Journal: bioRxiv

Article Title: Glutamate receptor-dependent cytosolic acidification in hippocampal neurons involves passive flux of protons from the extracellular space

doi: 10.1101/2024.11.17.624027

Figure Lengend Snippet: ( A ) Graphical depiction of the experimental design ( B ) Representation of the injection sites of the dual AAV strategy for expression of the jRGECO1a Ca 2+ sensor and cyto-pHluorin pH sensor; AAV.CaMKII.Cyto-pHluorin.WPRE.SV40 and AAV.hSyn.NES.jRGECO1a.WPRE.SV40. The viruses were injected bilateral on the CA1 region of the hippocampus. ( C ) Illustration of viral experimental setup. Acute hippocampal slices were prepared from C57bl6N mice with the AAV-mediated neuron-specific expression of the cytosolic biosensors jRGECO1a and cyto-pHluorin. Fluorescence intensities were recorded from the hippocampal CA1 region using 2-photon microscopy before, during and after LTP-inducing 20xTBS or 1xTBS stimulation to the Schaffer collaterals. Successful LTP establishment at CA3-CA1 synapses by 20xTBS stimulation was assessed by continuous recording of evoked field potentials in the CA1 region. The insert outlines the different hippocampal layers. ( D ) Representative median filtered 2-photon microscopy images of a part of the hippocampal CA1 region, showing the expression of the Ca 2+ -sensor, jRGECO1a, and the pH sensor, cyto-pHluorin. (Scale bar, 20 µM). The somatic region of CA1 neurons, defined by the dotted lines, were used in fluorescence analyses. The inserts (bottom right side for each image) show a magnification of a sample of analyzed somas. ( E ) Example trace of normalized field potential recordings before and after LTP induction through TBS stimulation. The inset displays representative fEPSP recordings before (black) and after (grey) LTP induction. ( F ) Heatmap representation of jRGECO1a and Cyto-pHluorin responses upon the 20XTBS stimulation. ( G ) Individual representative traces from neuronal responses within the same slice shown in (D) for jRGECO1a and Cyto-pHluorin. ( H ) Representative time course of the normalized mean fluorescence (mean F/F0) for jRGECO1a (red) and cyto-pHluorin (green) from the sample of CA1 somas shown in (D) stimulated with 20xTBS. The time course shows the change in fluorescence before, during and after LTP induction. Control traces from CA1 somas stimulated with 1xTBS shown at the bottom right of each of the 20xTBS representative traces. The shaded area illustrates the standard deviation from the mean response. ( I ) Individual maximal fluorescence responses induced by the LTP-inducing stimulation. The maximal response values reflect the maximal fold change in fluorescence for jRGECO1a and cyto-pHluorin in individual somas, recorded from three different slices. Ratio between both maximal responses is also plotted as (1/ΔCyto-pHluorin)/ ΔjRGECO1a. ( J ) Comparison of the maximal responses in individual experiments is shown in a dotplot. Data are shown on log10-scales. (N = 45 cells). SR: Stratum Radiatum, SP: Stratum Pyramidale, SO: Stratum Oriens.

Article Snippet: A bipolar tungsten stimulation electrode (TM33CCNON; World Precision Instruments, Sarasota, FL, USA) connected to a stimulus isolator (WPI A365, USA) was positioned in the stratum radiatum region at the CA3-CA1 border in the dorsal hippocampus.

Techniques: Injection, Expressing, Fluorescence, Microscopy, Control, Standard Deviation, Comparison

Journal: bioRxiv

Article Title: Glutamate receptor-dependent cytosolic acidification in hippocampal neurons involves passive flux of protons from the extracellular space

