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Muscles that move the retina augment compound eye vision in Drosophila

Nature volume 612, pages 116–122 (2022)Cite this article

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Abstract

Most animals have compound eyes, with tens to thousands of lenses attached rigidly to the exoskeleton. A natural assumption is that all of these species must resort to moving either their head or their body to actively change their visual input. However, classic anatomy has revealed that flies have muscles poised to move their retinas under the stable lenses of each compound eye1,2,3. Here we show that Drosophila use their retinal muscles to smoothly track visual motion, which helps to stabilize the retinal image, and also to perform small saccades when viewing a stationary scene. We show that when the retina moves, visual receptive fields shift accordingly, and that even the smallest retinal saccades activate visual neurons. Using a head-fixed behavioural paradigm, we find that Drosophila perform binocular, vergence movements of their retinas—which could enhance depth perception—when crossing gaps, and impairing the physiology of retinal motor neurons alters gap-crossing trajectories during free behaviour. That flies evolved an ability to actuate their retinas suggests that moving the eye independently of the head is broadly paramount for animals. The similarities of smooth and saccadic movements of the Drosophila retina and the vertebrate eye highlight a notable example of convergent evolution.

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Fig. 1: Drosophila have two muscles per eye that act to move the retina.
Fig. 2: Retinal movements yield the expected angular shifts in receptive fields of visual neurons.
Fig. 3: Drosophila perform vertical and horizontal retinal optokinetic responses and these responses are independently controlled in the two eyes.
Fig. 4: Drosophila spontaneously move their retina, often in a saccadic fashion.
Fig. 5: Drosophila make functionally relevant retinal movements when crossing gaps.

Data availability

The data shown in the main figures are available at https://doi.org/10.6084/m9.figshare.c.6145572. All other data generated in this study are available from the corresponding authors upon request.

Code availability

Custom-written software used to track the fly retina in real time is available at https://github.com/MaimonLab/EyeTrackerForm. Additional code is available upon request from the corresponding authors.

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Acknowledgements

We thank members of the Maimon lab for their input and constructive discussions. We thank A. Kim for help generating Supplementary Videos 5–8, A. Adachi for the immunostaining data shown in Extended Data Fig. 6, S. Thornquist for immunostaining in Fig. 5 and Extended Data Fig. 10, and A. Friedrich for immunostaining in Extended Data Fig. 10. We thank K. Fonselius for help with the pseudopupil tracking software and B. Solat for help with the behavioural experiments in Fig. 5. We thank J. Beetz, V. Bitsikas, M. Madhav, Y. Tanaka, A. Cario, P. Quinlan, C. Rusch, C. Herber, M. Ananth and L. Anneser—students in the Neural Systems and Behavior course in Woods Hole—for initial, inspiring work characterizing retinal movements at both behavioural and electrophysiological levels. We thank A. Straw and K. Panser for the GFP immunostaining data in Extended Data Fig. 2 and J. Tuthill for suggesting the use of a specific split-GAL4 line to silence early visual neurons (Extended Data Fig. 6). We thank H. Lacin for sending us the split-GAL4 lines used in Fig. 1, which also allowed us to ultimately make a more selective split-GAL4 line for retinal motor neurons. We thank W. Korff for insights into the biomechanics of the orbital ridge. We thank S. Sethi for advocating for the head-fixed gap-crossing paradigm and A. Janke for performing optomotor flight experiments that did not make it into the final paper. Fly stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) and antibodies from the Developmental Studies Hybridoma Bank at the University of Iowa (National Institute of Child Health and Human Development) were used in this study. Research reported in this publication was supported by a grant from the National Institute of Neurological Disorders and Stroke (R01NS121904) to G.M., a Leon Levy postdoctoral fellowship to L.M.F., a Kavli postdoctoral fellowship to L.M.F., and the Max Planck Society. G.M. is a Howard Hughes Medical Institute Investigator.

