Object-Detecting Neurons in Drosophila

Abstract

Many animals rely on vision to detect objects such as conspecifics, predators, and prey. Hypercomplex cells found in feline cortex and small target motion detectors found in dragonfly and hoverfly optic lobes demonstrate robust tuning for small objects, with weak or no response to larger objects or movement of the visual panorama [1-3]. However, the relationship among anatomical, molecular, and functional properties of object detection circuitry is not understood. Here we characterize a specialized object detector in Drosophila, the lobula columnar neuron LC11 [4]. By imaging calcium dynamics with two-photon excitation microscopy, we show that LC11 responds to the omni-directional movement of a small object darker than the background, with little or no responses to static flicker, vertically elongated bars, or panoramic gratings. LC11 dendrites innervate multiple layers of the lobula, and each dendrite spans enough columns to sample 75° of visual space, yet the area that evokes calcium responses is only 20° wide and shows robust responses to a 2.2° object spanning less than half of one facet of the compound eye. The dendrites of neighboring LC11s encode object motion retinotopically, but the axon terminals fuse into a glomerular structure in the central brain where retinotopy is lost. Blocking inhibitory ionic currents abolishes small object sensitivity and facilitates responses to elongated bars and gratings. Our results reveal high-acuity object motion detection in the Drosophila optic lobe.

Keywords: motion detection; object perception; visual object.

Figure 1. Anatomy and object selectivity of Lobula Columnar 11

A) Maximum intensity projection of the anterior view of a brain from a fly expressing membrane tethered GFP under the control of R22H02-Gal4 and labeled with anti-GFP (green) and nc82 (magenta). Dashed lines indicate the boundary of the ventrolateral protocerebrum. Arrowheads indicate cellular compartments; blue-cell bodies, red-terminals, yellow-dendrites. B) Dorsally mounted view of R22H02-Gal4>UAS-mCD8::GFP flies. Dashed rectangle indicates the unique foot shaped LC11 glomerulus. Me: Medulla, Lo: Lobula, LoP: Lobula Plate. Comparison of the labeling of ~50 LC11s innervating the glomerulus (Bi) vs. stochastic labeling of a single LC11 (Bii). Single LC11 shows the full glomerular innervation with no evidence of retinotopic organization. Blue dashed line indicates the glomerular boundary. C) Anterior view of a stochastic labeling of a single LC11. Dashed lines indicate individual columns within the lobula. The dendritic arbor of a single LC11 covers about 14–15 lobula retinotopic columns in this plane. D) Dorsal view of a single LC11. Dendritic arbors span 6–7 columns in this plane. E) Morphology of a single LC11. Yellow arrows indicate the bistratified dendritic morphology of LC11 within lobula. Blue and red arrowheads indicate cell body and terminals respectively. All scale bars are 25 um. F–H) Single confocal plane images of multicolor stochastic labeling of LC11s. Multiple cells were labeled and tagged with myristoylated smGFP attached to either HA (green) or FLAG (red) epitopes. Neuropile is labeled with nc82 (blue). The lobula is traced with a dashed line. Scale bar is 10 um. G,H) Red and green channels are displayed separately, and labeling is traced to highlight dendritic overlap (I). See also Figure S1. J–O) 2-photon imaging. J) The fly’s head is fixed and the surrounding LED arena covers 216° in azimuth and 63.2° in elevation. K) Image of LC11s expressing GCaMP6m under two-photon microscopy. Arrowheads indicate dendrites (cyan), axon terminals forming optic glomerulus (green) and cell bodies (orange). L) Mean GCaMP6m (±S.E.M. shading) signal from LC11 glomerulus in response to the movement of a 30°by 8.8° object (blue), a 30° by 70° bar (black) and a wide-field grating (red, n = 7 flies). M) Mean GCaMP6m (±S.E.M. shading) signal from LC11 glomerulus in response to the movement of a 30° by 70° bar (black), a 2.2° square object (red) and a 4.4° square object (purple) (n = 6 flies). Inset: From tethered flies, normalized mean steering responses to single pixel impulsive displacement of objects, sizes indicated (scale bar: t=0–100 ms, n = 10 flies). N) Normalized mean ΔF/F of cell body (orange), dendrites (cyan) and axon terminals (green) responses to the movement of a 30° by 8.8° object as in C. Visual midline indicated with a dashed grey line. n = 7 flies. O) Comparison of the peak onset delay between the dendrites (blue), terminal (red), and cell body (black) (*p<0.05, **p<0.01, paired t-test, n = 7 flies). All visual stimuli moved at 22 °/sec.

