Computational protocol: Diazepam effect during early neonatal development correlates with neuronal Cl−

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Protocol publication

[…] Two‐photon fluorescence imaging of neurons expressing Clomeleon was obtained using a Fluoview 1000MPE with prechirp optics and a fast acousto‐optical modulator mounted on a Olympus BX61WI upright microscope (Olympus Corporation, Tokyo, Japan) using a 20× water immersion objective (NA 0.95). A mode‐locked Ti:Sapphire laser (MaiTai, Spectra Physics, Fermont, CA) generated two‐photon fluorescence with 860 nm excitation. Emitted light passed through a dichroic mirror (460 nm cutoff) and was band‐pass filtered through 480 ± 15 nm filter (D480/30) for cyan fluorescent protein (CFP) and 535 ± 20 nm filter (D535/40) for yellow fluorescent protein (YFP). Two photomultiplier tubes (Hamamatsu Photonics, Hamamatsu City, Japan ) were used to simultaneously acquire CFP and YFP signals. Three‐dimensional stacks (3D) of raster scans in the XY plane were imaged at z‐axis intervals of 1–2 μm. Clomeleon‐expressing neurons were imaged in the neocortex in slices perfused with aCSF + 1 μM tetrodotoxin (TTX; selective inhibitor of Na+ channels) to prevent spontaneous epileptiform activity, held at 32–34°C and aerated with 95% O2–5% CO2. ImageJ 1.47 software (NIH, Bethesda, MD) was used for quantitative analysis. [...] [Cl−]i was expressed as a median ± SD as [Cl−]i follows a non‐Gaussian distribution. One‐way analysis of variance (ANOVA) on ranks with post hoc Dunn's method was used for multiple comparisons of nonparametric data. One‐way ANOVA with post hoc Holm–Sidak test was used for multiple comparisons of parametric data. The Wilcoxon Signed Rank Test was used for nonparametric paired data. Paired t‐tests were used for parametric paired comparisons. Chi‐square was used when comparing proportions. Statistical significance was set to P < 0.05. Igor Pro and SigmaPlot v11 (Systat Software, Inc, San Jose, CA) were used for data analysis. […]

Pipeline specifications

Software tools ImageJ, SigmaPlot
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Homo sapiens, Mus musculus
Diseases Epilepsy
Chemicals Benzodiazepines, Diazepam, gamma-Aminobutyric Acid