used for comparisons between normally distributed data, or a non-parametric paired Wilcoxon test was used otherwise. LDH inhibition partially rescued the Peredox responses in Isra+Cd 2+ and EGTA-AM, but not in 0Ca 2+ +EGTA, even though some pyruvate accumulation is also expected in the last condition due to less Ca 2+ -dependent pyruvate utilization in the mitochondria during stimulation. We do not have a definitive answer for this difference. It is possible that the Ca 2+ channel blockade or Ca 2+ chelation may not be complete, especially in dendrites, triggering a component of the metabolic responses that propagates to the soma, and is better revealed during LDH inhibition.

Techniques Used: Inhibition, Mouse Assay

6) Product Images from "The distinct roles of calcium in rapid control of neuronal glycolysis and the tricarboxylic acid cycle"

Article Title: The distinct roles of calcium in rapid control of neuronal glycolysis and the tricarboxylic acid cycle

Journal: eLife

doi: 10.7554/eLife.64821

DMSO control for the EGTA-AM experiments. Left: Representative trace of the effect of DMSO (0.1%) on the Peredox and RCaMP signals. Right: The change in Peredox lifetime elicited by stimulation was unaffected by the application of 0.1% DMSO for 30–60 min, contrary to the gradual decrease observed with EGTA-AM. The RCaMP spike decreased over time but it was not as attenuated as in EGTA-AM. The Peredox and RCaMP transients were compared using a Wilcoxon matched pairs test and a paired t-test, respectively (N neurons = 17, N slices = 4 and N mice = 3). Only neurons showing an initial ΔPeredox lifetime response ~0.05 ns or higher were included for analysis.
Figure Legend Snippet: DMSO control for the EGTA-AM experiments. Left: Representative trace of the effect of DMSO (0.1%) on the Peredox and RCaMP signals. Right: The change in Peredox lifetime elicited by stimulation was unaffected by the application of 0.1% DMSO for 30–60 min, contrary to the gradual decrease observed with EGTA-AM. The RCaMP spike decreased over time but it was not as attenuated as in EGTA-AM. The Peredox and RCaMP transients were compared using a Wilcoxon matched pairs test and a paired t-test, respectively (N neurons = 17, N slices = 4 and N mice = 3). Only neurons showing an initial ΔPeredox lifetime response ~0.05 ns or higher were included for analysis.

Techniques Used: Mouse Assay

7) Product Images from "The distinct roles of calcium in rapid control of neuronal glycolysis and the tricarboxylic acid cycle"

Article Title: The distinct roles of calcium in rapid control of neuronal glycolysis and the tricarboxylic acid cycle

Journal: bioRxiv

doi: 10.1101/2020.11.16.385526

Control experiment for 0.1% DMSO in the ACSF, as in the EGTA-AM experiments. Left: Representative trace of the effect of DMSO (0.1%) on the Peredox and RCaMP signals. Right: The change in Peredox lifetime elicited by stimulation was unaffected by the application of 0.1% DMSO for 30—60 min, contrary to the gradual decrease observed with EGTA-AM. The RCaMP spike decreased over time but it was not as attenuated as in EGTA-AM. The Peredox and RCaMP transients were compared using a Wilcoxon matched pairs test and a paired t-test, respectively (N neurons = 17, N slices = 4 and N mice = 3). Only neurons showing an initial ΔPeredox lifetime response ∼0.05 ns or higher were included for analysis.
Figure Legend Snippet: Control experiment for 0.1% DMSO in the ACSF, as in the EGTA-AM experiments. Left: Representative trace of the effect of DMSO (0.1%) on the Peredox and RCaMP signals. Right: The change in Peredox lifetime elicited by stimulation was unaffected by the application of 0.1% DMSO for 30—60 min, contrary to the gradual decrease observed with EGTA-AM. The RCaMP spike decreased over time but it was not as attenuated as in EGTA-AM. The Peredox and RCaMP transients were compared using a Wilcoxon matched pairs test and a paired t-test, respectively (N neurons = 17, N slices = 4 and N mice = 3). Only neurons showing an initial ΔPeredox lifetime response ∼0.05 ns or higher were included for analysis.

Techniques Used: Mouse Assay

8) Product Images from "Autocrine activation of neuronal NMDA receptors by aspartate mediates dopamine- and cAMP-induced CREB-dependent gene transcription"

Article Title: Autocrine activation of neuronal NMDA receptors by aspartate mediates dopamine- and cAMP-induced CREB-dependent gene transcription

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

doi: 10.1523/JNEUROSCI.1166-09.2009

Stimulation of CREB-dependent transcription by forskolin requires an increase in [Ca 2+ ] within a microdomain. One day after transfecting with 4x CRE reporter plasmid, cultures were preincubated with Bapta-AM or EGTA-AM (10 μM) for 1 hr and then
Figure Legend Snippet: Stimulation of CREB-dependent transcription by forskolin requires an increase in [Ca 2+ ] within a microdomain. One day after transfecting with 4x CRE reporter plasmid, cultures were preincubated with Bapta-AM or EGTA-AM (10 μM) for 1 hr and then

Techniques Used: Plasmid Preparation

9) Product Images from "Vesicular Zinc Regulates the Ca2+ Sensitivity of a Subpopulation of Presynaptic Vesicles at Hippocampal Mossy Fiber Terminals"

Article Title: Vesicular Zinc Regulates the Ca2+ Sensitivity of a Subpopulation of Presynaptic Vesicles at Hippocampal Mossy Fiber Terminals

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.4164-11.2011

Decay kinetics of ZnT3 KO mice are different from their WT littermates. A1 , Averaged sweeps of MF EPSCs evoked at various frequencies in control ACSF. Responses at 0.2 Hz (blue traces) and 1 Hz (red traces) were scaled to 0.05 Hz baseline value (black traces). A2 , Decay kinetics of evoked EPSCs recorded from WT ( n = 7) and KO ( n = 9) mice could be fitted with a single or double exponentials. A3 , EGTA-AM changes the decay kinetics by decreasing the occurrence of the slower component for both genotypes ( n = 7). B , Average values of the decay kinetics for WT and KO in control condition and after application of EGTA-AM. CTRL, Control.
Figure Legend Snippet: Decay kinetics of ZnT3 KO mice are different from their WT littermates. A1 , Averaged sweeps of MF EPSCs evoked at various frequencies in control ACSF. Responses at 0.2 Hz (blue traces) and 1 Hz (red traces) were scaled to 0.05 Hz baseline value (black traces). A2 , Decay kinetics of evoked EPSCs recorded from WT ( n = 7) and KO ( n = 9) mice could be fitted with a single or double exponentials. A3 , EGTA-AM changes the decay kinetics by decreasing the occurrence of the slower component for both genotypes ( n = 7). B , Average values of the decay kinetics for WT and KO in control condition and after application of EGTA-AM. CTRL, Control.