Figure 2. Individual LC11 receptive fields

A) Schematic of the experimental stimuli used to map individual LC11 receptive fields from individual cell body recordings. An 8.8° square dark object was scanned along non-overlapping trajectories along both horizontal and vertical paths at 33 °/sec. B) Reconstructed receptive field of a single LC11. Individual imaging responses from a single LC11 to the horizontal and vertical sweeps indicated in red and purple, respectively (scale bar inset represents 200% ΔF/F, 5 seconds). Reconstructed estimate of a single LC11 receptive field shown in blue (see Supplemental Experimental Procedures), the full-width at 25% max contour was drawn in white. C) Representative receptive field contours (25% max) from six preparations are mapped onto the projection of the visual display. D) 11 receptive fields from 6 flies are overlaid and color coded as in C. D′) a dot is plotted at the centroid of each receptive field to indicate the spatial distribution of sampled LC11 recordings. E) To analyze the retinotopy in the dendrites of neighboring LC11 columnar cells, ROIs from separate dendritic compartments are indicated by colored box. An object swept was horizontally at elevations indicated by the cartoon display. ΔF/F responses from all 10 ROIs are overlaid for each elevation. E′) To facilitate spatial comparisons, the responses are normalized to the maximum ΔF/F calcium signal at each ROI. Note that anterior (red) ROIs are activated by object motion across the top of the display, whereas posterior (blue) ROIs are activated by object motion across the bottom of the display. Dashed line indicates the earliest responses of anterior ROIs. Scale bar for the two-photon image represents 10 um. Abbreviations for anatomical directions; A: Anterior, P: Posterior, M: Medial and L: Lateral. See also Figure S2.

Figure 3. LC11 is contrast selective, omni-directional, and end-stopped on the spatial scale of one receptive field

A) Mean GCaMP6m signal from the LC11 terminal output glomerulus in response to a 30°by 8.8° moving ON object (red) and moving OFF object (black, n = 7 flies). A′). Pairwise comparison of maximum ΔF/F of responses from each preparation (***p<0.001, paired t-test, n = 7 flies). A″) Average maximum responses (±S.E.M.) of LC11 glomerulus to a stationary 30°by 8.8°OFF and ON object placed within the hotspot of the receptive field (n = 6 flies). B) Mean GCaMP6m signal from the LC11 glomerulus in response to varying contrast objects. Grayscale of the filled area is used to indicate the intensity of the visual background, whereas grayscale of the response line indicates intensity of the stimulus object (n = 9 flies). The most contrasting objects do not elicit the maximum responses from LC11. B′) Average of maximum responses (±S.E.M.) of the LC11 glomerulus to objects of varying contrast. Schematic on the x-axis shows the intensity of the background compared to each object. Weber contrast values are indicated numerically (see Supplemental Experimental Procedures, one-way ANOVA, n = 9 flies). C–F) LC11 glomerulus responses to parameterized direction (C, C′), vertical height (D, D′) horizontal width (E, E′), and two-object separation distance (F, F′). Time series responses shown in A–F, and color coded parameter values and maximum responses (±S.E.M) shown in C′–F′. C and C′) LC11 is omni-directional. An 8.8° square object was moved in 8 different directions in 45° steps as indicated by color-coded arrowheads (n = 7 flies, see single cell responses in Figure S3). D and D′) LC11 is vertically size tuned. A 30° wide object was moved on the same horizontal trajectory, with varied vertical heights: 2.2°, 4.4°, 8.8°, 18°, 35°, 73.2°, colors mapped to object size in B′ (n = 7 flies). Vertical gray line indicates average receptive field (RF) size (Figure 2). E and E′) LC11 is horizontally size tuned. An object of fixed height (8.8°) and varied width: 2.2°, 4.4°, 8.8°, 18°, 35°, 70°, 210°, was moved horizontally (n = 15 flies). The leading edge of each object appeared on the LED display at the same time. Vertical gray line indicates average estimated functional RF size (Figure 2). F and F′) LC11 is inhibited by a second object. Two 8.8° square objects moved on parallel trajectories. The distance between them was 0°, 2.2°, 4.4°, 8.8°, 18° and 32°, colors mapped to separation distance in C′ (n = 6 flies). Vertical gray line indicates average RF size (Figure 2). See also Figure S3.

Figure 4. Both sensitivity and selectivity for objects by LC11 requires inhibition

A and B) LC11 dendritic layer is enriched with the vesicular GABA transporter (VGAT), and the adjacent presumably presynaptic layer is enriched with choline acetyltransferase (ChAT). Dorsal view of GFP labeled LC11 neurons (green) co-labeled with either anti-ChAT (A, magenta) or VGAT (B, magenta). Dashed line indicates the border between the first and second lobula strata. Scale bars 25 um. C and D) Layering of VGAT and ChAT are highlighted with the same labeling as in A and B, but without LC11 overlaid. Scale bars are 25 um. E – G) Inhibition sculpts object responses and inhibits bar and grating responses. Time series glomerular LC11 responses from n = 7 flies in response to a 30°by 8.8° object (E), a 30° by 70° bar (F) and a wide-field grating (G) with (red) or without (black) 10 um picrotoxin. E′ – G′) Average maximum responses from each fly (E – G) with (red) or without (black) picrotoxin.