Journal: Neuroscience

Article Title: Extracellular Protons Mediate Presynaptic Homeostatic Potentiation at the Mouse Neuromuscular Junction

doi: 10.1016/j.neuroscience.2021.01.036

Figure Lengend Snippet: D-Tubocurarine (d-TC) induces presynaptic homeostatic potentiation (PHP) and this is blocked by ASIC inhibitors. A. mEPPs and EPPs were measured before and after a 10 min incubation in d-TC (n = 75 NMJs, 15 mice). Quantal Content (QC) was estimated from corrected EPP and mEPP amplitudes. The data are presented as violin plots, which show the probability density of the data at different values. The mean differences are shown as estimation plots (see Experimental Procedure: Statistical Analysis.) The insets are sample traces of EPPs and mEPPs before (black line) and after (grey line) d-TC. Each trace is the average of 10 measurements. Calibration bars indicate 0.2 mv, 2 ms for the mEPPs and 5 mv, 2 ms for the EPPs. B. QC was calculated from corrected mEPP and EPP amplitudes in the presence of 50 μM benzamil (n=25, 5 mice), 100 μM benzamil (n=15, 3 mice), 50 nM PcTx1 (n=73, 10 mice), 100 nM PcTx1 (n=25, 5 mice), and 50 nM Mamb3 (n=20, 4 mice) before and after a 10 min incubation in d-TC. For each condition, the ratios of QC after vs. before dTC were calculated and plotted as violin plots. The mean differences are shown as estimation plots. In both A and B, means are depicted as black dots; 95% confidence intervals are indicated by the ends of vertical error bars. In all figures, p values are indicated and were calculated from an ordinary one-way ANOVA.

Article Snippet: In and , benzamil hydrochloride hydrate (Sigma-Aldrich, St. Louis, MO), psalmotoxin-1 (PcTx1; Alomone Labs), or mambalgin-3 (Mamb3; Alomone Labs), were applied (in the presence of μ-Ctx) for 30 minutes prior to recording.

Techniques: Incubation

Journal: Neuroscience

Article Title: Extracellular Protons Mediate Presynaptic Homeostatic Potentiation at the Mouse Neuromuscular Junction

doi: 10.1016/j.neuroscience.2021.01.036

Figure Lengend Snippet: High concentrations of the ASIC inhibitors Benzamil and PcTx1 reduce mEPP and EPP amplitude. MEPPs, EPPs and Quantal Content are plotted from NMJs incubated in either control saline (n=75, same data show in 1A), 100 μM benzamil (n=15, 3 mice), and 100 nM PcTx1 (n=25, 5 mice). Data are plotted as in Fig. 1. P values are indicated and were calculated from Student’s t-test or ordinary one-way ANOVA.

Article Snippet: In and , benzamil hydrochloride hydrate (Sigma-Aldrich, St. Louis, MO), psalmotoxin-1 (PcTx1; Alomone Labs), or mambalgin-3 (Mamb3; Alomone Labs), were applied (in the presence of μ-Ctx) for 30 minutes prior to recording.

Techniques: Incubation

Journal: Neuroscience

Article Title: Extracellular Protons Mediate Presynaptic Homeostatic Potentiation at the Mouse Neuromuscular Junction

doi: 10.1016/j.neuroscience.2021.01.036

Figure Lengend Snippet: Lowering extracellular pH underlies the upregulation of QC triggered by d-TC. A. mEPPs and EPPs were measured and QC estimated before and after a 10 min incubation in pH 7.2 saline with 100nM PcTx1 present throughout (n=25, 5 mice). The data for mEPPs, EPPs and QC are presented as violin plots. The plot on the far right includes the data from Fig. 3B to depicts the effect of pH 7.2 saline under control conditions vs. in the presence of 100 nM PcTx1. The Mean differences are plotted as a bootstrap sampling distribution. Means are depicted as black dots; 95% confidence intervals are indicated by the ends of vertical error bars. In all figures, p values are indicated and were calculated from an ordinary one-way ANOVA. B. mEPPs and EPPs were measured and QC was estimated in pH 7.4 saline, after a 7 min incubation in pH 7.2 saline, and then after a 15 min incubation with d-TC, still in pH 7.2 saline (n = 5 mice). Although dTC had its normal effects on mEPP and EPP amplitude (compare to Fig. 1A), dTC did not alter QC when compared to pH 7.2 saline. The data is plotted as in panel A. C. The ratio of QC after application of dTC compared to before dTC was applied (post/pre dTC) is shown for pH 7.4 saline (this is the same data presented in Fig 1B, Control) and for pH 7.2 saline (the same data presented in Fig. 4B).