Techniques Used: Mouse Assay

10) Product Images from "Autocrine activation of neuronal NMDA receptors by aspartate mediates dopamine- and cAMP-induced CREB-dependent gene transcription"

Article Title: Autocrine activation of neuronal NMDA receptors by aspartate mediates dopamine- and cAMP-induced CREB-dependent gene transcription

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

doi: 10.1523/JNEUROSCI.1166-09.2009

Stimulation of CREB-dependent transcription by forskolin requires an increase in [Ca 2+ ] within a microdomain. One day after transfecting with 4x CRE reporter plasmid, cultures were preincubated with Bapta-AM or EGTA-AM (10 μM) for 1 hr and then
Figure Legend Snippet: Stimulation of CREB-dependent transcription by forskolin requires an increase in [Ca 2+ ] within a microdomain. One day after transfecting with 4x CRE reporter plasmid, cultures were preincubated with Bapta-AM or EGTA-AM (10 μM) for 1 hr and then

Techniques Used: Plasmid Preparation

11) Product Images from "Vesicular Zinc Regulates the Ca2+ Sensitivity of a Subpopulation of Presynaptic Vesicles at Hippocampal Mossy Fiber Terminals"

Article Title: Vesicular Zinc Regulates the Ca2+ Sensitivity of a Subpopulation of Presynaptic Vesicles at Hippocampal Mossy Fiber Terminals

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.4164-11.2011

Decay kinetics of ZnT3 KO mice are different from their WT littermates. A1 , Averaged sweeps of MF EPSCs evoked at various frequencies in control ACSF. Responses at 0.2 Hz (blue traces) and 1 Hz (red traces) were scaled to 0.05 Hz baseline value (black traces). A2 , Decay kinetics of evoked EPSCs recorded from WT ( n = 7) and KO ( n = 9) mice could be fitted with a single or double exponentials. A3 , EGTA-AM changes the decay kinetics by decreasing the occurrence of the slower component for both genotypes ( n = 7). B , Average values of the decay kinetics for WT and KO in control condition and after application of EGTA-AM. CTRL, Control.
Figure Legend Snippet: Decay kinetics of ZnT3 KO mice are different from their WT littermates. A1 , Averaged sweeps of MF EPSCs evoked at various frequencies in control ACSF. Responses at 0.2 Hz (blue traces) and 1 Hz (red traces) were scaled to 0.05 Hz baseline value (black traces). A2 , Decay kinetics of evoked EPSCs recorded from WT ( n = 7) and KO ( n = 9) mice could be fitted with a single or double exponentials. A3 , EGTA-AM changes the decay kinetics by decreasing the occurrence of the slower component for both genotypes ( n = 7). B , Average values of the decay kinetics for WT and KO in control condition and after application of EGTA-AM. CTRL, Control.

Techniques Used: Mouse Assay

12) Product Images from "The distinct roles of calcium in rapid control of neuronal glycolysis and the tricarboxylic acid cycle"

Article Title: The distinct roles of calcium in rapid control of neuronal glycolysis and the tricarboxylic acid cycle

Journal: bioRxiv

doi: 10.1101/2020.11.16.385526

Neuronal stimulation triggers glycolysis in response to energy demand from ion pumping. a. Left: Representative trace of Peredox and RCaMP1h lifetimes simultaneously recorded in a DGC from an acute hippocampal slice. The slice was superfused with 2 µM GSK-2837808A for at least 30 min before the experiment, and the LDH inhibitor was kept in the ACSF during the experiment. The ACSF also contained 1 mM EGTA to reinforce Ca 2+ removal in the nominal 0Ca 2+ condition (but [Ca 2+ ] in the control ACSF was accordingly adjusted to a free concentration of 2 mM, as in any other experiment). Effective Ca 2+ removal was confirmed by the absence of a RCaMP1h spike upon stimulation. The Peredox lifetime at baseline, and the metabolic transients in response to neuronal stimulation, were recorded after substituting the bath solution with a 0Ca 2+ ACSF (to obtain Na + -only NADH CYT responses), and the further application of 10 µM α-pompilidotoxin (α-Pmtx, a toxin that prevents voltage-gated Na + channel inactivation). Right: The Na + -only NADH CYT transient was increased in the presence of α-pompilidotoxin. The Peredox lifetime change in response to stimulation was diminished in the absence of Ca 2+ but bounced back to higher amplitudes by increasing Na + influx. The data were compared using a repeated measures ANOVA with a Student-Newman-Keuls post-test (N neurons = 35, N slices = 5 and N mice = 3). For all panels, only neurons showing an initial ΔPeredox lifetime response ≥0.05 ns were included for analysis. b. Representative trace of Peredox and RCaMP1h lifetimes in a DGC stimulated with trains of 100 and 200 electrical pulses. The two-stimulation protocol was also performed in 0Ca 2+ ACSF before and after the application of 3 µM α-pompilidotoxin. The latter condition was followed by the application of the Na + /K + ATPase inhibitor strophanthidin. As in (a) , the slices were exposed to the LDH inhibitor GSK-2837808A from 30 min prior, until the end of the experiment. Likewise, all the solutions contained 1 mM EGTA. c. Comparison of the Peredox lifetime changes in response to both stimulation paradigms (100 or 200 pulses) among the conditions in (b) . The NADH CYT transients in control condition was different from the other conditions (the discontinuous line for the associated p-value applies to all comparisons). The Na + -only NADH CYT responses recorded in 0Ca 2+ ACSF were increased slightly but significantly increased by the application of 3 µM α-pompilidotoxin, an effect that was reversed by strophanthidin. The data were compared using a non-parametric repeated measures ANOVA (Friedman test) with a Dunn post-test (N neurons = 66, N slices = 10 and N mice = 5).
Figure Legend Snippet: Neuronal stimulation triggers glycolysis in response to energy demand from ion pumping. a. Left: Representative trace of Peredox and RCaMP1h lifetimes simultaneously recorded in a DGC from an acute hippocampal slice. The slice was superfused with 2 µM GSK-2837808A for at least 30 min before the experiment, and the LDH inhibitor was kept in the ACSF during the experiment. The ACSF also contained 1 mM EGTA to reinforce Ca 2+ removal in the nominal 0Ca 2+ condition (but [Ca 2+ ] in the control ACSF was accordingly adjusted to a free concentration of 2 mM, as in any other experiment). Effective Ca 2+ removal was confirmed by the absence of a RCaMP1h spike upon stimulation. The Peredox lifetime at baseline, and the metabolic transients in response to neuronal stimulation, were recorded after substituting the bath solution with a 0Ca 2+ ACSF (to obtain Na + -only NADH CYT responses), and the further application of 10 µM α-pompilidotoxin (α-Pmtx, a toxin that prevents voltage-gated Na + channel inactivation). Right: The Na + -only NADH CYT transient was increased in the presence of α-pompilidotoxin. The Peredox lifetime change in response to stimulation was diminished in the absence of Ca 2+ but bounced back to higher amplitudes by increasing Na + influx. The data were compared using a repeated measures ANOVA with a Student-Newman-Keuls post-test (N neurons = 35, N slices = 5 and N mice = 3). For all panels, only neurons showing an initial ΔPeredox lifetime response ≥0.05 ns were included for analysis. b. Representative trace of Peredox and RCaMP1h lifetimes in a DGC stimulated with trains of 100 and 200 electrical pulses. The two-stimulation protocol was also performed in 0Ca 2+ ACSF before and after the application of 3 µM α-pompilidotoxin. The latter condition was followed by the application of the Na + /K + ATPase inhibitor strophanthidin. As in (a) , the slices were exposed to the LDH inhibitor GSK-2837808A from 30 min prior, until the end of the experiment. Likewise, all the solutions contained 1 mM EGTA. c. Comparison of the Peredox lifetime changes in response to both stimulation paradigms (100 or 200 pulses) among the conditions in (b) . The NADH CYT transients in control condition was different from the other conditions (the discontinuous line for the associated p-value applies to all comparisons). The Na + -only NADH CYT responses recorded in 0Ca 2+ ACSF were increased slightly but significantly increased by the application of 3 µM α-pompilidotoxin, an effect that was reversed by strophanthidin. The data were compared using a non-parametric repeated measures ANOVA (Friedman test) with a Dunn post-test (N neurons = 66, N slices = 10 and N mice = 5).