doi: 10.1101/2024.11.17.624027

Figure Lengend Snippet: (A) Representative image sequence (2 minutes interval for 30 minutes) of a hippocampal neuron co-expressing R-GECO1.2 (red) and cyto-pHluorin (green). (Scale bar, 20 μm). Images show the change in fluorescence in response to L-glutamate. (B) Representative magnified images from (A) before and after L-glutamate stimulation where dotted line shows the area measured. (C-G) Representative time course of the normalized fluorescence (F/F0) from the somatic region (see FigS1A), in response to chemical stimulation or depolarization, plotted on a log10-scale. L-glutamate (10 μM), GABA (50 µM for 10-15 min, or 200 µM for 10-20 min), NMDA (20 μM NMDA, 10 μM glycine), AMPA (100 μM AMPA, 100 μM APV) or high KCl (60 mM) was applied at the indicated time points for 3 min. (H) Comparison of the maximal responses induced by the stimulation protocols. The maximal response values reflect the maximal fold change in fluorescence for R-GECO1.2 and cyto-pHluorin in individual experiments. Data are shown on a log10-scale with mean ± SD. Log-transformed R-GECO1.2 or cyto-pHluorin responses from NMDA, AMPA and KCl experiments (log(F/F 0 )) are evaluated by Brown-Forsythe and Welch ONE-way ANOVA tests, with Dunnett’s T3 multiple comparisons test, *P≤0.1, ****P≤0.0001. AMPA data include AMPA responses in presence and absence of APV. KCl data include KCl responses in presence of APV and NBQX. (I) Comparison of cyto-pHluorin to R-GECO1.2 maximal response ratios for individual experiments. Ratios are calculated with inversed values of cyto-pHluorin responses (F(0)/F), and are shown on a log10-scale with medians and interquartile ranges. Data from NMDA, AMPA and KCl experiments are evaluated by Kruskal-Wallis test of column with Dunn’s multiple comparisons test, *P≤0.1, ****P≤0.0001. L-glutamate (N = 5 cells), GABA data (N = 4 cells), NMDA (N = 76 cells), AMPA (N = 33 cells), KCl (N = 28 cells). (J) x-y plot showing the maximal cyto-pHluorin response as a function of the maximal R-GECO1.2 response for each tested cell. The maximal response values reflect the maximal fold change in fluorescence for R-GECO1.2 and cyto-pHluorin in individual experiments, with cyto-pHluorin responses plotted as inversed values (F(0)/F). Data is a summary of the initial stimulations obtained throughout the rest of study and are shown on log10-scales.

Article Snippet: A bipolar tungsten stimulation electrode (TM33CCNON; World Precision Instruments, Sarasota, FL, USA) connected to a stimulus isolator (WPI A365, USA) was positioned in the stratum radiatum region at the CA3-CA1 border in the dorsal hippocampus.

Techniques: Sequencing, Expressing, Fluorescence, Comparison, Transformation Assay

Journal: bioRxiv

Article Title: Glutamate receptor-dependent cytosolic acidification in hippocampal neurons involves passive flux of protons from the extracellular space

doi: 10.1101/2024.11.17.624027

Figure Lengend Snippet: (A, D, G) Representative image sequences of hippocampal neurons co-expressing R-GECO1.2 (red) and cyto-pHluorin (green). (Scale bar, 20 μm). Images are plotted for the indicated time points and show the change in cell fluorescence in response to NMDA, AMPA or KCl in presence or absence of extracellular Ca 2+ . (B, E, H) Representative time courses of the normalized fluorescence (F/F0) in the somatic region of the neurons shown in (A, D, G), plotted on a log10-scale. NMDA (20 µM NMDA, 10 µM glycine), AMPA (100 µM AMPA, 100 µM APV), or KCl (60 mM) was applied at the indicated time points for 3 min before and after Ca 2+ exclusion from the extracellular medium. (C, F, I) Individual maximal responses to the stimulation protocols in presence or absence of extracellular Ca 2+ . Data are shown on log10-scales, and are evaluated by ratio paired t tests, *P≤0.05, **P≤0.01, ***P≤0.001 ****P≤0.0001. NMDA (N = 5 cells), AMPA (N = 5 cells), 60 mM KCl (N = 3 cells).

Article Snippet: A bipolar tungsten stimulation electrode (TM33CCNON; World Precision Instruments, Sarasota, FL, USA) connected to a stimulus isolator (WPI A365, USA) was positioned in the stratum radiatum region at the CA3-CA1 border in the dorsal hippocampus.

Techniques: Expressing, Fluorescence

Journal: bioRxiv

Article Title: Glutamate receptor-dependent cytosolic acidification in hippocampal neurons involves passive flux of protons from the extracellular space

doi: 10.1101/2024.11.17.624027

Figure Lengend Snippet: ( A ) Schematic representation of the expected mechanism of action of Ionomacyn incubation. ( B ) Representative image sequence of a hippocampal neuron co-expressing R-GECO1.2 (red) and cyto-pHluorin (green). (Scale bar, 20 μm). Images are plotted for every two minutes of recording from 0 to 20 min and show the change in fluorescence in response to bath administration of the Ca 2+ ionophore ionomycin. ( C ) Representative time course of the normalized fluorescence (F/F0) for the neuron shown in (B), plotted on a log10-scale. Ionomycin (10 µM) was applied at the indicated time point for 3 min. ( D ) Comparison of the maximal responses induced by ionomycin treatment (for 2-3 min) to the maximal responses induced by NMDA, AMPA and KCl. The maximal response values reflect the maximal fold change in fluorescence for R-GECO1.2 and cyto-pHluorin in individual experiments. Data are shown on a log10-scale with mean ± SD. Log-transformed R-GECO1.2 or cyto-pHluorin responses are evaluated by Brown-Forsythe and Welch ONE-way ANOVA tests, with Dunnett’s T3 multiple comparisons test, ****P≤0.0001. ( E ) x-y plot showing the maximal cyto-pHluorin response as a function of the maximal R-GECO1.2 response for individual cells treated with ionomycin for 1-3 min compared to stimulation with NMDA, AMPA and KCl. The maximal response values reflect the maximal fold change in fluorescence for R-GECO1.2 and cyto-pHluorin in individual experiments, with cyto-pHluorin responses plotted as inversed values (F(0)/F). Data are shown on log10-scales. ( F ) Comparison of cyto-pHluorin to R-GECO1.2 maximal response ratios for individual experiments shown in (E). Ratios are calculated with inversed values of cyto-pHluorin responses (F(0)/F), and are shown on a log10-scale with medians and interquartile ranges. Data are evaluated by Kruskal-Wallis test of column with Dunn’s multiple comparisons test, ****P≤0.0001. Ionomycin data are from 10 cells (N = 4-6 experiments for each treatment duration).

