For each pixel, we found the average fluorescence intensity (ranging from 0 to 216, arbitrary units) during the first two presentations of the angles closest to and diametrically opposite its preferred angle of polarization in the tuning experiment (Fpref). We then found the average intensity at orthogonal angles (Fortho) and calculated the polarization-selectivity index (PSI) as the difference between Fpref and Fortho, divided by their sum, with possible values ranging from 0 to 1. Where average PSI values are reported for a driver line, we used a broad ROI drawn around all labeled neurons in the brain area recorded, which we refer to as the ‘overall ROI’. To draw the overall ROI, we used an average intensity image from frames between stimulus sets as a guide. We also used this average intensity image to define additional regions: we defined regions of ‘neurons’ as the brightest 10% of pixels within the overall ROI, unless otherwise stated (e.g. Figure 7B,C), and ‘background’ as the dimmest 10% of pixels outside of the overall ROI. For the overall ROI, neurons and background regions, the distribution of PSI values within a recording tended to be non-normal; for average values we report the median value for an individual animal and the mean of the median values across animals. Where ΔPSI values are reported, we subtracted the mean PSI values within the same region across all tuning experiments recorded with the polarizer removed. Where we applied a PSI-threshold to filter polarization-selective pixels in a recording (e.g. tuning maps, polarotopy analysis), we used the mean + 1 SD of PSI values within its background. This typically resulted in a PSI threshold between 0.3 and 0.4. This threshold was modified for E-PG recordings in the protocerebral bridge where PSI values of neurons tended to be lower than the background when averaged over multiple presentations; instead we used the mean + 1 SD of PSI values within neurons across all tuning experiments with the polarizer removed.

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