Drosophila melanogaster is a model organism rich in genetic tools to manipulate and identify neural circuits involved in specific behaviors. Here we present a technique for two-photon calcium imaging in the central brain of head-fixed Drosophila walking on an air-supported ball. The ball's motion is tracked at high resolution and can be treated as a proxy for the fly's own movements. We used the genetically encoded calcium sensor, GCaMP3.0, to record from important elements of the motion-processing pathway, the horizontal-system lobula plate tangential cells (LPTCs) in the fly optic lobe. We presented motion stimuli to the tethered fly and found that calcium transients in horizontal-system neurons correlated with robust optomotor behavior during walking. Our technique allows both behavior and physiology in identified neurons to be monitored in a genetic model organism with an extensive repertoire of walking behaviors.
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We thank J. Simpson and S. Hampel for cloning GCaMPs into pMUH; D. Hall and K. Hibbard for fly crossing; members of Janelia's Fly Core and particularly G. Zhang for stock maintenance; K. Svoboda for donations of equipment and for advice in setting up our two-photon microscopes; D. Flickinger, S. Bassin, T. Tabachnik and C. Werner for contributions to the optical and mechanical design; V. Iyer for developing new ScanImage features and for support; T. Ofstad for assistance with free-walking experiments; N. Kladt for assistance with calibration experiments; M. Ahrens for software contributions and P. Coen for carrying out pilot experiments with the ball tracker. B. Pfeiffer and G. Rubin (Janelia Farm) provided pMUH. G. Rubin gifted us R27B03-Gal4. For pilot experiments, D. Reiff (Max Planck Institute, Martinsried) provided DB331-Gal4 and L. Luo (Stanford University) provided line 3A-Gal4. We received generous advice and support from others at Janelia Farm, including members of the Svoboda lab. This work was supported by the Howard Hughes Medical Institute.
The authors declare no competing financial interests.
Supplementary Figures 1–13 (PDF 1677 kb)
Real-time movie showing multiple views of the fly walking in response to visual stimulation. Note: the xvid codec is required to play all movies. (AVI 5276 kb)
Real-time movie showing fly-on-the-ball virtual two-dimensional trajectory during two-photon imaging trial in response to counterclockwise (blue) and then clockwise (red) global horizontal motion of vertical stripes (1-Hz spatial frequency). The frame rate was chosen to show fly's movements in real time. Frame size is 30 mm in the x dimension and 45 mm in the y dimension. (AVI 1580 kb)
Real-time movie showing a second example of fly-on-the-ball virtual two-dimensional trajectory during two-photon imaging trial in response to counterclockwise (blue) and then clockwise (red) global horizontal motion of vertical stripes (1-Hz spatial frequency). The frame rate was chosen to show fly's movements in real time. Frame size is 30 mm in the x dimension and 45 mm in the y dimension. (AVI 1636 kb)
Real-time movie showing (clockwise from top left): GCaMP signal from R27B03-Gal4 HS-neuron soma (shown in false color, linear intensity scale, window size: x = 62 μm, y = 67 μm); visual pattern presented to the fly (motion is first in the null direction for the HS neuron and then in the preferred direction); view of fly walking on ball from camera 3 (behind fly); traces of change in %ΔF/F (in green) and accumulated rotation (in black) as the motion stimuli are presented. Two-photon images were unfiltered (aside from xvid compression applied to entire movie) and not motion-corrected. (AVI 4623 kb)
Real-time movie shows (clockwise from top left): GCaMP signal from R27B03-Gal4 HS-neuron dendrites (false color, linear intensity scale, window size: 43 μm); visual pattern presented to the fly (motion is first in the null direction for the HS neuron, then in the preferred direction and this protocol is repeated); view of fly walking on ball from camera 3 (behind fly); traces of change in %ΔF/F (in green) and accumulated rotation (in black) as the motion stimuli are presented. Two-photon images are unfiltered (aside from xvid compression of the movie) and not motion-corrected. (AVI 5414 kb)
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Seelig, J., Chiappe, M., Lott, G. et al. Two-photon calcium imaging from head-fixed Drosophila during optomotor walking behavior. Nat Methods 7, 535–540 (2010). https://doi.org/10.1038/nmeth.1468
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