Article Snippet: In and , benzamil hydrochloride hydrate (Sigma-Aldrich, St. Louis, MO), psalmotoxin-1 (PcTx1; Alomone Labs), or mambalgin-3 (Mamb3; Alomone Labs), were applied (in the presence of μ-Ctx) for 30 minutes prior to recording.

Techniques: Incubation, Sampling

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

Article Title: Heteromeric ASIC channels composed of ASIC2b and ASIC1a display novel channel properties and contribute to acidosis-induced neuronal death

doi: 10.1523/JNEUROSCI.1665-11.2011

Figure Lengend Snippet: Knockdown of ASIC2b alters barium sensitivity and steady-state desensitization of hippocampal proton-gated currents. A, Quantification of anti-ASIC2b shRNA effects on genomic mRNA. The graph indicates the fold change in specified mRNAs between anti-ASIC2b shRNA vector-transfected cultures and empty vector-transfected cultures, as measured by quantitative RT-PCR (see Materials and Methods). **p < 0.01 (t test, unpaired); n = 3. B, Three representative traces showing dynamic sensitivity of acid-evoked current to pH 7.4 preconditioning, extracellular Ba2+, and PcTX1 in wild-type hippocampal neurons transfected with empty vector. Maximal current (left) was considered current evoked by perfusion of pH 6.0 (white bars) from a holding pH of 7.9 in the absence of Ba2+ and PcTX1. Colored bars indicate holding pH before acid application (white bars). Black bars (above trace) indicate perfusion of acidic solution containing either 10 mm Ba2+ or perfusion of pH 7.4 solutions containing 50 nm PcTX1 for 1.5 min before acid application as indicated. C, Comparison of pH 7.4 preconditioning, Ba2+ and PcTX1 effects on acid-evoked current amplitude in vector-transfected cells and anti-ASIC2b shRNA-transfected cells. Average current amplitude after the specified treatments was normalized to maximal current amplitude, and the percentage inhibition is the difference in normalized current amplitude. *p < 0.05; **p < 0.01 (t test, unpaired); n = 20. D, ASIC population analysis in empty vector- or anti-ASIC2b shRNA-transfected neurons. Individual neurons were classified as having a type ASIC1a, ASIC2b/1a, or ASIC2a/1a phenotype based on their response to extracellular Ba2+ and PcTX1 as before; n = 20 for both groups. Error bars are the SEM.

Article Snippet: Barium, tetraethylammonium (TEA), zinc, N , N , N ′, N ′-tetrakis(2-pyridylmethyl)ethylenediamine, and psalmotoxin 1 (PcTX1) experiments were done with Ringer's solution that also contained BaCl 2 , tetraethylammonium chloride, ZnCl 2 , TPEN, or purified PcTX1 peptide (Peptides International) at the concentrations indicated in the figures and figure legends.

Techniques: shRNA, Plasmid Preparation, Transfection, Quantitative RT-PCR, Inhibition

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

Article Title: Heteromeric ASIC channels composed of ASIC2b and ASIC1a display novel channel properties and contribute to acidosis-induced neuronal death