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Author notes

  1. Aditya Nair

    Present address: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA

  2. These authors contributed equally: Sofia C. Avritzer, Jazz L. Weisman

Authors and Affiliations

  1. Laboratory of Integrative Brain Function and Howard Hughes Medical Institute, The Rockefeller University, New York, NY, USA

    Lisa M. Fenk, Sofia C. Avritzer, Jazz L. Weisman, Aditya Nair, Thomas L. Mohren & Gaby Maimon

  2. Active Sensing, Max Planck Institute for Biological Intelligence (in foundation), Martinsried, Germany

    Lisa M. Fenk & Lucas D. Randt

  3. Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA

    Igor Siwanowicz

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Contributions

L.M.F. first conceived the project. L.M.F. and G.M. designed the experiments. I.S. collected the anatomical data in Fig. 1. L.M.F. collected all behavioural and electrophysiological data, unless otherwise indicated, and analysed these data. A.N. collected the electrophysiological data in Extended Data Fig. 8, with the guidance of L.M.F. and G.M.; L.M.F. analysed these data. S.C.A. collected preliminary data on retinal muscle anatomy and helped to create selective split-GAL4 fly lines for retinal motor neurons together with J.L.W. J.L.W. recorded and analysed optokinetic responses of silenced and control flies in Fig. 5. G.M. and L.M.F. collected the electrophysiological data in Fig. 2b and Extended Data Fig. 2; G.M. analysed these data. G.M. collected and analysed the tethered, gap-crossing data, with extensive help from T.L.M. in fabricating the wheel and T.L.M., S.C.A. and J.L.W. in data analysis. L.D.R. collected the free-behaviour gap-crossing data; L.M.F. and L.D.R. analysed those data. L.M.F. and G.M. wrote the paper.

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Correspondence to Lisa M. Fenk or Gaby Maimon.

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Extended data figures and tables

Extended Data Fig. 1 Retinal muscles that attach to the front of the orbital ridge can move the entire retinal sheet coherently.

(a) Schematic of the attachment of the retinal eye muscles to the orbital ridge in the Drosophila head. For an animated version see Supplementary Video 1. (b) Staining of a part of the Drosophila head including the eye and the orbital ridge in a cleared specimen using Calcofluor White. Soft tissue was removed proteolytically. It reveals the vesica piscis-shaped opening of the orbital ridge and shows strongly sclerotized parts in the frontal region, where the two muscles attach (red stars), as well as two discontinuities on the dorsal and ventral poles (arrows). One possibility is that these discontinuities decouple the front of the orbital ridge from the back, mechanically. This may allow muscles that pull the front of the retina to also move the rear by the same amount through internal cohesion within a stiff set of ommatidia, rather than through a force vector that dissipates over space from front to back. Alternatively, the inhomogeneities at the top and bottom of the orbital ridge could act as a fulcra or pivot points, leading to the rear part of the orbital ridge to move outward when the frontal part moves inward (towards the midline), which could aid coherent motion of the retina. We will test these models in biomechanical studies in the future. For an animated version of the Calcofluor White image stack see Supplementary Video 2. The same staining was also performed in a non-cleared specimen with equivalent results (data not shown). (c) We measured retinal movements using three cameras pointing at three different positions in one eye. To induce large movements, we optogenetically activated the retinal motoneurons. We expressed CsChrimson in a split-GAL4 line (w+;R44A07-AD;R13D09-DBD) and focused red light onto a spot between the fly eyes (Methods). The three plots to the right show the views of the three different cameras: we overlaid images before and after optogenetic activation and plotted the tracked centroid on top of the pseudopupil (white) to illustrate the retinal movements. (d) Optogenetic activation in this fly yielded movements roughly along the axis connecting photoreceptors 3–5, and we used this motion direction (dark arrow) for comparison across the different positions on the facet eye in (e). Schematic modified from Stavenga (1979)65 (e) Traces of the pseudopupil measured in front (blue), in the middle (orange) and in the back (green) of the fly eye. (f) Same traces as above but shifted in y (by hand) to illustrate the coherent motion of the pseudopupil across the eye.

Extended Data Fig. 2 Electrophysiological recordings from LC14 cells reveal that they have small, front facing receptive fields and that they respond strongly, in a non-direction-selective manner, to moving spots and bars.

(a) GFP expression in the VT37804-GAL4 line, characterized previously66, shows LC14 cells, which connect the lobula on one side to the contralateral lobula and medulla. Additional off-target expression in the anterior optic tubercle is also visible. (b) Example trace showing the LC14 membrane voltage (Vm) while it is modulated by a variety of visual stimuli (bars, spots and gratings). (c) Membrane potential responses from a single cell reveal strong, non-directional responses to moving spots and bars in a restricted portion of the visual field. Light gray: single trials. Black: mean response. (d) Single cell response to 100 ms spot flashes (gray region) on the LED screen. Light gray: single flashes. Black: mean response. (e) Heat map image representation of an LC14 cell’s receptive field (same cell as shown in d). The cell’s response for the heat map was determined by subtracting the mean, baseline Vm in the 100 ms before stimulus onset from the mean Vm in the 100 ms window starting 30 ms after stimulus onset. (f) Population-averaged responses to moving bars and gratings (six cells) reveal more consistent responses to bars than gratings. (g) Heat-map representations of four more LC14 cells’ receptive fields. Bottom left plot shows same data as Fig. 2b.

