Figure 3.

Using GtACR1 for optogenetic silencing of visual motion inputs to tangential cells. (A) Illustration of preparation for tangential cell recordings with GtACR1 expression in upstream direction-selective T4/T5 neurons (left schematic adapted with permission from ref.²²). (B) Confocal image showing expression of GtACR1-EYFP in T4/T5 neurons in a horizontal cross section. Me, medulla; Lo, lobula; LP, lobula plate. (C) Tangential cell responses in control (black traces) and T4/T5 > GtACR1 flies (red traces) to 615 nm illumination of indicated intensities. Note the different time scales. (D) Tangential cell responses in control (black traces) and T4/T5 > GtACR1 flies (red traces) to gratings of different sizes moving in the preferred direction. For the large pattern, cells in T4/T5 > GtACR1 flies show a reduced average response compared to control flies, presumably due to GtACR1 activation by the visual stimulus. Quantifications represent time-averaged and baseline-subtracted membrane potentials. (E) Tangential cell responses in control (black traces) and T4/T5 > GtACR1 flies (red traces) to combined visual and optogenetic stimulation. Visual stimuli were presented three times per trial and the second stimulation combined with 615 nm illumination (average voltage traces shown for 20 μW/mm²). Responses in control flies become progressively more reduced at increasing illumination intensities yet still reach ~50% at the highest intensity. In contrast, responses in T4/T5 > GtACR1 flies are eliminated already by weak illumination. For quantification, time-averaged membrane potentials were baseline-subtracted for the first and second visual stimulus. A normalized response was obtained by dividing the second by the first response. (F) Tangential cell responses to moving ON (red) and OFF edges (blue) in the same individuals expressing GtACR1 in ON-selective T4 cells. The stimulus is presented three times per trial and the second time combined with 615 nm light illumination. The OFF response is comparable to wild type while the ON response is almost absent. The third visual ON response is slightly reduced for unknown reasons. Quantification as in (E). Traces in C–F represent the membrane potential averaged across 4 trials and N cells. All data are shown as mean ± standard error of the mean. A two-tailed Wilcoxon ranksum test was performed to establish statistical significance: n.s., not significant; **p < 0.01; ***p < 0.001.