Quantitative Predictions Orchestrate Visual Signaling in Drosophila

Abstract

Vision influences behavior, but ongoing behavior also modulates vision in animals ranging from insects to primates. The function and biophysical mechanisms of most such modulations remain unresolved. Here, we combine behavioral genetics, electrophysiology, and high-speed videography to advance a function for behavioral modulations of visual processing in Drosophila. We argue that a set of motion-sensitive visual neurons regulate gaze-stabilizing head movements. We describe how, during flight turns, Drosophila perform a set of head movements that require silencing their gaze-stability reflexes along the primary rotation axis of the turn. Consistent with this behavioral requirement, we find pervasive motor-related inputs to the visual neurons, which quantitatively silence their predicted visual responses to rotations around the relevant axis while preserving sensitivity around other axes. This work proposes a function for a behavioral modulation of visual processing and illustrates how the brain can remove one sensory signal from a circuit carrying multiple related signals.

Keywords: Drosophila; action initiation; corollary discharge; efference copy; electrophysiology; eye movements; neural circuits; patch-clamp; vision; visuomotor processing.

Figure 1. Horizontal system (HS) and vertical system (VS) cells in the fly visual system may mediate optomotor responses and thus might need to be silenced with an efference copy during intended turns

(A) A schematic of the fly visual lobe with HS and VS cells in the lobula plate. (B) Immuno-amplified GFP signal of HS and VS cells, using the DB331-GAL4 driver, with nc82 anti-Bruchpilot neuropil counterstain in magenta. (C) Schematic illustrating head and body optomotor responses. A gust of wind causes the animal to turn, by accident. Visual neurons, V, (like HS and VS cells) respond to the optic-flow stimulus induced by this erroneous turn. These visual neurons activate motor neurons, M, which drive the body or head to rotate in the direction of experienced visual motion. A rotation of the body would stabilize straight flight (i.e., course stability), whereas a rotation of the head would stabilize the visual image on the retina (i.e., gaze stability). (D) Schematic of a voluntary turn with an efference copy to suppress the optomotor response. During voluntary turns, an internal decision center, D, sends an efference (motor command) to motor neurons, M, to cause the animal to turn, and a copy of the motor command, an efference copy, to the visual neurons, V. This motor-related input counteracts the expected self-generated visual input caused by the active motor command to turn the animal, thus minimizing or eliminating optomotor responses in the context of voluntary turns.

Figure 2. Expressing a K⁺ channel, Kir2.1, in HS and VS cells impairs optomotor head movements

(A) Average Vm traces of a wild-type HS cell, recorded in the right lobula plate (black) show direction-selective responses to leftward and rightward moving gratings. HS cells expressing Kir2.1 under the control of two GAL4 driver lines show strongly muted visual responses (orange). Average Vm traces for an example Kir2.1 expressing cell and the average of four Kir2.1 expressing cells are shown for each driver line (top traces: +/+;tsh-GAL80/UAS-Kir2.1::EGFP;VT058487-GAL4/+, bottom traces: w¹¹¹⁸/+;UAS-Kir2.1::EGFP/+; R24E09-GAL4/+). (B) Experimental apparatus. The fly’s thorax is tethered to a tungsten pin while its head is free to move during tethered flight. We track the yaw angle of the head using a camera capturing frames at 350 Hz (left image) and a wingbeat analyzer to simultaneously measure wing-steering responses (right image). The visual stimulus consisted of a wide-field stimulus with a naturalistic (1/f) intensity profile along the horizontal dimension (STAR Methods) moving at four different speeds. A sample time series of the right and left wing beat amplitudes together with the concomitant head yaw angles are shown. (C) Baseline subtracted head yaw angle and left-minus-right-wingbeat-amplitude (L-R WBA) signals on single presentations of the fastest stimulus tested (thin lines) and the mean response across all trials (thick lines) for one control fly (black) and one silenced fly (orange). All single trials shown. L-R WBA traces were low-pass filtered using a Gaussian kernel (σ = 2 ms) (D) Maximum z-projection of Kir2.1::EGFP expression in the VT058487-GAL4 line, used to silence HS and VS cells. The fly also carried a tsh-GAL80 transgene, minimizing GAL4 activity in the ventral nerve cord, which helped promote long flight bouts. (E) Mean head and wing responses (+/− SEM) to visual motion at four speeds of flies expressing Kir2.1 (tsh-Gal80;VT058487-GAL4 > Kir2.1) and of parental controls. We presented both rightward and leftward visual motion. Traces represent average baseline subtracted responses to leftward motion and inverted responses to rightward motion at a given speed. (F) Mean +/− SEM of the peak response and initial slope averaged across all conditions in panel E. Average responses of individual flies are shown as single dots. We calculated the peak of each signal by subtracting the mean L–R WBA and head-movement signals in a 100-ms baseline window immediately prior to visual motion onset from the peak signal in a window starting at visual motion onset and extending 200-ms after visual motion offset (peak). We calculated the initial slope of each signal by subtracting the mean signal in a 10-ms window immediately prior to visual motion onset from the mean signal in a 10-ms window starting 80 ms after visual motion onset, and dividing by the time difference between these windows (90 ms) (slope). Silenced flies show a head response that is both smaller and has a shallower slope than control flies (see Main Text for statistics). (G) Same as D, but for R24E09-GAL4. No tsh-GAL80 transgene was used with this driver. (H-I) Same as E-F, but for R24E09-GAL4. No tsh-GAL80 transgene was used with this driver.

