We used a custom polarized light stimulus device comprising a UV LED (M340D3, Thorlabs), a 7.5 mm diameter aperture, a ground glass diffuser (DGUV10-1500, Thorlabs), a low-pass filter (FGUV11, Thorlabs), and a removable linear polarizer (BVO UV, Bolder Optic). The UV LED was controlled through MATLAB 2017a (Mathworks, MA) via a DAQ (6259, NI) and LED driver (LEDD1B, Thorlabs). The polarizer was rotated with a bipolar stepper motor (ROB-10551, SparkFun) and spur gears (1:1), and a motor driver (ROB-12779, SparkFun) controlled through MATLAB (2017a, Mathworks) via a DAQ (USB1208, MCC), with a minimum step-size of 7.5°. The motor was operated in open-loop and a Hall effect sensor (A1324, Allegro) was used to detect the proximity of a magnet which passed once per revolution, in order to verify correct operation. Angles of polarization and directions of rotation are expressed from an external viewpoint looking toward the fly (Figure 1—figure supplement 1A). 0°/180° corresponds to a vertical orientation in the transverse plane and an alignment with the fly’s long-axis in the horizontal plane. We investigated the reproducibility of the polarizer’s angular positions and measured <1° variation over multiple revolutions and <1° of position hysteresis (backlash) after reversing the direction of rotation. The surface of the polarizer was positioned frontally, 110 mm from the fly’s head at an elevation of approximately 65° above the eye-equator (Figure 1—figure supplement 1A). The light subtended a solid angle of approximately 4° and the entirety of the fly, including the dorsal rim area of both eyes, was illuminated. We measured approximately 0.8 μW/cm2 irradiance at the fly’s head at the spectral peak of 342 nm (8.7 nm FWHM) with the polarizer attached (Figure 1—figure supplement 1B). We calibrated the LED power in order to maintain a similar irradiance value with the polarizer removed (Figure 1—figure supplement 1B). We measured a ± 5% modulation in light intensity over a full revolution of the device (Figure 1—figure supplement 1B), due to a slight off-axis tilt of the diffuser and polarizer. This intensity modulation was of similar magnitude both with the polarizer attached and removed, and was therefore unlikely to be an effect of polarization. We reasoned that if calcium activity in neurons was modulated by the rotation of the device with the polarizer attached, but not with the polarizer removed, then the varying angle of polarization throughout the revolution was its cause, rather than the varying light intensity. To quantify the difference in modulation between these two polarizer conditions, we report the change in polarization-selectivity index (ΔPSI) throughout (see Polarization-selectivity index).

We verified that the polarized light stimulus elicited an expected response in the dorsal rim photoreceptors by recording calcium signals in R7/R8 terminals in the medulla dorsal rim area (MEDRA) (Figure 1—figure supplement 1C–E). We observed preferential responses to different angles of polarized light across the MEDRA and approximately orthogonal preferred angles within R7/R8 pairs in individual columns (Figure 1—figure supplement 1C–E). Moving anterior to posterior across the right MEDRA, the preferred angle of polarization rotated counter-clockwise (Figure 1—figure supplement 1E), matching a previous characterization (Weir et al., 2016). We estimated that at least 80% of MEDRA columns were stimulated and conveyed polarization tunings that matched predictions based on the anatomy of photoreceptors at corresponding positions (Weir et al., 2016; Figure 1—figure supplement 1E–G), with weak responses or deviations observed only in the anterior-most columns (Figure 1—figure supplement 1E,F) likely due to their posterior receptive fields which faced away from the stimulus. With the polarizer removed, we observed no spatial organization of tunings in photoreceptor terminals and PSI values close to zero (Figure 1—figure supplement 1J), indicating reduced modulation of activity by the stimulus.

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