Extended Data Fig. 6: Imaging ASAP4b and ASAP2f in awake flies during visual stimulation.

(A) From top to bottom: (i) Experimental setup for two-photon imaging of visually evoked responses in the Drosophila brain using a flickering visual stimulus on a gray background. (ii) Drawing depicting L2 non-spiking interneurons. L2 interneurons are directly postsynaptic to photoreceptors R1–R6, and depolarize to dark flashes and hyperpolarize to light flashes. Multiple L2 interneurons send axons in parallel through the lamina into the medulla, tiling visual space. (iii) Sample two-photon image of ASAP4b expression in the terminal arbors of four L2 axons. (B) Stimulus-evoked GEVI responses in L2 terminal arbors (n = 43 neurons from five ASAP2f flies, or 45 neurons from four ASAP4b flies). The ASAP2f response is shown inverted to facilitate comparison. Flies were stimulated with a repeated sequence of 20-ms light and dark flashes starting from a mean gray background, and then, for each arbor, stimuli-aligned responses were extracted and averaged. The solid line in the graph represents the mean waveform across all cells sampled, and the shaded area represents SEM. (C) Left, mean responses of ASAP4b or ASAP2f to depolarizing or hyperpolarizing stimuli. Error bars represent SEM. *** p < 0.001 for depolarization (dark) and for hyperpolarization (light), using a two-sided t-test. Right, ASAP4b produces responses with faster kinetics than ASAP2f. Plots represent mean ± SEM. * p = 0.0406 for depolarization (dark) and 0.0268 for hyperpolarization (light), by two-sided t-test.