Computational protocol: Large Scale In Vivo Recording of Sensory Neuron Activity with GCaMP6

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Protocol publication

[…] Drift in time-lapse recordings were corrected using NIS Elements AR 4.30.01 (Nikon, align application). Further image processing was done using Fiji/ImageJ version 1.48v, and graphing and statistical analysis was undertaken with a combination of Microsoft Office Excel 2013, IBM SPSS Statistics 23 package and RStudio 0.99.893. All tests conducted were two-tailed, and p < 0.05 was considered significant. Statistical tests and sample sizes are fully recorded in the appropriate figure legends.To generate traces of calcium signals from time lapse images, regions of interest (ROIs) surrounding cell bodies were chosen using a free hand selection tool in Fiji. ROIs were chosen with minimal overlap to ensure less interference from surrounding somata.A region of background was selected and its signal subtracted from each ROI. To generate normalised data, a baseline period of fluorescence was recorded for each ROI and changes from this baseline fluorescence were calculated as ΔF/F.ΔF/F=Ft-F0F0 Where Ft is the fluorescence at time t and F0 is the fluorescence average over a baseline period. Unless otherwise stated ΔF/F is expressed as a percentage.The threshold for a positive response was taken as an average signal of 70% above baseline fluorescence plus 4 SD.For tests of polymodality, a high level of stringency was used. To ensure that overlapping cell bodies were not contaminating the signal, and that low levels of response were not missed, all thermally responsive neurons were double checked visually for their response to pinch.For histologic analysis, to assess the success of labeling with AAV9, six to eight neuronal cell bodies with the lowest fluorescence were selected and their average intensity plus 1 SEM was considered the cutoff for a positive signal against which all other neurons were assessed. […]

Pipeline specifications

Software tools ImageJ, SPSS
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Homo sapiens, Mus musculus