Article Snippet: A bipolar tungsten stimulation electrode (TM33CCNON; World Precision Instruments, Sarasota, FL, USA) connected to a stimulus isolator (WPI A365, USA) was positioned in the stratum radiatum region at the CA3-CA1 border in the dorsal hippocampus.

Techniques: Incubation, Sequencing, Expressing, Fluorescence, Comparison, Transformation Assay

Journal: bioRxiv

Article Title: Glutamate receptor-dependent cytosolic acidification in hippocampal neurons involves passive flux of protons from the extracellular space

doi: 10.1101/2024.11.17.624027

Figure Lengend Snippet: (A,E) Schematic representation of the expected mechanism of action of Verapamil in the two different stimulation paradigms ( B, F ) Representative image sequences of hippocampal neurons co-expressing R-GECO1.2 (red) and cyto-pHluorin (green). (Scale bar, 20 μm). Images are plotted for the indicated time points and show the change in fluorescence in response to AMPA or KCl before and after addition of the voltage-gated Ca 2+ channel inhibitor verapamil. (C, G) Representative time courses of the normalized fluorescence (F/F0) for the neurons shown in (B, F), plotted on a log10-scale. AMPA (100 µM) or high KCl (60 mM) was applied at the indicated time points for 3 min before and after incubation with verapamil (100 µM) for 30 min. ( D, H ) Individual maximal responses induced by AMPA or KCl before and after verapamil incubation, shown on a log10-scale. Data are evaluated by ratio paired t tests, **P≤0.01, ***P≤0.001 ****P≤0.0001. AMPA (N = 7 cells), KCl (N = 8 cells). AMPA data include responses in presence of APV.

Article Snippet: A bipolar tungsten stimulation electrode (TM33CCNON; World Precision Instruments, Sarasota, FL, USA) connected to a stimulus isolator (WPI A365, USA) was positioned in the stratum radiatum region at the CA3-CA1 border in the dorsal hippocampus.

Techniques: Expressing, Fluorescence, Incubation

Journal: bioRxiv

Article Title: Glutamate receptor-dependent cytosolic acidification in hippocampal neurons involves passive flux of protons from the extracellular space

doi: 10.1101/2024.11.17.624027

Figure Lengend Snippet: ( A,G ) Schematic representation of Ca 2+ chelation by BAPTA-AM in the two different stimulation paradigms ( B, H ) Representative image sequences of a hippocampal neuron co-expressing R-GECO1.2 (red) and cyto-pHluorin (green). (Scale bar, 20 μm). Images are plotted for every two minutes of recording in the indicated time intervals and show the change in fluorescence in response to AMPA or KCl after pre-incubation with the Ca 2+ chelator BAPTA-AM. ( C, I ) Representative time courses of the normalized fluorescence (F/F0) in the somatic region of the neurons shown in (A, F), plotted on log10-scales. AMPA (100 µM) or high KCl (60 mM) was applied at the indicated time points for 3 min, after pre-incubation with BAPTA-AM (20 µM) for 20 min. ( D, J ) Comparison of the maximal responses induced by AMPA or KCl in presence of BAPTA-AM (15-20 min pre-incubation) compared to the maximal responses induced by AMPA or KCl in absence of BAPTA-AM. The maximal response values reflect the maximal fold change in fluorescence for R-GECO1.2 and cyto-pHluorin in individual experiments. Data are shown on a log10-scale with mean ± SD. Log-transformed R-GECO1.2 or cyto-pHluorin responses are evaluated by unpaired t-tests, ***P≤0.001, ****P≤0.0001. ( E, K ) x-y plot showing the maximal cyto-pHluorin response as a function of the maximal R-GECO1.2 response for individual cells treated with AMPA or KCl in presence of BAPTA-AM (15-20 min pre-incubation) compared to the maximal responses induced by AMPA or KCl in absence of BAPTA-AM. The maximal response values reflect the maximal fold change in fluorescence for R-GECO1.2 and cyto-pHluorin in individual experiments, with cyto-pHluorin responses plotted as inversed values (F(0)/F). Data are shown on log10-scales. ( F, L ) Comparison of cyto-pHluorin to R-GECO1.2 maximal response ratios for individual experiments shown in (E, K). Ratios are calculated with inversed values of cyto-pHluorin responses (F(0)/F), and are shown on a log10-scale with medians and interquartile ranges. Data are evaluated by Mann-Whitney tests, ns: P>0.1. AMPA (N = 3 cells), KCl (N = 3 cells).

