Computational protocol: Rate and Temporal Coding Convey Multisensory Information in Primary Sensory Cortices

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Protocol publication

[…] Retrograde tracer injections were performed as previously described (). In brief, ketamine/xylazine anesthetized rats were immobilized into a preformed mold fixed into the stereotaxic apparatus and received unilateral injections of the retrograde tracer Fluorogold (FG; Fluorochrome) in S1 barrel field (2.4-2.6 mm posterior and 5.5-5.8 mm lateral to bregma) or V1 (6.9-7.1 mm posterior and 3.4-3.7 mm lateral to bregma). A total volume of 100 nl FG (5% in dH2O) was injected (30 nl/min) via a 26-G needle attached to a pump controller (Micro4, World Precision Instruments) at a cortical depth of 300 µm. The syringe was left in place for 3 min to ensure an optimal diffusion of the tracer. The surgical opening was sealed with fibrin glue (Surgibond, SMI sutures) and postsurgery analgesic therapy was given (Meloxicam; 0.1-0.2 mg/kg). After a survival time of 4-8 d, the animals were deeply anesthetized with ketamine/xylazine and perfused transcardially with 4% paraformaldehyde (PFA). Brains were removed and postfixed in 4% PFA for 24 h. Coronal slices were sectioned at 50 µm and treated with PBS containing 0.2% Triton X-100 (Sigma-Aldrich), 10% normal bovine serum (Jackson ImmunoResearch), and 10% donkey serum (Millipore). The sections were incubated 2-4 d with mouse monoclonal Alexa Fluor 488-conjugated antibody against NeuN (1:100, MAB377X, Millipore) and rabbit polyclonal primary antibody against GABA (1:1000, #A2052, Sigma-Aldrich) followed by a 2-h incubation with Alexa Fluor 568 donkey anti-rabbit IgG secondary antibody (1:1000, A10042, Invitrogen).Fluorescent images were obtained with a Axioskop 2 Mot microscope (Zeiss) equiped with a fluorescence camera. For quantification of retrogradely backlabeled cells, five 50 µm thick sections spanning S1 and V1 were selected and regions of interests (ROIs; height: 150 µm, width: 300 µm) were defined using ImageJ software. FG- and GABA-positive neurons were counted within each ROI and normalized to the number of NeuN-positive cells detected within supragranular, granular, and infragranular layers.Fluorescent Nissl staining was performed as previously described () using NeuroTrace 500/525 green fluorescent Nissl stain (Invitrogen). Coronal sections were incubated for 20 min with 1:100 diluted NeuroTrace (Thermo Fisher), washed, and coverslipped with Fluoromount-G (SouthBiotech). To precisely detect the position of DiI-stained electrode, the sections were examined using the green (460-488 nm) and red (535-555 nm) excitation filters of the fluorescence microscope (Imager M1, Zeiss). The photographs were adjusted for brightness and contrast using Adobe Photoshop (version CS6). [...] Statistical analyses were performed using Matlab or IBM SPSS Statistics version 22.0 (IBM). Gaussian distribution of the data were assessed using the Kolmogorov-Smirnov test. Normally distributed data were tested for significant differences (*p < 0.05, **p < 0.01, and ***p < 0.001) using unrelated t test. Data that did not follow a Gaussian distribution were tested with Wilcoxon signed-rank test for paired data or with the Mann-Whitney U test for nonpaired data. Count data were analyzed with the two proportion z test. Nonuniformity of circular data were assessed using the Rayleigh test. Significant differences in the preferred phase of neuronal firing to oscillatory activity were assessed using the nonparametric circ_cm test of the Matlab circular statistics toolbox (). Data are shown as mean ± SEM. […]

Pipeline specifications

Software tools ImageJ, SPSS, CircStat
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Rattus norvegicus