Computational protocol: The corticothalamocortical circuit drives higher-order cortex in the mouse

Similar protocols

Protocol publication

[…] We performed flavoprotein autofluorescence imaging (Figs. –) as previously described. We captured green light (520–560 nm) generated by mitochondrial flavoproteins in the presence of blue light (472–488 nm), using a high sensitivity camera (QImaging Retiga-SRV; QImaging Corporation, Surrey, BC Canada) with a Firewire interface. We acquired all images using 4×4 binning at 8-bits with 2.5x magnification. Final image resolution was 348 × 260 pixels, with 60 pixels spanning 1 mm in both the x- and y- dimensions. We suspended slices above the chamber bottom with a piece of titanium mesh mounted on a hand-bent platinum wire to maintain adequate slice oxygenation and perfusion on both sides of the slice. This has been shown by others, and in our experience, to significantly increase the flavoprotein autofluorescence signal amplitude. We typically captured images for 14 second-long sweeps, with stimuli during the second second of each sweep. We stored the resulting images on a custom-built computer running a commercially available software package (Streampix 3; Norpix, Inc., Montreal, Quebec, Canada) and analyzed them with programs that we generated in-house made to run on Matlab. In the case of simultaneous whole-cell recordings (), we selected a box of pixels located immediately over the group of cells near the electrode tip in ImageJ and exported them to Excel, where we produced line graphs.We typically acquired flavoprotein autofluorescence images at a rate of 7–12 frames per second. We manually adjusted exposure times to yield image brightness (i.e., baseline flavoprotein autofluorescence) that was subjectively similar across experiments. For Figs. and : after acquisition and processing (details above), we uniformly adjusted the contrast, brightness and transparency of flavoprotein autofluorescence images and then overlaid onto raw images from Streampix 3 in CorelDRAW X4 (Corel Corporation, Ottawa, Ontario Canada). We added all drawn items (arrows, axes, drawings etc.) with CorelDRAW for Figs. –. We generated was in Microsoft Excel 2003, then exported to CorelDRAW for font modification and standardization. […]

Pipeline specifications

Software tools ImageJ, CorelDraw
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Mus musculus