Techniques Used: Concentration Assay, Mouse Assay

Spontaneous oscillations in the Peredox signal may occur during the prolonged application of zero Ca 2+ -ACSF, in the presence of EGTA and LDH inhibition. Prolonged exposure to a nominal Ca 2+ -free solution, plus LDH inhibition, caused spontaneous elevations of the Peredox lifetime in 25 ± 13% of the cells in each slice (slices=10, mice=5), contrasting with only ∼3% of cells exposed to a brief exposure as in Figure 3c . The spontaneous elevations in Peredox lifetime occurred at any time after removing Ca 2+ from the ACSF. Although these neurons were not included in the analysis, once the spontaneous transients cleared, the experiment resumed as usual. The continuous presence of 1 mM EGTA and 2 µM GSK-2837808 (LDH inhibitor) is not indicated in the figure for simplicity. In the control ACSF, the [Ca 2+ ] was adjusted accordingly to yield a free concentration of 2 mM.
Figure Legend Snippet: Spontaneous oscillations in the Peredox signal may occur during the prolonged application of zero Ca 2+ -ACSF, in the presence of EGTA and LDH inhibition. Prolonged exposure to a nominal Ca 2+ -free solution, plus LDH inhibition, caused spontaneous elevations of the Peredox lifetime in 25 ± 13% of the cells in each slice (slices=10, mice=5), contrasting with only ∼3% of cells exposed to a brief exposure as in Figure 3c . The spontaneous elevations in Peredox lifetime occurred at any time after removing Ca 2+ from the ACSF. Although these neurons were not included in the analysis, once the spontaneous transients cleared, the experiment resumed as usual. The continuous presence of 1 mM EGTA and 2 µM GSK-2837808 (LDH inhibitor) is not indicated in the figure for simplicity. In the control ACSF, the [Ca 2+ ] was adjusted accordingly to yield a free concentration of 2 mM.

Techniques Used: Inhibition, Mouse Assay, Concentration Assay

A major Ca 2+ -dependent component of the NADH CYT also occurs under LDH inhibition. a. Comparison of the ΔPeredox/ΔRCaMP values obtained in the continuous presence of the LDH inhibitor GSK-2837808A, before and after the blockade of voltage gated Ca 2+ channels with a combination of isradipine and cadmium (Isra+Cd 2+ ; N neurons = 13, N slices = 3 and N mice = 3). The slices were exposed to GSK-2837808A for at least 30 min prior to the experiment. LDH inhibition should improve the ability to detect the cytosol-only component of Ca 2+ actions on the NADH CYT transients by increasing the control responses to stimulation, as well as by preventing the potential impact of pyruvate accumulation on these transients due to lower Ca 2+ influx into the mitochondria. The effects of the manipulation on the Peredox baseline and the RCaMP spike are also included for the experiments with or without the LDH inhibitor (the sample size is reported in Figure 3 for the latter). b—c. Comparisons for the application of the cell-permeable Ca 2+ chelator EGTA-AM (N neurons = 15, N slices = 3 and N mice = 3 for experiments with LDHi, sample sizes for the other group as in Figure 3 ), or the removal of Ca 2+ from the ACSF (N neurons = 136, N slices = 22 and N mice = 13 for experiments with LDHi, sample sizes for the other group as in Figure 3 ). For all panels, only neurons showing an initial ΔPeredox lifetime response ≥0.05 ns were included for analysis. A paired Student’s t test was used for comparisons between normally distributed data, or a non-parametric paired Wilcoxon test was used otherwise. LDH inhibition partially rescued the Peredox responses in Isra+Cd 2+ and EGTA-AM, but not in 0Ca 2+ +EGTA, even though some pyruvate accumulation is also expected in the last condition due to less Ca 2+ -dependent pyruvate utilization in the mitochondria during stimulation. We do not have a definitive answer for this difference. It is possible that the Ca 2+ channel blockade or Ca 2+ chelation may not be complete, especially in dendrites, triggering a component of the metabolic responses that propagates to the soma, and is better revealed during LDH inhibition.
Figure Legend Snippet: A major Ca 2+ -dependent component of the NADH CYT also occurs under LDH inhibition. a. Comparison of the ΔPeredox/ΔRCaMP values obtained in the continuous presence of the LDH inhibitor GSK-2837808A, before and after the blockade of voltage gated Ca 2+ channels with a combination of isradipine and cadmium (Isra+Cd 2+ ; N neurons = 13, N slices = 3 and N mice = 3). The slices were exposed to GSK-2837808A for at least 30 min prior to the experiment. LDH inhibition should improve the ability to detect the cytosol-only component of Ca 2+ actions on the NADH CYT transients by increasing the control responses to stimulation, as well as by preventing the potential impact of pyruvate accumulation on these transients due to lower Ca 2+ influx into the mitochondria. The effects of the manipulation on the Peredox baseline and the RCaMP spike are also included for the experiments with or without the LDH inhibitor (the sample size is reported in Figure 3 for the latter). b—c. Comparisons for the application of the cell-permeable Ca 2+ chelator EGTA-AM (N neurons = 15, N slices = 3 and N mice = 3 for experiments with LDHi, sample sizes for the other group as in Figure 3 ), or the removal of Ca 2+ from the ACSF (N neurons = 136, N slices = 22 and N mice = 13 for experiments with LDHi, sample sizes for the other group as in Figure 3 ). For all panels, only neurons showing an initial ΔPeredox lifetime response ≥0.05 ns were included for analysis. A paired Student’s t test was used for comparisons between normally distributed data, or a non-parametric paired Wilcoxon test was used otherwise. LDH inhibition partially rescued the Peredox responses in Isra+Cd 2+ and EGTA-AM, but not in 0Ca 2+ +EGTA, even though some pyruvate accumulation is also expected in the last condition due to less Ca 2+ -dependent pyruvate utilization in the mitochondria during stimulation. We do not have a definitive answer for this difference. It is possible that the Ca 2+ channel blockade or Ca 2+ chelation may not be complete, especially in dendrites, triggering a component of the metabolic responses that propagates to the soma, and is better revealed during LDH inhibition.

