Visual stimuli were presented in sets as described below. Between each stimulus set, 10 s of spontaneous activity was recorded in darkness with no visual stimulation. The polarizer could only be removed or attached between recordings, but could be done so while maintaining the same imaging parameters and field-of-view under both conditions.

To characterize responses to different angles of polarization, we rotated the polarizer discontinuously in 30° steps with the UV LED on throughout. Each of the 12 positions (six unique angles of polarization) was maintained for 4–4.5 s and we used 4 s of imaging data collected during this period in our analysis. With a volume-rate of at least 1 Hz, this ensured that at least four time-points were captured for every plane in the imaging volume. The polarizer was then rotated through 30° in 0.5 s. At least two complete revolutions of the polarizer were made. For recordings with the polarizer removed, the procedure was repeated and one revolution of the stimulus was made. The discontinuous rotation of the polarizer differs from most previous studies of polarization-sensitive neurons in insects, which have typically analyzed spike frequency modulated by continuously rotating stimuli, and as such the data obtained using this protocol may not be directly comparable.

To characterize responses to individual wide-field flashes of polarized light, the polarizer was first rotated to 0° (vertical) in darkness. A series of three flashes of the UV LED were presented, 4 s on:4 s off. After 10 s, the same procedure was repeated with the polarizer at 90° (horizontal). The light was the same used in the angle of polarization tuning protocol. For recordings with the polarizer removed, the procedure was repeated with flashes at the 0° position.

To characterize responses to individual wide-field flashes of unpolarized light, the entire LED display was illuminated following the same procedure as for polarized light flashes.

To characterize retinotopic responses to unpolarized stimuli, a single bright, vertical bar was presented on the LED display (32 × 1 pixel) with all other LEDs off (0.78 Weber contrast). Bars initially remained stationary for 3 s, then jittered left and right (±1 pixel) for 3 s, followed by an inter-trial period of 4 s with all LEDs off. Bars were presented at five equally spaced azimuth positions spanning ±90°, presented sequentially from left to right around the fly. This procedure was repeated twice.

To characterize responses to unpolarized motion stimuli, a sparse random dot pattern was presented on the LED display that simulated forward translational optic-flow (thrust), with the frontal point of expansion approximately at the eye-equator. Approximately 1% of LEDs in the display were illuminated in each frame of the pattern, with all other LEDs off (0.83 Weber contrast). Windowed regions of this pattern were presented sequentially (lateral-left: −90°:−50° azimuth; frontal: −40°:+40° azimuth; lateral-right: +50°:+90° azimuth; each covering the full elevation extent of ±30°) followed by the whole pattern (−90°:+90° azimuth). Motion was presented in each region for 4 s, with an inter-trial period of 4 s with all LEDs off. This procedure was repeated twice.

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