Figure 3. During rapid flight turns, or saccades, Drosophila perform an active head rotation against gaze stability about the yaw axis

(A) Schematic of head stabilization movements about the yaw axis. (B) Schematic of head stabilization movements about the roll axis. (C) Head movements expected during a banked turn saccade if all head stabilizing reflexes remain intact, emphasizing that the head yaw stability reflex (red) is maladaptive during saccades. (D) Schematic of the magnetic tether apparatus. A fly, tethered to a ferromagnetic steel pin, is suspended within a magnetic field, and is thus free to perform saccadic rotations of its body around the yaw axis. A camera records the fly’s head and body movements from below. (E) Sample images of the body and head of a magnetically tethered fly performing a leftward saccade. By 30 ms into the saccade (green arrow), the head has performed a leftward yaw rotation relative to the thorax (dotted line) and a clockwise roll rotation (yellow regions highlight the changing visible areas of the left and right compound eye, as viewed from below the fly, indicating a roll movement of the head). (F) Sample traces of body and head angles during leftward saccades. The green arrowhead indicates the saccade illustrated in panel E. (G) Head and body angles associated with saccades from a single fly. Individual saccades are shown in gray. Averages are shown in black. Rightward saccades were inverted and combined with leftward saccades. (H) Head and body angles associated with saccades from all flies. Average traces from individual flies are shown in gray. Averages across all flies are shown in black.

Figure 4. Yaw optic flow elicits strong responses in HS cells, intermediate responses in VS4-6 cells and weak responses in VS1-3 cells

(A) Electrophysiological setup. We angled our visual display by 66° as schematized, so that vertically and horizontally moving stimuli on the display were aligned to the vertical and horizontal motion-sensitive axes of the fly’s retina. We chose 66° based on the average, measured, eye angles of 11 flies placed in our setup (Figure S3). (B) Sample starfield frame (top) and vector fields representing local motion associated with yaw, pitch and roll stimuli (i.e., optic flow fields). (C) Sample Vm trace of one VS5 cell in response to four rotational optic flow stimuli: downward pitch, clockwise roll, counterclockwise roll, and rightward yaw. Stimulus intervals are indicated by gray rectangles. This cell responds strongest to roll, but also shows intermediate responses to yaw. (D–F) Responses of HS, VS1–3, VS4-6 cells to the rotational stimuli experienced at the start of a leftward saccade: rightward yaw, downward pitch, and clockwise roll. Top row: Averaged Vm from a single cell. Bottom row: Population-averaged Vm (colored) +/− SEM (grey band), with the mean baseline Vm indicated (arrows). (G–I) Mean visual responses to yaw, pitch and roll visual motion. We calculated these responses by subtracting the mean Vm in a 500-ms baseline window prior to stimulus onset from the mean Vm during the stimulus presentation (excluding the first 100 ms). Individual dots represent single cells; bars indicate mean +/− SEM. (J) Visual responses to horizontally moving gratings (1 Hz temporal frequency), measured during non-flight. Responses are large in HS, small in VS1-3 and intermediate in VS4-6, similar to the pattern observed for yaw starfield stimuli. (K) Visual responses to a wide-field stimulus mimicking natural scene statistics (1/f spatial-frequency weighting). The stimulus moved with a saccadic velocity profile over 130 ms with a peak velocity of 1000°/s. Like in panels G and J, responses are large in HS, small in VS1-3 and intermediate in VS4-6. All error bars indicate SEM.

