Computational protocol: Osmotic Edema Rapidly Increases Neuronal Excitability Through Activation of NMDA Receptor-Dependent Slow Inward Currents in Juvenile and Adult Hippocampus

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Protocol publication

[…] Electrophysiological recordings of neuronal currents or membrane potential were analyzed using Clampfit 10.2–10.4 software (Molecular Devices, Sunnyvale, CA). Baselines were manually adjusted prior to event detection in order to offset the hyperpolarizing shift (outward current in voltage-clamp) observed in hACSF. All events were detected using threshold searches with parameters adjusted accordingly. SICs were detected semi-automatically as inward currents ≥20 pA, with rise times slower than 10 ms (; , ). Events detected twice were manually rejected. Excitatory postsynaptic potentials (EPSPs) were detected semi-automatically as events ≥2 mV, and APs were detected automatically as positive deflections ≥65 mV above baseline. APs were classified as bursts (bAPs) according to the following criteria: (a) Two or more spikes must ride atop a longer, plateau-like EPSP (; ; ) and (b) Interspike intervals (see ) for spikes in a burst must fall within 2 standard error lengths of the interspike interval distribution for all cells (). Figure 3.Statistical analysis of event amplitude, frequency, and kinetics was performed using IBM SPSS Statistics 22. Split plot repeated measures ANOVAs were used to account for between-group factors (e.g., hACSF dose) as well as within-cell factors (baseline, hACSF, or wash) in each experiment and followed by planned comparisons using the Student’s t-test. Holm-Bonferroni or Tukey’s honest significant difference correction for multiple comparisons was used to adjust α as necessary. Statistical significance for the p values is as follows: (*p < .05), (**p < .01), and (***p < .001). N = 8 to 10 cells for all experiments, with an equal number of cells per group for parametric comparison.Analysis of astrocyte volume was performed off-line using Fiji/ImageJ. Image stacks at each time point were z-adjusted to correct for cell “drift” caused by slice swelling during hypoosmolar treatment. Stacks were then concatenated into an x-y-z-t hyperstack and filtered to remove noise (median filter, 2 pixel diameter). Next, a max-intensity projection was taken through the z-plane to produce a 2D time series, where each image showed the full extent of the soma at that time point. After registration and background subtraction, the finalized time series was binarized using the “mean” thresholding algorithm. An elliptical region of interest (ROI) was drawn to encompass the soma as closely as possible throughout the time series, and the resulting thresholded area encompassed by the ROI was quantified. Soma area was normalized to baseline for each astrocyte and measured as percent change over baseline for each time point in hACSF and wash. Percent changes in astrocyte volume were analyzed by repeated measures ANOVA with posthoc tests and adjusted for multiple comparisons by Bonferroni correction (*p < .05, ***p < .001). N = 7 to 8 cells per condition. […]

Pipeline specifications

Software tools SPSS, ImageJ
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Mus musculus
Diseases Brain Diseases, Epilepsy, Hyponatremia
Chemicals N-Methylaspartate