Feature detection and orientation tuning in the Drosophila central complex

Abstract

Many animals, including insects, are known to use visual landmarks to orient in their environment. In Drosophila melanogaster, behavioural genetics studies have identified a higher brain structure called the central complex as being required for the fly's innate responses to vertical visual features and its short- and long-term memory for visual patterns. But whether and how neurons of the fly central complex represent visual features are unknown. Here we use two-photon calcium imaging in head-fixed walking and flying flies to probe visuomotor responses of ring neurons--a class of central complex neurons that have been implicated in landmark-driven spatial memory in walking flies and memory for visual patterns in tethered flying flies. We show that dendrites of ring neurons are visually responsive and arranged retinotopically. Ring neuron receptive fields comprise both excitatory and inhibitory subfields, resembling those of simple cells in the mammalian primary visual cortex. Ring neurons show strong and, in some cases, direction-selective orientation tuning, with a notable preference for vertically oriented features similar to those that evoke innate responses in flies. Visual responses were diminished during flight, but, in contrast with the hypothesized role of the central complex in the control of locomotion, not modulated during walking. Taken together, these results indicate that ring neurons represent behaviourally relevant visual features in the fly's environment, enabling downstream central complex circuits to produce appropriate motor commands. More broadly, this study opens the door to mechanistic investigations of circuit computations underlying visually guided action selection in the Drosophila central complex.

Figure 1. Drosophila ellipsoid body ring neurons arborize in visually responsive LTr microglomeruli that show a retinotopic organization

a, Schematic of fly central brain showing antennal lobe (AL), mushroom bodies calyces (MB) and optic lobes along with sub-structures of the central complex: ellipsoid body (EB), fan-shaped body (FB), protocerebral bridge (PB), and noduli (NO). Inset: EB ring neurons (R1–R4) and LTr. b, Frame of a confocal stack showing LTr microglomeruli labeled by pan-neuronal GFP expression. c, Projection of two-photon calcium imaging video of LTr with overlay of microglomeruli selected based on responses to moving visual stimuli. 20±5 microglomeruli delineated in individual planes of focus; 30±4 microglomeruli over multiple planes of focus (n = 11 flies) (see Methods). d, Schematic of RF mapping setup with fly positioned in center of curved visual display. e, Sample frame from trial showing responses in selected glomeruli (red outlines). f, Calcium transients of three LTr microglomeruli in response to visual stimulus moving left to right in front of the fly at different elevations. g, Two-dimensional response maps (two-trial averages) for all microglomeruli shown in c. h, LTr microglomeruli from left and right hemisphere of same fly, colored according to center of RF in azimuth, and i, elevation. j, Histogram of correlation coefficients between RF center and anatomical position. For n = 42 focal planes with 20±5 glomeruli, correlation is significantly different than for randomly arranged microglomeruli (r = 0.27±0.17, p = 2.3·10⁻¹⁷, n = 11 flies), indicating retinotopy in the organization of microglomeruli across flies. k, Histogram of primary retinotopic axis of LTr map as found by principal component analysis (see Methods, n = 42 focal planes, 11 flies). All scale bars: 5µm.

Figure 2. Ring neurons are tuned to specific visual features and orientations

a, Multicolor FLP-out of R2 neurons showing three cell bodies on each side along with their color-matched microglomeruli (green, light green and purple at left; two green and one red at right). Red and yellow stars mark two cell bodies (one on each side, lateral) and arrows of like color their respective LTr microglomeruli (medial). All neurons send processes throughout EB rings. Scale bar: 30µm. b, Example frame of white noise stimulus used for RF mapping using reverse correlation. c, Sample RFs of R2 microglomeruli. Red subfields: excitatory responses > 30% of maximum; blue subfields: inhibitory responses < 30% of minimum of mean-subtracted weighted average. See Extended Data Fig. 8 for all RFs. d, Bright bars with four different orientations used as test features. e, Modeled and actual (black) ΔF/F changes of an R2 microglomerulus in response to differently oriented bars (fly 2 in Extended Data Fig. 7). In red: (i) trial used for fitting parameters, and, (ii)–(iv) tests. f, Orientation tuning curves for R2 neurons (two-trial average, fit in red). 90° corresponds to back-to-front movement of vertical bar, 270° to front-to-back movement of vertical bar. Error bars: standard deviation. g, Polar plots of orientation tuning data and fits (red) for data shown in f. h, Microglomeruli of R2 neurons colored by orientation preference (collapsed to 0°–180°) and orientation selectivity (two-trial average). i, Same microglomeruli as in h colored by azimuth and elevation of center of their excitatory RFs, measured using horizontally moving bars as described in Extended Data Fig. 1 (two-trial average). j, Direction selectivity of same microglomeruli (two-trial average). k, Preferred orientation (collapsed to 0°–180°) and l, direction selectivity of microglomeruli in pan-neuronal line (both four-trial average). See Methods for analysis details. Scale bar for h–k: 5µm.

Figure 3. Ring neuron LTr microglomeruli show stereotyped RF properties across flies

a, Subset of RFs measured in R4d neurons across seven flies aligned by similarity (see Extended Data Fig. 8b for full set). Numbers below RFs are cross-correlations with top RF in column as template. b, Histogram of cross-correlation values calculated for R4d neuron RFs with best-matched template. c, Histogram of cross-correlation values for R2 neurons (n = 6 flies, see Extended Data Fig. 8a for RFs).

Figure 4. Ring neuron visual responses are not significantly modulated during walking but diminished during flight

a, Setup for two-photon imaging in behaving flies. Insets: Schematic of fly tethered in flying holder, and positioned on air-supported ball in walking holder. b, Subset of simultaneously recorded R2 neurons during walking. Starred boxes: responses to identical visual stimuli when fly is stationary versus walking. Azimuthal position of visual stimulus shown in e (bottom). c, Distributions of R4d neuron visual responses during walking and non-walking conditions are not significantly different (n = 14 flies, trials_walking = 1722, trials_walking<50% = 42, mean_walking = 0±44.3, mean_walking<50% = 1.1±49.7, p = 0.45). d, Same as c for R2 neurons (n = 8 flies, trials_walking = 2015, trials_walking<50% = 245, mean_walking = 0±28.1, mean_walking<50% = −2.2±24.4, p = 0.37). e, Subset of simultaneously recorded R4d microglomeruli during flight. Starred boxes: diminished responses to identical visual stimuli during flight. f, Distributions of all R4d microglomeruli recorded shows significant shift towards lower responses during flight (n = 13 flies, trials_flying = 759, trials_flying<50% = 481, mean_flying = 0±42.2, mean_flying<50% = 31.2±72.3, p = 6·10⁻¹⁵). All p-values: two-sample Kolmogorov-Smirnov test.