Fitmaster software

Primary cortical neurons cultured on glass coverslips were recorded at DIV15 in voltage clamp mode. The external solution was kept at 25 °C and contained 120 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 1.5 mM CaCl₂, 10 mM 4-(2-hydroxyethyl)-1-piperazineethane-sulfonic acid (HEPES) and 15 mM glucose. The external solution (pH 7.35) supplemented with AP5 (100 μM), tetrodotoxin (1 μM), and bicuculline (50 μM) to isolate AMPAR currents. Patch recording pipettes (3–6 MΩ) were filled with an internal solution (pH 7.2). The internal solution contained 120 mM K-gluconate, 6 mM NaCl, 1 mM MgCl₂, 10 mM HEPES, and 0.2 mM ethylene glycol tetraacetic acid. Data were acquired using an HEKA EPC-10 patch-clamp amplifier interfaced with PatchMaster software and analyzed using FitMaster software (HEKA Elektronik, Holliston, MA) and MiniAnalysis (Synaptosoft, Fort Lee, NJ). Miniature EPSCs were recorded for a minimum of 2 min at a holding potential of −80 mV. Analysis of synaptic event data was performed using MiniAnalysis software with event detection threshold set at maximum root mean squared noise level (5 pA). Recordings of synaptic events were assessed for frequency (Hz) and mean amplitude (pA).

Studies were designed to generate groups of almost equal size (n = 5), using randomization and blinded analysis. Normalization was performed in order to control the variations in sodium channel expression and inward current amplitude and in order to be able to fit the recorded data with Boltzmann function (for voltage-dependences) or an exponential function (for time courses of inactivation). Fitting and graphing were done using FitMaster software (HEKA Elektronik, Lambrecht, Germany) and Igor Pro (Wavemetrics, Lake Oswego, OR, United States). Statistical analysis consisted of one-way ANOVA (endpoint data) along with post hoc testing of significant findings along with Student’s t-test and Tukey’s test using Prism 7 software (Graphpad Software Inc., San Diego, CA, United States). Values are presented as mean ± SEM with probability levels less than 0.05 considered significant. Statistical analysis was undertaken only for studies where each group size was at least “n = 5.” The declared group size is the number of independent values, and that statistical analysis was done using these independent values. In the electrophysiological experiments, we randomized the different treatments under the different conditions (e.g., control vs. high glucose or inflammatory mediators), so that five cells in each treatment or condition came from five different randomized cell passages.

Whole-cell patch-clamp recordings were performed at room temperature. The resistance of the recording pipettes ranged between 3 and 5 MΩ. Currents were acquired at 10 kHz and filtered at 3 kHz using an EPC-10 amplifier with Patchmaster software (HEKA, Elektronik GmbH, Germany) in combination with a Zeiss 200 inverted microscope and were analyzed using FitMaster software (HEKA, Elektronik GmbH). The bath solution contained (in mM) 140 NaCl, 4 KCl, 2 CaCl₂, 1 MgCl₂, 4 glucose and 10 HEPES, adjusted to pH 7.4 with NaOH. The internal solution contained (in mM) 110 KCl, 10 NaCl, 1 MgCl₂, 1 EGTA and 10 HEPES, adjusted to pH 7.3 with KOH. The membrane potential was held at −40 mV in chondrocyte measurements (Sánchez and Wilkins, 2003 (link); Sánchez et al., 2006 (link)) and −60 mV for HEK-293 cell measurements. GSK205 (Calbiochem, Billerica, MA, 616522) was used at a concentration of 10 µM and cells were treated for 3 min. ACA (Calbiochem, 104550) was used at a concentration of 20 µM and applied directly via the patch pipette. We allowed solution exchange for at least 3 min before collecting data.

Analysis and graphing were done using FitMaster software (HEKA Elektronik) and Igor Pro (Wavemetrics, Lake Oswego, OR, USA). All data acquisition and analysis programs were run on an Apple iMac (Apple Computer). Statistical analysis was performed in JMP version 13. Confocal images were analyzed in ImageJ.

Data analysis was performed using FitMaster software (HEKA Elektronik, Lambrecht, Germany) and Igor Pro (WaveMetrics, Lake Oswego, OR, United States). Data points are presented as means ± s.e.m. (n) with n being the number of independent experimental replicates. Data sets were tested for statistical significance with an unpaired 2-tailed Mann–Whitney U-test for averaged data or Fisher’s exact test for proportions when appropriate.

Data were analyzed and graphed using Fitmaster software (HEKA Elektronik), Igor Pro software (Wavemetrics), GraphPad Prism 5 or 6 (GraphPad Software), SPSS (IBM SPSS Statistics Version 25), and Corel Draw X6 (Corel Corporation).
For statistical testing, two groups were compared with a Student’s t test for parametric testing or a Mann–Whitney U test, depending on normal distribution. More than two groups were compared by an ANOVA, followed by a Bonferroni’s multiple comparison post hoc analysis. The exact value of n (number of cells) is indicated in the figure legends. Data are presented as mean and error bars denote 95% confidence interval (CI). No outliers were defined or eliminated.