Sun Navigation Requires Compass Neurons in Drosophila

Abstract

Despite their small brains, insects can navigate over long distances by orienting using visual landmarks [1], skylight polarization [2-9], and sun position [3, 4, 6, 10]. Although Drosophila are not generally renowned for their navigational abilities, mark-and-recapture experiments in Death Valley revealed that they can fly nearly 15 km in a single evening [11]. To accomplish such feats on available energy reserves [12], flies would have to maintain relatively straight headings, relying on celestial cues [13]. Cues such as sun position and polarized light are likely integrated throughout the sensory-motor pathway [14], including the highly conserved central complex [4, 15, 16]. Recently, a group of Drosophila central complex cells (E-PG neurons) have been shown to function as an internal compass [17-19], similar to mammalian head-direction cells [20]. Using an array of genetic tools, we set out to test whether flies can navigate using the sun and to identify the role of E-PG cells in this behavior. Using a flight simulator, we found that Drosophila adopt arbitrary headings with respect to a simulated sun, thus performing menotaxis, and individuals remember their heading preference between successive flights-even over several hours. Imaging experiments performed on flying animals revealed that the E-PG cells track sun stimulus motion. When these neurons are silenced, flies no longer adopt and maintain arbitrary headings relative to the sun stimulus but instead exhibit frontal phototaxis. Thus, without the compass system, flies lose the ability to execute menotaxis and revert to a simpler, reflexive behavior.

Keywords: central complex; navigation; sun compass.

Figure 1.. Flies Navigate Using a Sun Stimulus and Retain Memory of Their Heading

(A) A tethered fly, backlit with infrared light, is surrounded by a cylindrical LED display; a single 2.4° spot simulates the sun. (B) Example trace showing closed-loop behavior.After ~90 s, the fly stabilized the sun stimulus at a heading of −92° (dashed red line). (C) Heading during a stripe presentation. (D) Polar representation of data for flies presented with a sun stimulus, with a stripe, and in the dark. Angular position indicates a fly’s mean heading; radial distance indicates vector strength. Red line indicates population mean heading with a circular 95% confidence interval; a histogram of mean headings is plotted around each circle. Due to high variance, we could not calculate a 95% confidence interval for the dark data and do not present a population mean heading, as it is not meaningful with such low vector strengths. A Rayleigh test indicates that headings in the dark are uniformly distributed (p = 0.158), whereas this test rejects uniformity for all datasets with visual feedback throughout the study (p < 0.008 in all cases). (E) Sun versus stripe headings for data shown in (D). Data are repeated on the vertical axis to indicate their circular nature. Diagonal line indicates identical heading over both trials. Error bars indicate circular variance multiplied by an arbitrary scale factor, 36, for visibility. Distributions of mean headings for the sun and stripe are different (Mardia-Watson-Wheeler, W = 13.916, p = 0.001). (F) Heading in first trial plotted against second trial heading for increasing inter-trial intervals; plotting conventions are as in (E). Only flies for which both trials had a vector strength >0.2 are plotted. The black lines again indicate exact correspondence between the first and second sun flight headings, which we refer to as the fixed memory (FM) model. Blue lines indicate the expected shift in headings if flies performed a full time compensation (TC) model, assuming a sun movement of 15° hr⁻¹. (G) Headings for first and second sun presentations for flies in which both trials had a vector strength greater than 0.2; plotting conventions are as in (D). Note that the first sun trial for the 5-min dataset is the same data as in (D), except with the 0.2 vector strength cutoff applied. The bottom row of plots indicates the change in heading, with black and blue lines indicating expected values for FM and TC models, respectively. (H) Distribution of 10,000 bootstrapped heading differences between random pairings of first and second trials from (F). Red line indicates mean heading difference of observed data; p value, proportion of resampled differences that are smaller than the observed mean heading difference. (I) Statistical comparison of FM and TC models. Gray histogram shows the distribution of the difference in mean squared residuals (ΔMSR) between the TC and FM models calculated from 10,000 random samples of 20 data pairs. Blue shading and p values indicate the proportion of subsampled ΔMSRs in which the TC model performed better than the FM model.

Figure 2.. E-PG Neuron Activity Correlates with Both Sun and Stripe Positions

(A) Ca²⁺ imaging schematic. (B) Glomeruli assignment in protocerebral bridge based on an SD of GCaMP6f fluorescence in E-PG terminals. (C) Continuous circular representation of angular position based on glomeruli positions in (B). (D) Example trial from functional imaging experiment. GCaMP6f fluorescence (ΔF/F), shown in grayscale, is plotted in each glomerular position (from C) during 45 s of a sun stimulus presentation. Azimuthal position of the E-PG activity bump (blue trace, computed as in [29]) and sun position (red trace) co-vary. Histograms showing angular distributions for each trace are shown at the right, as are regressions plotting sun position against bump position. The dark gray regions in the regression plots indicate the sectors of the arena in which the stimulus was not visible to the fly. (E) Same as in (D), but showing data from the second sun presentation. (F) Same as in (D), but showing data from the stripe presentation. (G) Heading during the second sun trial plotted against heading in the first sun trial, with a minimum 5-min inter-trial interval (n = 20; plotted as in Figure 1E). (H) Polar representation of second sun-bout headings, plotted as in Figure 1D. Shaded area indicates sector that was not visible to fly. (I) E-PG bump-to-stimulus offset for the second sun trial plotted against the mean sun heading. (J) Regression of the median bump-to-stimulus offset for the second sun trial plotted against the offset for the first sun trial. (K) Bump-to-stimulus offset for stripe plotted against the offset for the sun. See also Videos S1 and S2.

Figure 3.. E-PG Neuron Activity Is Necessary for Sun Menotaxis

(A) Fluorescence labeling of GFP expressed in E-PG neurons in three experimental split-GAL4 lines, maximum-intensity projections. The exact number of E-PG neurons is not known, but we counted a range of 53–68 cells in the three driver lines used. Collectively, these cells tile all 16 medial glomeruli of the protocerebral bridge and all wedges of the ellipsoid body. (B) Sun menotaxis and stripe fixation in genetic controls (Kir; empty vectorsplit-GAL4). Left: first 5-min trial of sun fixation (n = 111 flies). Center: second 5-min trial of sun fixation (n = 108). Right: 5 min of stripe fixation (n = 38). (C) Results from the same experimental paradigm for Kir; SS00096 (ns = 54, 49, and 28). (D) Sun and stripe fixation for Kir; SS00408 (ns = 64, 66, and 28). (E) Headings from Kir; SS00131 (ns = 60, 60, and 19). (F) Flies with silenced E-PG neurons have smaller variances than the geneticcontrol. Distribution of 10,000 bootstrapped circular variances subsampled from the empty-vector control second sun trial are indicated in gray (n = 50 each). Black lines depict the observed heading variance of the entire dataset (n = 108). Colored lines indicate population heading variance of the second sun trial for each experimental group. The p values indicate the proportion of bootstrapped variances that are smaller than the observed variance for each experimental group.