Extended Data Fig. 3 Optokinetic retinal tracking is interspersed with (nystagmus-like) counter-saccades and the largest counter-saccade magnitudes are observed in flight.

(a) We isolated large saccades of the right retina with a simple, threshold-crossing algorithm and plotted these alongside the concomitant retinal movements of the left retina in 10 quiescent (i.e., non-flying) flies. Data are shown in the context of full-field rightward grating motion (top) (87 saccades), rightward motion in the right visual hemisphere (middle) (56 saccades), and bilateral front-back-motion (bottom) (66 saccades). (b) Same as panel a but during tethered flight. We also show the left minus right wing beat amplitude (L–R WBA) of the flies, with rightward deflections indicating a rightward steering response and vice versa (top, middle and bottom traces include 122, 136 and 152 saccades respectively).

Extended Data Fig. 4 Retinal saccade magnitudes and peak velocities are tightly correlated, akin to the main sequence in human saccades.

(a) Saccade peak velocity plotted against saccade amplitude of the saccades of the right retina from Extended Data Fig. 3a. Data are shown in the context of full-field rightward grating motion (top) (87 saccades), rightward motion in the right visual hemisphere (middle) (56 saccades), and bilateral front-back-motion (bottom) (66 saccades). (b) Same as panel a but for the saccades of the right retina from Extended Data Fig. 3b, which occurred during tethered flight (top, middle and bottom traces include 122, 136 and 152 saccades respectively). The mean saccade amplitudes for flight and quiescence were 2.6° and 1.1° for full-field rightward motion, 1.6° and 0.7° for rightward motion in the right hemisphere and 2.7° vs 0.6° deg for bilateral front-to-back motion. All saccade-magnitude differences between flight and quiescence were highly significant (two-sided Welch-test: p = 9·10−14, 7·10−16, 5·10−55 for top, middle, and bottom panels respectively).

Extended Data Fig. 5 The gain of optokinetic tracking is below unity and was significantly modulated by flight for bilateral front-to-back motion.

(a) The initial pseudopupil velocity for full-field 15°/s leftward grating motion when analyzing the left eye (orange) and for full-field 15°/s rightward grating motion when analyzing the right eye (blue). Data for optokinetic responses made during quiescence and tethered flight are shown separately. Each point represents data from one fly. (b) Initial pseudopupil velocities for unilateral motion stimuli: 15°/s grating motion in the left side for the left eye and right side for the right eye. (c) Initial pseudopupil velocities for bilateral front-back-motion. The initial pseudopupil velocities during flight and quiescence were not significantly different for rotational stimuli (a, b) but they were significantly bigger in flight for bilateral front-to-back motion, which simulates forward translation (two-sided Wilcoxon signed-rank test, left eye: p = 0.005, right eye: p = 0.007).

Extended Data Fig. 6 Silencing neurons in the early fly visual system abolishes optokinetic responses to visual motion but preserves spontaneous retinal movements in flying flies.

(a) Immuno-stain of the split-GAL4 line R34G07-AD;R9B08-DBD, characterized previously36, labelling L1–L4 visual neurons (green) and neuropil (anti-brp, magenta). (b) Example traces showing retinal movement in a control fly, expressing inactive tetanus toxin in the L1–L4 cells, and an experimental fly, expressing active tetanus toxin in the same neurons, in flight and non-flight. All behavioral responses were made in the context of a full-field grating rotating at 15°/s (1 Hz temporal frequency). (c) Single fly averaged (thin lines) and population averaged (thick line) retinal responses for 8 control flies and 10 experimental flies during non-flight/quiescence. Right panel: Optomotor index (Methods ) quantifying the response strength to visual motion in the expected optokinetic direction. (d) Same as panel c, but in flying flies. Black: Left minus right wingbeat amplitude (L–R WBA).

Extended Data Fig. 7 In both D. melanogaster and D. suzukii, the sign and magnitude of retinal optokinetic responses and walking optomotor responses are consistent with non-hyperacute sampling of the visual world by the fly retina.