Techniques Used: Inhibition, Mouse Assay

The rise in [Ca 2+ ] CYT , mainly caused by the activity of high voltage-activated Ca 2+ channels, makes a major contribution to the NADH CYT transients in response to stimulation. a. Left: Representative trace from a DGC expressing Peredox and RCaMP1h. The slice was superfused for ∼20 min with the L-type Ca 2+ channel inhibitor isradipine (Isra, 3 µM), and then stimulated. In the continuous presence of isradipine, 20 µM of CdCl 2 (Cd 2+ , a non-selective blocker of voltage-activated Ca 2+ channels) was added to the ACSF. Inhibition of Ca 2+ influx was evident from the progressive reduction of the stimulus associated RCaMP1h spike. Right: The amplitude of the metabolic responses to stimulation (Peredox lifetime change) mirrored the decrease in the RCaMP spikes ( Figure 3 – Supplement 3a ). The data were compared using a non-parametric repeated measures ANOVA (Friedman test) with a Dunn post-test (N neurons = 86, N slices = 11 and N mice = 6). For all panels, only neurons showing an initial ΔPeredox lifetime response ≥0.05 ns were included for analysis. b. Left: Representative trace of a DGC superfused with EGTA-AM (100 µM), a cell-permeable Ca 2+ chelator. As expected, the stimulus-induced RCaMP transients gradually diminished over time, typically stabilizing after ∼1h of treatment. Right: NADH CYT transients are strongly attenuated after effective Ca 2+ buffering by EGTA-AM ( Figure 3 – Supplement 3b ). The data were compared using a Wilcoxon matched pairs test (N neurons = 45, N slices = 5 and N mice = 5). c. Left: Representative trace for the effect of Ca 2+ removal from the bath solution on the metabolic transients in the cytosol. The cell-impermeant Ca 2+ chelator EGTA (1 mM) was added to the ACSF to reinforce Ca 2+ removal after switching to a nominal 0Ca 2+ solution. A modified control ACSF also contained 1 mM EGTA and an adjusted total [Ca 2+ ] resulting in a free concentration of 2 mM, as in any other control experiment. Effective Ca 2+ removal was confirmed by the absence of a RCaMP1h spike upon stimulation. Right: The NADH CYT transients were diminished in a Ca 2+ -deprived ACSF. The data were compared using a Wilcoxon matched pairs test (N neurons = 31, N slices = 7 and N mice = 6).
Figure Legend Snippet: The rise in [Ca 2+ ] CYT , mainly caused by the activity of high voltage-activated Ca 2+ channels, makes a major contribution to the NADH CYT transients in response to stimulation. a. Left: Representative trace from a DGC expressing Peredox and RCaMP1h. The slice was superfused for ∼20 min with the L-type Ca 2+ channel inhibitor isradipine (Isra, 3 µM), and then stimulated. In the continuous presence of isradipine, 20 µM of CdCl 2 (Cd 2+ , a non-selective blocker of voltage-activated Ca 2+ channels) was added to the ACSF. Inhibition of Ca 2+ influx was evident from the progressive reduction of the stimulus associated RCaMP1h spike. Right: The amplitude of the metabolic responses to stimulation (Peredox lifetime change) mirrored the decrease in the RCaMP spikes ( Figure 3 – Supplement 3a ). The data were compared using a non-parametric repeated measures ANOVA (Friedman test) with a Dunn post-test (N neurons = 86, N slices = 11 and N mice = 6). For all panels, only neurons showing an initial ΔPeredox lifetime response ≥0.05 ns were included for analysis. b. Left: Representative trace of a DGC superfused with EGTA-AM (100 µM), a cell-permeable Ca 2+ chelator. As expected, the stimulus-induced RCaMP transients gradually diminished over time, typically stabilizing after ∼1h of treatment. Right: NADH CYT transients are strongly attenuated after effective Ca 2+ buffering by EGTA-AM ( Figure 3 – Supplement 3b ). The data were compared using a Wilcoxon matched pairs test (N neurons = 45, N slices = 5 and N mice = 5). c. Left: Representative trace for the effect of Ca 2+ removal from the bath solution on the metabolic transients in the cytosol. The cell-impermeant Ca 2+ chelator EGTA (1 mM) was added to the ACSF to reinforce Ca 2+ removal after switching to a nominal 0Ca 2+ solution. A modified control ACSF also contained 1 mM EGTA and an adjusted total [Ca 2+ ] resulting in a free concentration of 2 mM, as in any other control experiment. Effective Ca 2+ removal was confirmed by the absence of a RCaMP1h spike upon stimulation. Right: The NADH CYT transients were diminished in a Ca 2+ -deprived ACSF. The data were compared using a Wilcoxon matched pairs test (N neurons = 31, N slices = 7 and N mice = 6).

Techniques Used: Activity Assay, Expressing, Inhibition, Mouse Assay, Modification, Concentration Assay

13) Product Images from "Ca2+/H+ exchange by acidic organelles regulates cell migration in vivo"

Article Title: Ca2+/H+ exchange by acidic organelles regulates cell migration in vivo

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201510019

CAX regulates cell substrate attachment. (a and b) Effect of CAX knockdown on chemotaxis. (a) Tracks of individual explants migrating toward beads soaked in Sdf-1 over the entire period of recording (left) or the initial 100 min (right). (b) Summary data (from three knockdowns) quantifying forward motion index, persistence, and velocity of migration. (c–g) Effect of CAX knockdown on cell dispersion. (c) Summary data from three knockdowns quantifying the size of control and morphant explants 30 min after plating. (d) Representative color-coded triangulation diagrams at time 0 and 6 h from control or morphant explants. (e) Time courses (from three knockdowns) quantifying triangle area. Data are normalized to control explants at time 0. (f) Transmitted light micrographs showing spreading of control or morphant explants over a 20-min period. Panels on the right show an overlay of the area occupied by the explants where red represents the extent of spreading. Bar, 500 µm. (g) Summary data from three knockdowns quantifying spread area. (h and i) Effect of Ca 2+ buffering on cell dispersion. Triangulation (h) and spreading (i) analysis of explants treated with cell-permeable Ca 2+ chelators (50 µM BAPTA/EGTA-AM) or DMSO 30 min before imaging. (j and k) Live cell imaging of CAX. Stills from time-lapse confocal imaging of explants coexpressing mRFP-CAX and either membrane GFP (j; bar, 10 µm) or focal adhesion kinase–GFP (k). Arrowheads mark small CAX-positive vesicles. (k) Bars: (top) 4 µm; (bottom) 1 µm. Heat map summarizes the mean projection for CAX-positive vesicles relative to the centroid of focal adhesions (intersection of white lines) within 5 × 5–µm regions of interest. Data are from 28 focal adhesions from two expression experiments. (l–n) Effect of CAX knockdown on focal adhesions. (l) Expression of focal adhesion kinase–GFP (left) and immunocytochemistry analysis using an antibody to phosphopaxillin (middle) in control and morphant explants. Overlays (right) show costaining of phalloidin. Bar, 20 µm. (m) Summary data (94–422 cells from two to three knockdowns) quantifying the number and size of labeled structures. (n) Lifetime analysis of focal adhesion kinase–GFP in control and morphant explants (30–58 focal adhesions from two knockdowns). 10 ng AMO1 was used for all knockdowns. Error bars represent SEM. *, P
Figure Legend Snippet: CAX regulates cell substrate attachment. (a and b) Effect of CAX knockdown on chemotaxis. (a) Tracks of individual explants migrating toward beads soaked in Sdf-1 over the entire period of recording (left) or the initial 100 min (right). (b) Summary data (from three knockdowns) quantifying forward motion index, persistence, and velocity of migration. (c–g) Effect of CAX knockdown on cell dispersion. (c) Summary data from three knockdowns quantifying the size of control and morphant explants 30 min after plating. (d) Representative color-coded triangulation diagrams at time 0 and 6 h from control or morphant explants. (e) Time courses (from three knockdowns) quantifying triangle area. Data are normalized to control explants at time 0. (f) Transmitted light micrographs showing spreading of control or morphant explants over a 20-min period. Panels on the right show an overlay of the area occupied by the explants where red represents the extent of spreading. Bar, 500 µm. (g) Summary data from three knockdowns quantifying spread area. (h and i) Effect of Ca 2+ buffering on cell dispersion. Triangulation (h) and spreading (i) analysis of explants treated with cell-permeable Ca 2+ chelators (50 µM BAPTA/EGTA-AM) or DMSO 30 min before imaging. (j and k) Live cell imaging of CAX. Stills from time-lapse confocal imaging of explants coexpressing mRFP-CAX and either membrane GFP (j; bar, 10 µm) or focal adhesion kinase–GFP (k). Arrowheads mark small CAX-positive vesicles. (k) Bars: (top) 4 µm; (bottom) 1 µm. Heat map summarizes the mean projection for CAX-positive vesicles relative to the centroid of focal adhesions (intersection of white lines) within 5 × 5–µm regions of interest. Data are from 28 focal adhesions from two expression experiments. (l–n) Effect of CAX knockdown on focal adhesions. (l) Expression of focal adhesion kinase–GFP (left) and immunocytochemistry analysis using an antibody to phosphopaxillin (middle) in control and morphant explants. Overlays (right) show costaining of phalloidin. Bar, 20 µm. (m) Summary data (94–422 cells from two to three knockdowns) quantifying the number and size of labeled structures. (n) Lifetime analysis of focal adhesion kinase–GFP in control and morphant explants (30–58 focal adhesions from two knockdowns). 10 ng AMO1 was used for all knockdowns. Error bars represent SEM. *, P

