Optogenetic control of fly optomotor responses

Abstract

When confronted with a large-field stimulus rotating around the vertical body axis, flies display a following behavior called "optomotor response." As neural control elements, the large tangential horizontal system (HS) cells of the lobula plate have been prime candidates for long. Here, we applied optogenetic stimulation of HS cells to evaluate their behavioral role in Drosophila. To minimize interference of the optical activation of channelrhodopsin-2 with the visual perception of the flies, we used a bistable variant called ChR2-C128S. By applying pulses of blue and yellow light, we first demonstrate electrophysiologically that lobula plate tangential cells can be activated and deactivated repeatedly with no evident change in depolarization strength over trials. We next show that selective optogenetic activation of HS cells elicits robust yaw head movements and yaw turning responses in fixed and tethered flying flies, respectively.

Figure 1.

Schematic of the experimental approach and analysis procedures. A, Setup used to analyze head movement. B, Apparatus for tethered flight experiments. 1, Thin light fiber; 2, matched achromatic doublet pair; 3, tether; 4, infrared LED; 5, camera; 6, head positioner; 7, wing beat analyzer consisting of a mask, two prisms (white), and two photodetectors (not shown). Ci, Head movement analysis from raw images of fly head. The light spot for unilateral optogenetic stimulation is indicated with an arrow. Cii, A box is fitted around the contours (green) of the thresholded image shown in Ci. D, Example wing beat trace for clockwise and counter-clockwise visual rotation (stimulus time interval indicated by gray boxes). The LWB is in red, the RWB is in blue, and the LWB–RWB is in green. E, ChR2–C128S-EYFP expression using R27B03-Gal4. The micrograph shows a collapsed confocal image stack of one brain hemisphere (posterior view). In the optic lobe, expression is largely confined to the three HS cells: HSN (top), HSE (middle), and HSS (bottom).

Figure 2.

Electrophysiological recordings from HS cells expressing ChR2–C128S. Ai, Four consecutive recordings (separated by ∼2 min; each trace represents an average from 5 cells) of ChR2–C128S-expressing HS cells in blind norpA⁷ mutant flies. One second, 472 nm (30 nm bandpass) light elicits a prolonged depolarization that is terminated 9 s later by 3 s, 565/30 nm light. Amplitude and dynamics of the membrane potential changes remain comparable across trials. Aii, Mean membrane potential changes ± SEM of the traces shown in Ai calculated from the integrated responses 3 to 5 s after stimulus offset (indicated by shaded area in Ai) relative to the baseline (2 to 0 s before stimulus onset). B, Averaged responses (4 cells with four traces each per condition) of combined optogenetic and visual HS cell stimulation in visually intact flies (red trace). Blue light pulses evoke transient visual and prolonged optogenetic depolarizing responses that are terminated by 3 s yellow–green light pulses. Presenting vertical still gratings to the fly (light gray bars) leads to depolarizing flicker responses, while gratings moving back to front (3 to 5 s after blue/yellow–green light offset; dark gray bars) leads to robust hyperpolarization in all conditions. Hence, HS cell photoactivation does not notably interfere with visual motion processing. The black trace shows the responses of the same cells to visual stimulation only. C, Same experiments as in B performed on control flies raised without ATR. Blue and yellow–green light pulses lead to transient visual responses, but not prolonged depolarizing voltage changes as in the +ATR condition. Blue bars in all panels indicate optical stimulation with 1 s 472/30 nm light at ∼0.1 mW/mm². Yellow–green bars indicate stimulation with 3 s 565/30 nm light at ∼0.5–0.8 mW/mm². Light and dark gray bars in B and C indicate visual stimulation with still and moving square wave gratings in the HS cell null direction, respectively. Shaded areas (omitted in Ai and Aii for clarity) represent the SD.

Figure 3.

Head movement analysis. Ai, Bi, Mean traces ± SEM. Gray boxes indicate the time of visual stimulation, and blue and yellow boxes show timing of optogenetic light stimulation. Aii, Bii, Mean ± SEM response to the blue light pulses for R27B03-Gal4 (Aii) and norpA7;+; R27B03-Gal4 (Bii) experimental and control flies. Experimental flies expressed ChR2–C128S and were fed with ATR (red traces). Control flies had either the same genotype, but were not fed ATR (blue traces) or did not carry the ChR2–C128S transgene (green trace). *p < 0.01; **p < 0.001; ***p < 0.0001.

Figure 4.

Wing beat analysis. Ai, Bi, Mean traces ± SEM. Gray boxes indicate the time of visual stimulation, blue and yellow boxes show timing of optogenetic light stimulation. Aii, Bii, Mean ± SEM for R27B03-Gal4 (Aii) and norpA⁷;+; R27B03-Gal4 (Bii) experimental and control flies. Experimental flies expressed ChR2–C128S and were fed with ATR (red traces). Control flies had either the same genotype, but were not fed ATR (blue traces) or did not carry the ChR2–C128S transgene (green trace). *p < 0.01.