Article Snippet: A bipolar tungsten stimulation electrode (TM33CCNON; World Precision Instruments, Sarasota, FL, USA) connected to a stimulus isolator (WPI A365, USA) was positioned in the stratum radiatum region at the CA3-CA1 border in the dorsal hippocampus.

Techniques: Expressing, Fluorescence, Incubation, Comparison, Transformation Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: Glutamate receptor-dependent cytosolic acidification in hippocampal neurons involves passive flux of protons from the extracellular space

doi: 10.1101/2024.11.17.624027

Figure Lengend Snippet: ( A, B ) Representative image sequence and time course of the normalized fluorescence for a hippocampal neuron expressing cyto-pHluorin (green). Images are plotted for every minute of recording from 0-11 min and show the cell fluorescence during the gradual decrease of extracellular pH from 9.0 to 7.4 (pH 9.0, 8.5, 8.0, 7.4). The time course shows the normalized fluorescence (F/F0) in the somatic region of the cell, plotted on a log10-scale. The extracellular buffers were each applied for 1 min at the indicated time points. N = 1 cell. ( C, D ) Representative image sequence and time course of the normalized fluorescence for a hippocampal neuron expressing cyto-pHluorin (green). Images are plotted for every minute of recording from 0-11 min and show the cell fluorescence during the gradual decrease of extracellular pH from 9.0 to 7.4 (pH 9.0, 8.5, 8.0, 7.4) in presence of the protonophore CCCP (5 µM). The time course shows the normalized fluorescence (F/F0) in the somatic region of the cell, plotted on a log10-scale. The extracellular buffers were each applied for 1 min in presence of CCCP. N = 5 cells. ( E,K ) Schematic representation of the influence of extracellular pH 9 in the proton gradient in the two different stimulation paradigms ( F, L ) Representative image sequences of a hippocampal neuron co-expressing R-GECO1.2 (red) and cyto-pHluorin (green). (Scale bar, 20 μm). Images are plotted for every two minutes of recording from 0 to 20 min and show the change in fluorescence in response to AMPA or KCl at extracellular pH 9. ( G, M ) Representative time courses of the normalized fluorescence (F/F0) in the somatic region of the neurons shown in (F, L), plotted on log10-scales. AMPA (100 µM) or KCl (60 mM) was applied at the indicated time points for 3 min in extracellular buffers adjusted to pH 9. ( H, N ) Comparison of the maximal responses induced by AMPA or KCl at extracellular pH 9 compared to the maximal responses induced by AMPA or KCl at extracellular pH 7.4. The maximal response values reflect the maximal fold change in fluorescence for R-GECO1.2 and cyto-pHluorin in individual experiments. Data are shown on a log10-scale with mean ± SD. Log-transformed R-GECO1.2 or cyto-pHluorin responses are evaluated by unpaired t-test, **P≤0.01. ( I, O ) x-y plot showing the maximal cyto-pHluorin response as a function of the maximal R-GECO1.2 response for individual cells treated with AMPA or KCl in at extracellular pH 9 compared to the maximal responses induced by AMPA or KCl at extracellular pH 7.4. The maximal response values reflect the maximal fold change in fluorescence for R-GECO1.2 and cyto-pHluorin in individual experiments, with cyto-pHluorin responses plotted as inversed values (F(0)/F). Data are shown on log10-scales. ( J, P ) Comparison of cyto-pHluorin to R-GECO1.2 maximal response ratios for individual experiments shown in (H, M). Ratios are calculated with inversed values of cyto-pHluorin responses (F(0)/F), and are shown on a log10-scale with medians and interquartile ranges. Data are evaluated by Mann-Whitney tests, **P≤0.01. AMPA/pH9 (N = 6 cells), KCl/pH9 (N = 4 cells).

Article Snippet: A bipolar tungsten stimulation electrode (TM33CCNON; World Precision Instruments, Sarasota, FL, USA) connected to a stimulus isolator (WPI A365, USA) was positioned in the stratum radiatum region at the CA3-CA1 border in the dorsal hippocampus.

Techniques: Sequencing, Fluorescence, Expressing, Comparison, Transformation Assay, MANN-WHITNEY