Techniques Used: Chemotaxis Assay, Migration, Imaging, Live Cell Imaging, Expressing, Immunocytochemistry, Labeling

14) Product Images from "The distinct roles of calcium in rapid control of neuronal glycolysis and the tricarboxylic acid cycle"

Article Title: The distinct roles of calcium in rapid control of neuronal glycolysis and the tricarboxylic acid cycle

Journal: bioRxiv

doi: 10.1101/2020.11.16.385526

Neuronal stimulation triggers glycolysis in response to energy demand from ion pumping. a. Left: Representative trace of Peredox and RCaMP1h lifetimes simultaneously recorded in a DGC from an acute hippocampal slice. The slice was superfused with 2 µM GSK-2837808A for at least 30 min before the experiment, and the LDH inhibitor was kept in the ACSF during the experiment. The ACSF also contained 1 mM EGTA to reinforce Ca 2+ removal in the nominal 0Ca 2+ condition (but [Ca 2+ ] in the control ACSF was accordingly adjusted to a free concentration of 2 mM, as in any other experiment). Effective Ca 2+ removal was confirmed by the absence of a RCaMP1h spike upon stimulation. The Peredox lifetime at baseline, and the metabolic transients in response to neuronal stimulation, were recorded after substituting the bath solution with a 0Ca 2+ ACSF (to obtain Na + -only NADH CYT responses), and the further application of 10 µM α-pompilidotoxin (α-Pmtx, a toxin that prevents voltage-gated Na + channel inactivation). Right: The Na + -only NADH CYT transient was increased in the presence of α-pompilidotoxin. The Peredox lifetime change in response to stimulation was diminished in the absence of Ca 2+ but bounced back to higher amplitudes by increasing Na + influx. The data were compared using a repeated measures ANOVA with a Student-Newman-Keuls post-test (N neurons = 35, N slices = 5 and N mice = 3). For all panels, only neurons showing an initial ΔPeredox lifetime response ≥0.05 ns were included for analysis. b. Representative trace of Peredox and RCaMP1h lifetimes in a DGC stimulated with trains of 100 and 200 electrical pulses. The two-stimulation protocol was also performed in 0Ca 2+ ACSF before and after the application of 3 µM α-pompilidotoxin. The latter condition was followed by the application of the Na + /K + ATPase inhibitor strophanthidin. As in (a) , the slices were exposed to the LDH inhibitor GSK-2837808A from 30 min prior, until the end of the experiment. Likewise, all the solutions contained 1 mM EGTA. c. Comparison of the Peredox lifetime changes in response to both stimulation paradigms (100 or 200 pulses) among the conditions in (b) . The NADH CYT transients in control condition was different from the other conditions (the discontinuous line for the associated p-value applies to all comparisons). The Na + -only NADH CYT responses recorded in 0Ca 2+ ACSF were increased slightly but significantly increased by the application of 3 µM α-pompilidotoxin, an effect that was reversed by strophanthidin. The data were compared using a non-parametric repeated measures ANOVA (Friedman test) with a Dunn post-test (N neurons = 66, N slices = 10 and N mice = 5).
Figure Legend Snippet: Neuronal stimulation triggers glycolysis in response to energy demand from ion pumping. a. Left: Representative trace of Peredox and RCaMP1h lifetimes simultaneously recorded in a DGC from an acute hippocampal slice. The slice was superfused with 2 µM GSK-2837808A for at least 30 min before the experiment, and the LDH inhibitor was kept in the ACSF during the experiment. The ACSF also contained 1 mM EGTA to reinforce Ca 2+ removal in the nominal 0Ca 2+ condition (but [Ca 2+ ] in the control ACSF was accordingly adjusted to a free concentration of 2 mM, as in any other experiment). Effective Ca 2+ removal was confirmed by the absence of a RCaMP1h spike upon stimulation. The Peredox lifetime at baseline, and the metabolic transients in response to neuronal stimulation, were recorded after substituting the bath solution with a 0Ca 2+ ACSF (to obtain Na + -only NADH CYT responses), and the further application of 10 µM α-pompilidotoxin (α-Pmtx, a toxin that prevents voltage-gated Na + channel inactivation). Right: The Na + -only NADH CYT transient was increased in the presence of α-pompilidotoxin. The Peredox lifetime change in response to stimulation was diminished in the absence of Ca 2+ but bounced back to higher amplitudes by increasing Na + influx. The data were compared using a repeated measures ANOVA with a Student-Newman-Keuls post-test (N neurons = 35, N slices = 5 and N mice = 3). For all panels, only neurons showing an initial ΔPeredox lifetime response ≥0.05 ns were included for analysis. b. Representative trace of Peredox and RCaMP1h lifetimes in a DGC stimulated with trains of 100 and 200 electrical pulses. The two-stimulation protocol was also performed in 0Ca 2+ ACSF before and after the application of 3 µM α-pompilidotoxin. The latter condition was followed by the application of the Na + /K + ATPase inhibitor strophanthidin. As in (a) , the slices were exposed to the LDH inhibitor GSK-2837808A from 30 min prior, until the end of the experiment. Likewise, all the solutions contained 1 mM EGTA. c. Comparison of the Peredox lifetime changes in response to both stimulation paradigms (100 or 200 pulses) among the conditions in (b) . The NADH CYT transients in control condition was different from the other conditions (the discontinuous line for the associated p-value applies to all comparisons). The Na + -only NADH CYT responses recorded in 0Ca 2+ ACSF were increased slightly but significantly increased by the application of 3 µM α-pompilidotoxin, an effect that was reversed by strophanthidin. The data were compared using a non-parametric repeated measures ANOVA (Friedman test) with a Dunn post-test (N neurons = 66, N slices = 10 and N mice = 5).

Techniques Used: Concentration Assay, Mouse Assay

Spontaneous oscillations in the Peredox signal may occur during the prolonged application of zero Ca 2+ -ACSF, in the presence of EGTA and LDH inhibition. Prolonged exposure to a nominal Ca 2+ -free solution, plus LDH inhibition, caused spontaneous elevations of the Peredox lifetime in 25 ± 13% of the cells in each slice (slices=10, mice=5), contrasting with only ∼3% of cells exposed to a brief exposure as in Figure 3c . The spontaneous elevations in Peredox lifetime occurred at any time after removing Ca 2+ from the ACSF. Although these neurons were not included in the analysis, once the spontaneous transients cleared, the experiment resumed as usual. The continuous presence of 1 mM EGTA and 2 µM GSK-2837808 (LDH inhibitor) is not indicated in the figure for simplicity. In the control ACSF, the [Ca 2+ ] was adjusted accordingly to yield a free concentration of 2 mM.
Figure Legend Snippet: Spontaneous oscillations in the Peredox signal may occur during the prolonged application of zero Ca 2+ -ACSF, in the presence of EGTA and LDH inhibition. Prolonged exposure to a nominal Ca 2+ -free solution, plus LDH inhibition, caused spontaneous elevations of the Peredox lifetime in 25 ± 13% of the cells in each slice (slices=10, mice=5), contrasting with only ∼3% of cells exposed to a brief exposure as in Figure 3c . The spontaneous elevations in Peredox lifetime occurred at any time after removing Ca 2+ from the ACSF. Although these neurons were not included in the analysis, once the spontaneous transients cleared, the experiment resumed as usual. The continuous presence of 1 mM EGTA and 2 µM GSK-2837808 (LDH inhibitor) is not indicated in the figure for simplicity. In the control ACSF, the [Ca 2+ ] was adjusted accordingly to yield a free concentration of 2 mM.