Figure 5. All HS and VS cells show saccade-related potentials, but the magnitude of these potentials differs across cell classes

(A) Setup for electrophysiology in tethered, flying Drosophila. We recorded from HS and VS cells in flying Drosophila and measured their wing steering behavior. Left and right wingbeat amplitudes (WBAs) were estimated on each frame (100 Hz frame rate) using image analysis, and these signals were used to calculate the left-minus-right-wingbeat-amplitude (L-R WBA). (B) Sample Vm and L-R WBA traces from an HSN-cell recording session in the context of a uniformly lit screen and during presentation of yaw visual motion. L-R WBA traces were low-pass-filtered with a 10 Hz cut-off frequency. Stimulus interval is highlighted by a gray rectangle; dashed lines facilitate comparisons of baseline changes and SRP magnitudes over the entire time interval. (C) Same as B, but data are shown for a VS5 cell with a clockwise roll visual stimulus. (D) Average baseline-subtracted saccade-related potentials (SRPs) during leftward saccades for all stimulus conditions. Average SRPs from single cells are shown in gray. Average SRPs across all cells are shown in color. The average stimulus-driven (or blank screen) Vm immediately preceding each SRP is indicated in mV (arrows). (E) Distribution of average SRP amplitudes across all seven visual conditions. The SRP amplitude was calculated as the mean Vm in a 50-ms window starting 75 ms after saccade onset from which we subtracted the mean Vm in a 150-ms window, starting 200-ms before saccade onset. (F) Baseline subtracted saccadic L-R WBA signals, averaged across all flies, with all stimulus conditions overlaid.

Figure 6. Amplitudes of saccade-related potentials in HS/VS cells show a strong negative correlation with yaw visual responses, but not with pitch and roll responses, across the population

(A) Mean visual-response amplitudes (in mV) to the yaw-right optic-flow stimulus (black) and mean SRP amplitudes (in mV) for leftward saccades generated during presentation of this stimulus (maroon), for HS, VS1-3 and VS4-6 cells (left) and all cell classes, individually (right). SRP amplitudes measured in the context of a blank screen are shown in orange. Inset: representation of visual response (black arrow) and SRP magnitudes (orange and magenta arrows) (see Main Text for details). (B) The data in panel A, right, are shown here plotted one against the other. Magenta points show amplitudes of yaw-right visual responses plotted against amplitudes of SRPs during the yaw-right starfield stimulus. Orange points show amplitudes of yaw-right visual responses plotted against amplitudes of SRPs during a blank screen. Linear fits are shown for plots with statistically significant, negative correlations. Data points indicate mean +/− SEM. (C–D) Same as panels A-B, but for downward pitch. Positive correlations in panel D are not statistically significant. (E–F) Same as panels A-B, but for clockwise roll. Positive correlations in panel F are not statistically significant.

Figure 7. SRP amplitudes are anti-correlated with pre-saccade Vm for contraversive saccades and different HS/VS cell classes show a different linear relationship between these variables, suggesting different underlying conductance mechanisms

(A) Population-averaged SRPs for contraversive saccades (same data as in Figure 5D), ordered by the stimulus-induced pre-saccadic Vm, so as to highlight its influence on SRP amplitude. Horizontal dashed lines indicate the resting membrane voltage, estimated as the pre-saccade membrane voltage in the context of a blank screen. Solid lines indicate the zero-crossing Vm calculated from the linear fits in panel B. (B) SRP amplitude plotted against the pre-saccadic Vm. Lines indicate linear fits. SRP amplitude was calculated as in Figure 5E. Horizontal and vertical lines indicate +/− SEM of pre-saccadic Vm and SRP amplitude, respectively. (C) A graphical representation of the distance between the in-flight resting potential of each cell class (square) in comparison to the zero crossing point of the linear fits in panel B (arrows). The relative distances of these voltages suggest strong hyperpolarizing inhibition in HS cells, intermediate-strength hyperpolarizing inhibition in VS4-6 and weak shunting/dampening inhibition in VS1-3. (D) Summary diagram. Visual responses of HS and VS cells contribute to driving head stability movements. Cell-type tailored saccade-related inputs to these cells are tuned to match their yaw optic-flow sensitivity. Our working model is that these inputs serve as efference copies to silence the head’s yaw optomotor stability response, while leaving head stability responses around other axes intact.