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: Science Advances

Article Title: Extracellular electrical recording of pH-triggered bursts in C6 glioma cell populations

doi: 10.1126/sciadv.1600516

Figure Lengend Snippet: ( A ) Time evolution of current noise of C6 glioma cells upon adding PcTX-1, up to a concentration of 100 nM in acidified cell culture medium. The black line represents the original state. The red line is recorded after adding PcTX-1 to a concentration of only 100 nM. The blue curve represents the recovery plot after thrice washing. ( B ) The corresponding current noise spectra of . ( C ) The mean of 300 spike amplitudes of acidified cells as depicted in . PcTX-1 data quantification recorded over two inhibitory experiments.

Article Snippet: PcTX-1 (Merck Chemicals GmbH) was directly dissolved in cell culture medium (F-12K) to a working concentration of 0.1 M. Final dilutions of 1 μM TTX and 100 nM PcTX-1 were obtained by diluting the working solution in the cell culture medium.

Techniques: Concentration Assay, Cell Culture

Journal: Science signaling

Article Title: The acid-sensing ion channel ASIC1a mediates striatal synapse remodeling and procedural motor learning

doi: 10.1126/scisignal.aar4481

Figure Lengend Snippet: (A to D) Representative immunoblots and statistical analysis of phosphorylated (“p-”) and total protein abundance of CaMKII (A and B) and ERK1 and ERK2 (C and D) in striata from WT (+/+) and Asic1a null (−/−) mice. n = 4 to 6 for each group. *P < 0.05, **P < 0.01, and ***P < 0.001, unpaired Student’s t test. (E to H) Effects of treatment with acid (pH 6.0) on the phosphorylation of CaMKII (E and F) and ERK (G and H). (E and G) Representative immunoblots and analysis showing phosphorylated and total protein abundances of CaMKII (E and F) and ERK1 and ERK2 (G and H) in cultured striatal neurons prepared from WT mice and maintained at pH 7.4 only or exposed to a pH 6.0 external solution for 2 min without or with pretreatment (30 min) of PcTX1 (20 nM) or KN93 (10 μM), as indicated. n = 6 to 15 for each group. **P < 0.01 and ***P < 0.001, compared with pH 7.4 only; #P < 0.05, compared with pH 6.0 alone, unpaired Student’s t test. (I to L) Representative immunoblots and statistical analysis of the effects of the same acid treatment on the phosphorylation of CaMKII (I and J) and ERK (K and L) in cultured striatal neurons prepared from Asic1a null (Asic1a−/−) mice. n = 3 for each group.

Article Snippet: For PcTX1 or KN93 blockage experiments, 20 nM PcTX1 (Peptide Institute) or 10 μM KN93 was first added into the culture medium 30 min before acidic treatment and then co-applied with the pH 6.0 solution.

Techniques: Western Blot, Cell Culture