doi: 10.1523/JNEUROSCI.1665-11.2011

Figure Lengend Snippet: Psalmotoxin 1 inhibits ASIC2b/1a current. A–C, Representative traces showing PcTX1 (0.2 μm) effects on mouse ASIC1a (A), ASIC2b/1a (B), and ASIC2a/1a (C) current in oocytes. Activation of ASIC current was achieved by applying pH 4.5 (white bar) from holding pH 7.9. The black bar indicates application of 0.2 μm PcTX1 for 3 min before application of pH 4.5. D, Quantification of PcTX1 effect on ASIC current. % Inhibition, Difference in current amplitude after PcTX1 treatment compared with maximal current amplitude in the absence of PcTX1. **p < 0.01, compared with ASIC1a group with ANOVA (1-way). n = 6 for ASIC1a, n = 7 for ASIC2b/1a, and n = 5 for ASIC2a/1a. E, Concentration dependence of PcTX1 inhibition. PcTX1 was applied at the indicated concentrations, and ASIC current was measured as above. The IC50 shown is the average value calculated from individual cells (n = 4–7 for ASIC1a and ASIC2b/1a). Error bars are the SEM.

Article Snippet: Barium, tetraethylammonium (TEA), zinc, N , N , N ′, N ′-tetrakis(2-pyridylmethyl)ethylenediamine, and psalmotoxin 1 (PcTX1) experiments were done with Ringer's solution that also contained BaCl 2 , tetraethylammonium chloride, ZnCl 2 , TPEN, or purified PcTX1 peptide (Peptides International) at the concentrations indicated in the figures and figure legends.

Techniques: Activation Assay, Inhibition, Concentration Assay

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

Article Title: Heteromeric ASIC channels composed of ASIC2b and ASIC1a display novel channel properties and contribute to acidosis-induced neuronal death

doi: 10.1523/JNEUROSCI.1665-11.2011

Figure Lengend Snippet: ASIC2 is required for dynamic barium sensitivity in hippocampal neurons. A, B, Quantification of Ba2+ (A) and PcTX1 (B) inhibition in wild-type and ASIC2 knock-out neurons. The whole-cell patch-clamp technique was used to record pH 6.0-evoked currents (holding pH was 7.9) in day 14–21 cultured hippocampal neurons. Current was evoked in the presence of 10 mm Ba2+ or 0.2 μm PcTX1 and normalized to current in the absence of either compound. % Inhibition, Difference in normalized current amplitude after treatment compared with maximal current amplitude. Note the different y-axis scale in A and B. n = 22 for ASIC2+/+, and n = 14 for ASIC2−/−. **p < 0.01, compared with ASIC2+/+ group with t test analysis (2-tailed, unpaired data). C, Representative traces showing dynamic sensitivity of acid-evoked current to extracellular Ba2+ and PcTX1 in individual neurons. Black bars indicate perfusion of extracellular solution containing 10 mm Ba2+ before and during acid application or perfusion of extracellular solution containing 0.2 μm PcTX1 for 1.5 min before acid application. D, Representative trace showing a loss of Ba2+ inhibition in hippocampal neurons lacking the ASIC2 gene (ACCN1). The effects of Ba2+ and PcTX1 on acid-evoked current were assessed as above. E, Comparison of Ba2+ and PcTX1 inhibition of acid-evoked current among individual neurons. Cells were categorized based on the percentage of inhibition observed (compared with maximal current) in the presence of 10 mm Ba2+ and 0.2 μm PcTX1. The graph shows the relative percentage of individual cells within each group that displayed the indicated phenotype: ASIC2b/1a like, >20% Ba2+ inhibition, >20% PcTX1 inhibition; ASIC1a like, <20% Ba2+ inhibition, >20% PcTX1 inhibition; ASIC2a/1a, <20% Ba2+ inhibition, <20% PcTX1 inhibition; mixed, >20% Ba2+ inhibition, <20% PcTX1 inhibition. n = 22 for ASIC2+/+, and n = 14 for ASIC2−/−. Error bars are the SEM.

Article Snippet: Barium, tetraethylammonium (TEA), zinc, N , N , N ′, N ′-tetrakis(2-pyridylmethyl)ethylenediamine, and psalmotoxin 1 (PcTX1) experiments were done with Ringer's solution that also contained BaCl 2 , tetraethylammonium chloride, ZnCl 2 , TPEN, or purified PcTX1 peptide (Peptides International) at the concentrations indicated in the figures and figure legends.

Techniques: Inhibition, Knock-Out, Patch Clamp, Cell Culture