(a) Experimental setup: pin-tethered flies walked on floating ball. We recorded the ball’s rotations as a readout of the flies’ turning velocity (black) alongside the displacements of the deep pseudopupil (left: orange, right: blue). Visual stimuli were presented on a conical screen using a projector and consisted of full-field rotating gratings at varying spatial wavelengths (always 4 Hz temporal frequency) for 5 s. (b) The inter-ommatidial angle Δϕ of the compound eye limits the spatial wavelength λ of a grating that can be properly resolved (modified from Land 199767). Below the cut-off wavelength of λL = 2·Δϕ, direction-selective motion responses of the visual system are predicted to invert due to spatial aliasing67,68. (c) Top and middle: displacements of the left and right pseudopupils during the stimulus period as a function of the grating wavelength, λ (1 to 40°) for left- and rightward motion. Bottom: concomitant average walking velocity during the stimulus period. Thin lines are the trial-averages from 11 single flies. Thick lines are population averages. Note the sign inversion in the range of λ = 5–10° for both the retinal optokinetic reflex and the walking optomotor response. (d) Average responses for both retinas for front-to-back (thicker line) and back-to-front motion for Drosophila melanogaster (top, data as in b) in comparison to Drosophila suzukii (bottom). D. suzukii showed inverted responses at smaller wavelengths than D. melanogaster, i.e. had a higher spatial acuity, consistent with the fact that D. suzukii have approximately twice the number of ommatidia as D. melanogaster69. The sign-inversion of behavioral responses around the critical wavelength inferred from the optics of the eye argues that any retinal movements flies are making in the context of moving gratings are not allowing them to perceive the motion direction of fine gratings better than would be expected from the first-order optics of the eye. The lack of evidence for hyperacuity in this context does not exclude it existing in other circumstances.

Extended Data Fig. 8 Visual neurons activate during spontaneous saccades with direction-selective responses.

(a) Left: sample Vm responses of an HS cell on the left side of the brain to rightward followed by leftward grating motion (1 Hz temporal frequency). (b) Vm of the same cell, alongside the x-movements of the left retina (orange), in the context a stationary vertical grating. Arrows indicate moments of spontaneous saccades. (c) Trial-averaged Vm of single flies (gray, N = 12 HS or VS cells) and population averaged Vm (black) for left-eye retinal saccades (orange). Data from a dark arena (left), a uniformly lit arena (middle), and a stationary grating (right) are shown. Top plots shown downward retinal movements for VS cells and leftward retinal movements for HS cells, which should produce visual motion in the preferred direction. Bottom plots show the opposite, null-direction retinal movements. The direction-selective responses to gratings argue that HS and VS cells respond to the visual motion induced on the retina by < 1° eye movements. The weak response to eye movements in darkness, or with a uniformly lit screen, is opposite in sign to that observed with a grating, which may represent an efference copy of the predicted motion signal arriving to HS/VS cells with each eye movement. This efference copy is potentially superseded by the actual, grating motion input with a high contrast grating, in the rightmost column.

Extended Data Fig. 9 Evidence that flies are genuinely in the dark during the lights-off epoch of the gap crossing experiments.

(a) Flies walking on the gap-crossing wheel (Fig. 5) were presented with a grating printed on paper that was physically moved back and forth in front of the right eye with a motorized manipulator. A small slit in the printed grating allowed us to slide an InfiniStix lens through it, abutting the fly’s right eye, to track the deep pseudopupil. (b) Example traces showing the horizontal shift of the right retina in two flies (blue) together with the grating position (black). (c) We observed a clear optokinetic response with the lights on, but not during darkness, demonstrating that there was genuinely no light available for flies to see with the lights off, even after being dark adapted for 30 min. Light blue: single flies. Dark blue: population mean. Five repetitions of the grating’s movement were presented and averaged for each fly, in each 5-min. epoch shown. (d) We quantified the number of gap crossings from the data presented in Fig. 5 and observed a ~30%, statistically significant, drop in the rate of gap crossing during the dark period (two-tailed paired t-test, p = 0.013 when comparing lights on #1 with darkness and p = 0.0056 when comparing lights on #2 with darkness).

Extended Data Fig. 10 Anatomical characterization of two the split GAL4 lines used for silencing retinal motor neurons.