Techniques Used: Inhibition, Mouse Assay, Concentration Assay

A major Ca 2+ -dependent component of the NADH CYT also occurs under LDH inhibition. a. Comparison of the ΔPeredox/ΔRCaMP values obtained in the continuous presence of the LDH inhibitor GSK-2837808A, before and after the blockade of voltage gated Ca 2+ channels with a combination of isradipine and cadmium (Isra+Cd 2+ ; N neurons = 13, N slices = 3 and N mice = 3). The slices were exposed to GSK-2837808A for at least 30 min prior to the experiment. LDH inhibition should improve the ability to detect the cytosol-only component of Ca 2+ actions on the NADH CYT transients by increasing the control responses to stimulation, as well as by preventing the potential impact of pyruvate accumulation on these transients due to lower Ca 2+ influx into the mitochondria. The effects of the manipulation on the Peredox baseline and the RCaMP spike are also included for the experiments with or without the LDH inhibitor (the sample size is reported in Figure 3 for the latter). b—c. Comparisons for the application of the cell-permeable Ca 2+ chelator EGTA-AM (N neurons = 15, N slices = 3 and N mice = 3 for experiments with LDHi, sample sizes for the other group as in Figure 3 ), or the removal of Ca 2+ from the ACSF (N neurons = 136, N slices = 22 and N mice = 13 for experiments with LDHi, sample sizes for the other group as in Figure 3 ). For all panels, only neurons showing an initial ΔPeredox lifetime response ≥0.05 ns were included for analysis. A paired Student’s t test was used for comparisons between normally distributed data, or a non-parametric paired Wilcoxon test was used otherwise. LDH inhibition partially rescued the Peredox responses in Isra+Cd 2+ and EGTA-AM, but not in 0Ca 2+ +EGTA, even though some pyruvate accumulation is also expected in the last condition due to less Ca 2+ -dependent pyruvate utilization in the mitochondria during stimulation. We do not have a definitive answer for this difference. It is possible that the Ca 2+ channel blockade or Ca 2+ chelation may not be complete, especially in dendrites, triggering a component of the metabolic responses that propagates to the soma, and is better revealed during LDH inhibition.
Figure Legend Snippet: A major Ca 2+ -dependent component of the NADH CYT also occurs under LDH inhibition. a. Comparison of the ΔPeredox/ΔRCaMP values obtained in the continuous presence of the LDH inhibitor GSK-2837808A, before and after the blockade of voltage gated Ca 2+ channels with a combination of isradipine and cadmium (Isra+Cd 2+ ; N neurons = 13, N slices = 3 and N mice = 3). The slices were exposed to GSK-2837808A for at least 30 min prior to the experiment. LDH inhibition should improve the ability to detect the cytosol-only component of Ca 2+ actions on the NADH CYT transients by increasing the control responses to stimulation, as well as by preventing the potential impact of pyruvate accumulation on these transients due to lower Ca 2+ influx into the mitochondria. The effects of the manipulation on the Peredox baseline and the RCaMP spike are also included for the experiments with or without the LDH inhibitor (the sample size is reported in Figure 3 for the latter). b—c. Comparisons for the application of the cell-permeable Ca 2+ chelator EGTA-AM (N neurons = 15, N slices = 3 and N mice = 3 for experiments with LDHi, sample sizes for the other group as in Figure 3 ), or the removal of Ca 2+ from the ACSF (N neurons = 136, N slices = 22 and N mice = 13 for experiments with LDHi, sample sizes for the other group as in Figure 3 ). For all panels, only neurons showing an initial ΔPeredox lifetime response ≥0.05 ns were included for analysis. A paired Student’s t test was used for comparisons between normally distributed data, or a non-parametric paired Wilcoxon test was used otherwise. LDH inhibition partially rescued the Peredox responses in Isra+Cd 2+ and EGTA-AM, but not in 0Ca 2+ +EGTA, even though some pyruvate accumulation is also expected in the last condition due to less Ca 2+ -dependent pyruvate utilization in the mitochondria during stimulation. We do not have a definitive answer for this difference. It is possible that the Ca 2+ channel blockade or Ca 2+ chelation may not be complete, especially in dendrites, triggering a component of the metabolic responses that propagates to the soma, and is better revealed during LDH inhibition.

Techniques Used: Inhibition, Mouse Assay

15) Product Images from "Vesicular Zinc Regulates the Ca2+ Sensitivity of a Subpopulation of Presynaptic Vesicles at Hippocampal Mossy Fiber Terminals"

Article Title: Vesicular Zinc Regulates the Ca2+ Sensitivity of a Subpopulation of Presynaptic Vesicles at Hippocampal Mossy Fiber Terminals

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.4164-11.2011

Decay kinetics of ZnT3 KO mice are different from their WT littermates. A1 , Averaged sweeps of MF EPSCs evoked at various frequencies in control ACSF. Responses at 0.2 Hz (blue traces) and 1 Hz (red traces) were scaled to 0.05 Hz baseline value (black traces). A2 , Decay kinetics of evoked EPSCs recorded from WT ( n = 7) and KO ( n = 9) mice could be fitted with a single or double exponentials. A3 , EGTA-AM changes the decay kinetics by decreasing the occurrence of the slower component for both genotypes ( n = 7). B , Average values of the decay kinetics for WT and KO in control condition and after application of EGTA-AM. CTRL, Control.
Figure Legend Snippet: Decay kinetics of ZnT3 KO mice are different from their WT littermates. A1 , Averaged sweeps of MF EPSCs evoked at various frequencies in control ACSF. Responses at 0.2 Hz (blue traces) and 1 Hz (red traces) were scaled to 0.05 Hz baseline value (black traces). A2 , Decay kinetics of evoked EPSCs recorded from WT ( n = 7) and KO ( n = 9) mice could be fitted with a single or double exponentials. A3 , EGTA-AM changes the decay kinetics by decreasing the occurrence of the slower component for both genotypes ( n = 7). B , Average values of the decay kinetics for WT and KO in control condition and after application of EGTA-AM. CTRL, Control.

Techniques Used: Mouse Assay

Related Articles

Mouse Assay:

Article Title: Vesicular Zinc Regulates the Ca2+ Sensitivity of a Subpopulation of Presynaptic Vesicles at Hippocampal Mossy Fiber Terminals
Article Snippet: .. The selective effect of EGTA-AM on ZnT3 KO mice raises the question whether the observed changes are caused by the absence of vesicular zinc or by the absence of the zinc transporter. ..

other:

Article Title: Vesicles derived via AP-3 dependent recycling contribute to asynchronous release and influence information transfer
Article Snippet: EGTA-AM was dissolved in properly oxygenated solution at pH 7.4.

Article Title: Autocrine activation of neuronal NMDA receptors by aspartate mediates dopamine- and cAMP-induced CREB-dependent gene transcription
Article Snippet: Antagonists were added 30 minutes before stimulation except for Bapta-AM and EGTA-AM which were added 60 before stimulation.

