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Voltage-sensitive dye imaging (VSDI) can simultaneously monitor the spatiotemporal electrical dynamics of thousands of neurons and is often used to identify functional differences in models of neurological disease. While the chief advantage of VSDI is the ability to record spatiotemporal activity, there are no tools available to visualize and statistically compare activity across the full spatiotemporal range of the VSDI dataset. Investigators commonly analyze only a subset of the data, and a majority of the dataset is routinely excluded from analysis. We have developed a software toolbox that simplifies visual inspection of VSDI data, and permits unaided statistical comparison across spatial and temporal dimensions. First, the three-dimensional VSDI dataset (x,y,time) is geometrically transformed into a two-dimensional spatiotemporal map of activity. Second, statistical comparison between groups is performed using a non-parametric permutation test. The result is a 2D map of all significant differences in both space and time. Here, we used the toolbox to identify functional differences in activity in VSDI data from acute hippocampal slices obtained from epileptic  Arx  conditional knock-out and control mice. Maps of spatiotemporal activity were produced and analyzed to identify differences in the activity evoked by stimulation of each of two axonal inputs to the hippocampus: the perforant pathway and the temporoammonic pathway. In mutant hippocampal slices, the toolbox identified a widespread decrease in spatiotemporal activity evoked by the temporoammonic pathway. No significant differences were observed in the activity evoked by the perforant pathway. The VSDI toolbox permitted us to visualize and statistically compare activity across the spatiotemporal scope of the VSDI dataset. Sampling error was minimized because the representation of the data is standardized by the toolbox. Statistical comparisons were conducted quickly, across the spatiotemporal scope of the data, without  a priori  knowledge of the character of the responses or the likely differences between them.

A Toolbox for Spatiotemporal Analysis of Voltage-Sensitive Dye Imaging Data in Brain Slices