We visualized expression in these two split-Gal4 lines by driving CsChrimson-tdTomato in R44A07-AD;R13D09-DBD (a) and R14B04-AD;R13D09-DBD (b). Maximum z-projections of the brain are shown over roughly the posterior and anterior halves to better visualize the branching. VNC maximum projections are shown over the full stack. The green cells on the right and left side of the SEZ (arrows) are retinal motor neurons, based on their dendritic arborization, location of cell body and their axons leaving the brain just below the antennal lobe. We could optogenetically induce retinal movements via expression of CsChrimson expression in both split lines. Expression levels were low and stochastic, particularly in line R44A07-AD;R13D09-DBD. We could clearly detect motoneurons in on both sides of the brain in 4/14 imaged brains and one motoneuron in 7/14 brains. For the lines shown in (b) we could detect motoneurons bilaterally in 16/16 brains. The stacks shown here and in in Fig. 5f are particularly clear examples chosen to best illustrate the location branching pattern if the neurons. (c) We checked for Kir2.1/Kir2.1-mutant expression in our experimental flies and could detect the motoneurons in both split GAL4 lines, again with low expression. We stained three brains for R44A07-AD;R13D09-DBD and detected the motoneurons bilaterally in 2/3 (one shown) and unilaterally in one brain (not shown). For line R14B04-AD;R13D09-DBD, we detected motoneurons bilaterally in 3/3 imaged brains (only one is shown).

Supplementary information

Supplementary Information

Supplementary Discussion and References.

Reporting Summary

Supplementary Video 1

Schematic of the attachment of the retinal eye muscles to the orbital ridge in the Drosophila head. Animated schematic of the retinal muscles in the fly head.

Supplementary Video 2

3D rendering of the orbital ridge. Cuticular stain of a part of the Drosophila head, including the eye and the orbital ridge, in a cleared specimen using Calcofluor white.

Supplementary Video 3

3D rendering of the retinal muscles. 3D rendering of a portion of the fly head, near one eye, showing immunohistochemical labelling of both retinal muscles using phalloidin (red) and chitin using Calcofluor white (blue).

Supplementary Video 4

Simultaneous tracking of photoreceptor tips using a water immersion objective and the deep pseudopupil using an air lens. Simultaneous imaging and tracking of the deep pseudopupil seen through an air objective (top left) and photoreceptor tips seen through water immersion (top right).

Supplementary Video 5

Retinal movements in response to panoramic visual motion in a pin-tethered, non-flying fly (4× speed). Top panel: visual stimulus. Top inner panels: right and left side of the fly head. Top outer panels: Zoom-in on the two deep pseudopupils. The tracked centroids of the right and left deep pseudopupils are marked with blue and orange dots, respectively. Lower traces show the horizontal movements of the left (orange) and right (blue) deep pseudopupil over time.

Supplementary Video 6

Retinal movements and a wing-steering signal in response to panoramic visual motion in a pin-tethered, flying fly (4× speed). Same as Supplementary Video 5, but for a flying fly. The lower trace shows the L–R WBA signal.

Supplementary Video 7

Retinal movements in response to unilateral visual motion in a pin-tethered, non-flying fly (4× speed). Same as Supplementary Video 5, but for visual motion presented to only one eye at a time.

Supplementary Video 8

Spontaneous retinal movements in a pin-tethered, flying fly in the context of varying, stationary visual scenes (2× speed). Same as Supplementary Video 6, but in the context of stationary visual stimuli (dark screen, uniformly lit screen, and vertical grating).

Supplementary Video 9

First example video showing retinal movements during tethered gap crossing (0.25× speed). Top left: tracked centroids of the right (blue) and left (orange) deep pseudopupil. Grey regions reflect a 2D histogram of pseudopupil positions for each eye (100 × 100 bins), showing all bins with more than 25 counts as grey. Below: video of the tethered fly walking on a 3D-printed wheel with two gaps (one with a horizontal grating painted on the wall and one with a vertical grating). Traces on the right show the x movement of the right (blue) and left (orange) pseudopupil and the angular position of the wheel (black) over time.

Supplementary Video 10

Second example video showing retinal movements during tethered gap crossing (0.25× speed). Same as Supplementary Video 9, but for a second example fly.

Supplementary Video 11

Third example video showing retinal movements during tethered gap crossing (0.25× speed). Same as Supplementary Video 9, but for a third example fly.

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Fenk, L.M., Avritzer, S.C., Weisman, J.L. et al. Muscles that move the retina augment compound eye vision in Drosophila. Nature 612, 116–122 (2022). https://doi.org/10.1038/s41586-022-05317-5

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