Article Title: Autocrine activation of neuronal NMDA receptors by aspartate mediates dopamine- and cAMP-induced CREB-dependent gene transcription
Article Snippet: Unlike Bapta-AM, EGTA-AM had only a small inhibitory effect on cAMP-induced CREB-dependent gene transcription , suggesting that the relevant increases in [Ca2+ ] occur within distinct microdomains near the site of Ca2+ entry ( ; ; ; ) rather than throughout the cytoplasm.

Article Title: Vesicular Zinc Regulates the Ca2+ Sensitivity of a Subpopulation of Presynaptic Vesicles at Hippocampal Mossy Fiber Terminals
Article Snippet: In contrast, EGTA-AM has significantly reduced glutamate release from ZnT3 KO synaptosomes (KCl, 24.9 ± 2.70 vs 17.4 ± 0.61 ng/μg; glutamate, 8.08 ± 0.79 vs 5.28 ± 0.65 ng/μg) ( B , C ).

Article Title: Vesicles derived via AP-3 dependent recycling contribute to asynchronous release and influence information transfer
Article Snippet: EGTA-AM has previously been shown to reduce the synchronous component under certain circumstances , , , this effect is most probably associated with multivesicular neurotransmitter release from mossy fiber boutons.

Article Title: Autocrine activation of neuronal NMDA receptors by aspartate mediates dopamine- and cAMP-induced CREB-dependent gene transcription
Article Snippet: To investigate this possibility, we compared the effects of Bapta-AM and EGTA-AM on cAMP-induced CREB-dependent transcription.

Inhibition:

Article Title: Autocrine activation of neuronal NMDA receptors by aspartate mediates dopamine- and cAMP-induced CREB-dependent gene transcription
Article Snippet: Consequently, unlike Bapta, EGTA will not buffer Ca2+ fast enough to prevent activation of Ca2+ -dependent processes within the microdomain in the vicinity of Ca2+ entry ( ; ; ; ). .. When EGTA-AM was substituted for Bapta-AM, stimulation of transcription by forskolin was only reduced by about 30% in contrast to 95% inhibition by Bapta , suggesting that increased Ca2+ within such microdomains mediates the stimulation of CREB-dependent gene transcription by cAMP. .. Since the results in suggested that 4x CRE activation by cAMP requires Ca2+ influx, we examined the effects of inhibitors of the two principal Ca2+ entry pathways in neurons, the NMDA class of ionotropic glutamate receptors (NMDARs) and voltage-gated Ca2+ channels (Cav s).

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    AnaSpec egta am
    Control experiment for 0.1% <t>DMSO</t> in the ACSF, as in the <t>EGTA-AM</t> experiments. Left: Representative trace of the effect of DMSO (0.1%) on the Peredox and RCaMP signals. Right: The change in Peredox lifetime elicited by stimulation was unaffected by the application of 0.1% DMSO for 30—60 min, contrary to the gradual decrease observed with EGTA-AM. The RCaMP spike decreased over time but it was not as attenuated as in EGTA-AM. The Peredox and RCaMP transients were compared using a Wilcoxon matched pairs test and a paired t-test, respectively (N neurons = 17, N slices = 4 and N mice = 3). Only neurons showing an initial ΔPeredox lifetime response ∼0.05 ns or higher were included for analysis.
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    Control experiment for 0.1% DMSO in the ACSF, as in the EGTA-AM experiments. Left: Representative trace of the effect of DMSO (0.1%) on the Peredox and RCaMP signals. Right: The change in Peredox lifetime elicited by stimulation was unaffected by the application of 0.1% DMSO for 30—60 min, contrary to the gradual decrease observed with EGTA-AM. The RCaMP spike decreased over time but it was not as attenuated as in EGTA-AM. The Peredox and RCaMP transients were compared using a Wilcoxon matched pairs test and a paired t-test, respectively (N neurons = 17, N slices = 4 and N mice = 3). Only neurons showing an initial ΔPeredox lifetime response ∼0.05 ns or higher were included for analysis.

    Journal: bioRxiv

    Article Title: The distinct roles of calcium in rapid control of neuronal glycolysis and the tricarboxylic acid cycle

    doi: 10.1101/2020.11.16.385526

    Figure Lengend Snippet: Control experiment for 0.1% DMSO in the ACSF, as in the EGTA-AM experiments. Left: Representative trace of the effect of DMSO (0.1%) on the Peredox and RCaMP signals. Right: The change in Peredox lifetime elicited by stimulation was unaffected by the application of 0.1% DMSO for 30—60 min, contrary to the gradual decrease observed with EGTA-AM. The RCaMP spike decreased over time but it was not as attenuated as in EGTA-AM. The Peredox and RCaMP transients were compared using a Wilcoxon matched pairs test and a paired t-test, respectively (N neurons = 17, N slices = 4 and N mice = 3). Only neurons showing an initial ΔPeredox lifetime response ∼0.05 ns or higher were included for analysis.

    Article Snippet: The final concentration of DMSO in the experiments was kept ≤0.04%, except for EGTA-AM (0.1%), for which control experiments with 0.1% DMSO-only solution were performed to rule out interferences from the organic solvent in the recordings.

    Techniques: Mouse Assay

    Stimulation of CREB-dependent transcription by forskolin requires an increase in [Ca 2+ ] within a microdomain. One day after transfecting with 4x CRE reporter plasmid, cultures were preincubated with Bapta-AM or EGTA-AM (10 μM) for 1 hr and then

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: Autocrine activation of neuronal NMDA receptors by aspartate mediates dopamine- and cAMP-induced CREB-dependent gene transcription

    doi: 10.1523/JNEUROSCI.1166-09.2009

    Figure Lengend Snippet: Stimulation of CREB-dependent transcription by forskolin requires an increase in [Ca 2+ ] within a microdomain. One day after transfecting with 4x CRE reporter plasmid, cultures were preincubated with Bapta-AM or EGTA-AM (10 μM) for 1 hr and then

    Article Snippet: Antagonists were added 30 minutes before stimulation except for Bapta-AM and EGTA-AM which were added 60 before stimulation.

    Techniques: Plasmid Preparation

    Spontaneous oscillations in the Peredox signal may occur during the prolonged application of zero Ca 2+ -ACSF, in the presence of EGTA and LDH inhibition. Prolonged exposure to a nominal Ca 2+ -free solution, plus LDH inhibition, caused spontaneous elevations of the Peredox lifetime in 25 ± 13% of the cells in each slice (slices = 10, mice = 5), contrasting with only ~3% of cells exposed to a brief exposure as in Figure 4c . The spontaneous elevations in Peredox lifetime occurred at any time after removing Ca 2+ from the ACSF. Although these neurons were not included in the analysis, once the spontaneous transients cleared, the experiment resumed as usual. The continuous presence of 1 mM EGTA and 2 µM GSK-2837808 (LDH inhibitor) is not indicated in the figure for simplicity. In the control ACSF, the [Ca 2+ ] was adjusted accordingly to yield a free concentration of 2 mM.

    Journal: eLife

    Article Title: The distinct roles of calcium in rapid control of neuronal glycolysis and the tricarboxylic acid cycle

    doi: 10.7554/eLife.64821

    Figure Lengend Snippet: Spontaneous oscillations in the Peredox signal may occur during the prolonged application of zero Ca 2+ -ACSF, in the presence of EGTA and LDH inhibition. Prolonged exposure to a nominal Ca 2+ -free solution, plus LDH inhibition, caused spontaneous elevations of the Peredox lifetime in 25 ± 13% of the cells in each slice (slices = 10, mice = 5), contrasting with only ~3% of cells exposed to a brief exposure as in Figure 4c . The spontaneous elevations in Peredox lifetime occurred at any time after removing Ca 2+ from the ACSF. Although these neurons were not included in the analysis, once the spontaneous transients cleared, the experiment resumed as usual. The continuous presence of 1 mM EGTA and 2 µM GSK-2837808 (LDH inhibitor) is not indicated in the figure for simplicity. In the control ACSF, the [Ca 2+ ] was adjusted accordingly to yield a free concentration of 2 mM.

    Article Snippet: Stock solutions of MgCl2 (1M) were purchased from Teknova (Hollister, CA). dorsomorphin dihydrochloride (Compound C) and GSK-2837808A were obtained from Tocris (Bristol, UK), EGTA-AM from Anaspec Inc (Fremont, CA) and α-pompilidotoxin from Alomone Labs (Jerusalem, Israel).

    Techniques: Inhibition, Mouse Assay, Concentration Assay

    A major Ca 2+ -dependent component of the NADH CYT also occurs under LDH inhibition. ( a ) Comparison of the ΔPeredox/ΔRCaMP values obtained in the continuous presence of the LDH inhibitor GSK-2837808A, before and after the blockade of voltage gated Ca 2+ channels with a combination of isradipine and cadmium (Isra+Cd 2+ ; N neurons = 13, N slices = 3 and N mice = 3). The slices were exposed to GSK-2837808A for at least 30 min prior to the experiment. LDH inhibition should improve the ability to detect the cytosol-only component of Ca 2+ actions on the NADH CYT transients by increasing the control responses to stimulation, as well as by preventing the potential impact of pyruvate accumulation on these transients due to lower Ca 2+ influx into the mitochondria. The effects of the manipulation on the Peredox baseline and the RCaMP spike are also included for the experiments with or without the LDH inhibitor (the sample size is reported in Figure 4 for the latter). ( b–c ) Comparisons for the application of the cell-permeable Ca 2+ chelator EGTA-AM (N neurons = 15, N slices = 3 and N mice = 3 for experiments with LDHi, sample sizes for the other group as in Figure 4 ), or the removal of Ca 2+ from the ACSF (N neurons = 136, N slices = 22 and N mice = 13 for experiments with LDHi, sample sizes for the other group as in Figure 4 ). For all panels, only neurons showing an initial ΔPeredox lifetime response ≥0.05 ns were included for analysis. A paired Student’s t test was used for comparisons between normally distributed data, or a non-parametric paired Wilcoxon test was used otherwise. LDH inhibition partially rescued the Peredox responses in Isra+Cd 2+ and EGTA-AM, but not in 0Ca 2+ +EGTA, even though some pyruvate accumulation is also expected in the last condition due to less Ca 2+ -dependent pyruvate utilization in the mitochondria during stimulation. We do not have a definitive answer for this difference. It is possible that the Ca 2+ channel blockade or Ca 2+ chelation may not be complete, especially in dendrites, triggering a component of the metabolic responses that propagates to the soma, and is better revealed during LDH inhibition.

    Journal: eLife

    Article Title: The distinct roles of calcium in rapid control of neuronal glycolysis and the tricarboxylic acid cycle

    doi: 10.7554/eLife.64821

    Figure Lengend Snippet: A major Ca 2+ -dependent component of the NADH CYT also occurs under LDH inhibition. ( a ) Comparison of the ΔPeredox/ΔRCaMP values obtained in the continuous presence of the LDH inhibitor GSK-2837808A, before and after the blockade of voltage gated Ca 2+ channels with a combination of isradipine and cadmium (Isra+Cd 2+ ; N neurons = 13, N slices = 3 and N mice = 3). The slices were exposed to GSK-2837808A for at least 30 min prior to the experiment. LDH inhibition should improve the ability to detect the cytosol-only component of Ca 2+ actions on the NADH CYT transients by increasing the control responses to stimulation, as well as by preventing the potential impact of pyruvate accumulation on these transients due to lower Ca 2+ influx into the mitochondria. The effects of the manipulation on the Peredox baseline and the RCaMP spike are also included for the experiments with or without the LDH inhibitor (the sample size is reported in Figure 4 for the latter). ( b–c ) Comparisons for the application of the cell-permeable Ca 2+ chelator EGTA-AM (N neurons = 15, N slices = 3 and N mice = 3 for experiments with LDHi, sample sizes for the other group as in Figure 4 ), or the removal of Ca 2+ from the ACSF (N neurons = 136, N slices = 22 and N mice = 13 for experiments with LDHi, sample sizes for the other group as in Figure 4 ). For all panels, only neurons showing an initial ΔPeredox lifetime response ≥0.05 ns were included for analysis. A paired Student’s t test was used for comparisons between normally distributed data, or a non-parametric paired Wilcoxon test was used otherwise. LDH inhibition partially rescued the Peredox responses in Isra+Cd 2+ and EGTA-AM, but not in 0Ca 2+ +EGTA, even though some pyruvate accumulation is also expected in the last condition due to less Ca 2+ -dependent pyruvate utilization in the mitochondria during stimulation. We do not have a definitive answer for this difference. It is possible that the Ca 2+ channel blockade or Ca 2+ chelation may not be complete, especially in dendrites, triggering a component of the metabolic responses that propagates to the soma, and is better revealed during LDH inhibition.

    Article Snippet: Stock solutions of MgCl2 (1M) were purchased from Teknova (Hollister, CA). dorsomorphin dihydrochloride (Compound C) and GSK-2837808A were obtained from Tocris (Bristol, UK), EGTA-AM from Anaspec Inc (Fremont, CA) and α-pompilidotoxin from Alomone Labs (Jerusalem, Israel).

    Techniques: Inhibition, Mouse Assay

    DMSO control for the EGTA-AM experiments. Left: Representative trace of the effect of DMSO (0.1%) on the Peredox and RCaMP signals. Right: The change in Peredox lifetime elicited by stimulation was unaffected by the application of 0.1% DMSO for 30–60 min, contrary to the gradual decrease observed with EGTA-AM. The RCaMP spike decreased over time but it was not as attenuated as in EGTA-AM. The Peredox and RCaMP transients were compared using a Wilcoxon matched pairs test and a paired t-test, respectively (N neurons = 17, N slices = 4 and N mice = 3). Only neurons showing an initial ΔPeredox lifetime response ~0.05 ns or higher were included for analysis.

    Journal: eLife

    Article Title: The distinct roles of calcium in rapid control of neuronal glycolysis and the tricarboxylic acid cycle

    doi: 10.7554/eLife.64821

    Figure Lengend Snippet: DMSO control for the EGTA-AM experiments. Left: Representative trace of the effect of DMSO (0.1%) on the Peredox and RCaMP signals. Right: The change in Peredox lifetime elicited by stimulation was unaffected by the application of 0.1% DMSO for 30–60 min, contrary to the gradual decrease observed with EGTA-AM. The RCaMP spike decreased over time but it was not as attenuated as in EGTA-AM. The Peredox and RCaMP transients were compared using a Wilcoxon matched pairs test and a paired t-test, respectively (N neurons = 17, N slices = 4 and N mice = 3). Only neurons showing an initial ΔPeredox lifetime response ~0.05 ns or higher were included for analysis.

    Article Snippet: The final concentration of DMSO in the experiments was kept ≤0.04%, except for EGTA-AM (0.1%), for which control experiments with 0.1% DMSO-only solution were performed to rule out interferences from the organic solvent in the recordings.

    Techniques: Mouse Assay