Fine-grained descending control of steering in walking Drosophila

Abstract

Locomotion involves rhythmic limb movement patterns that originate in circuits outside the brain. Purposeful locomotion requires descending commands from the brain, but we do not understand how these commands are structured. Here, we investigate this issue, focusing on the control of steering in walking Drosophila. First, we describe different limb "gestures" associated with different steering maneuvers. Next, we identify a set of descending neurons whose activity predicts steering. Focusing on two descending cell types downstream of distinct brain networks, we show that they evoke specific limb gestures: one lengthens strides on the outside of a turn, while the other attenuates strides on the inside of a turn. Our results suggest that a single descending neuron can have opposite effects during different locomotor rhythm phases, and we identify networks positioned to implement this phase-specific gating. Together, our results show how purposeful locomotion emerges from specific, coordinated modulations of low-level patterns.

Keywords: DNa02; DNg13; Drosophila; descending neurons; locomotion; steering; walking.

Figure 1.. Different steering maneuvers use different leg gestures

(A) During the power stroke, the leg pushes backward. During the return stroke, the leg swings forward to a new position. Schematics below show each leg at the endpoint of its power and return strokes. Stride length is the difference between the endpoints in the anterior-posterior axis. (B) Left: example steering bout, showing the path of the fly’s body (gray) and the positions of the leg tips at the end of the return stroke. Right: the fly’s rotational and forward body velocity and the anterior-posterior position of the leg tips in body-centric coordinates. Dotted line marks the peak of the turn. (C) When flies rotate, stride length decreases for the inside legs, while increasing for the outside legs. For each turning bout, we measured the bout’s peak rotational velocity and the stride length for the step that occurred at that peak. Stride lengths are expressed as the change from the mean when the fly was not turning. Stride length varies significantly with rotational velocity for both outside and inside legs. Violin plots are Gaussian kernel estimates fit to all data points. n = 138 (55), 454 (68), 434 (68), and 151 (50) bouts (flies) for 20–50, 50–100, 100–150, and 150–200 °/s, respectively. Asterisks mark legs with significant changes. (D) Top: paths for example steering bouts, corresponding to a pivot and a swerve. Bottom: rotational and forward velocity for these examples. Pivots are bouts where forward velocity decreases by >1 mm/s; swerves are bouts where forward velocity increases by >1 mm/s. In swerves, because forward velocity is high, the curvature of the path is more subtle than it is for pivots. (E) Pivots and swerves produce different changes in stride length (n = 389 (64) and 155 (53) bouts (flies), respectively). See also Figure S1. See Table S1 for statistics.

Figure 2.. Many descending neuron types correlate with the body’s rotational velocity

DNa01, DNa02, DNb05, DNb06, and DNg13 are represented by a single right-left cell pair; calcium imaging shows that all 5 correlate with rotational velocity. (A) Left: schematic of the left cell for each DN type. Right: in example flies, the mean right-left difference in ΔF/F, as a heatmap over rotational and forward velocity bins. Here and in (B), we excluded times when the fly was not walking. Gray bins had <20 time points. (B) Rotational velocity is related to the right-left difference in ΔF/F. Means of individual flies are in gray, with the mean across flies in black. The mean ± SEM across flies of the correlation between rotational velocity and ΔF/F is shown. For (B-D), n (flies) is 10 (DNa01), 8 (DNa02), 7 (DNb05), 7 (DNb06), 5 (DNg13). (C) Filters describing the mean rotational velocity around an impulse change in the right-left difference in ΔF/F. Here and in (D), we did not exclude times when the fly was not walking. (D) Filters describing the mean right-left difference in ΔF/F around an impulse change in rotational velocity. See also Figures S2 and S3.

Figure 3.. DNa02 and DNg13 have distinct inputs in the brain and distinct effects on leg movement

(A) Left: Schematic of the left and right DNa02 and DNg13 cells in the brain. Right: The full brain connectome shows that DNa02 and DNg13 neurons in the same hemisphere share relatively few presynaptic cells (numbers are cell counts). (B) Summary of the cells presynaptic to each DN in the brain. These DNs also receive axo-axonic inputs in the ventral nerve cord, which are not included here. (C) The fly walks on a spherical treadmill coated with IR long-pass ink and painted with spots of far-red fluorescent ink. The camera used for tracking the sphere (and thus the fictive body velocity) captures only far-red light, making the ball appear black with white spots (top). The camera used for tracking the legs captures only IR light; because the sphere is transparent to IR light, it is not visible in this view (bottom). Key points tracked by Animal Part Tracker are marked. (D) Example trials for unilateral optogenetic stimulation of DNa02 with CsChrimson (left) and unilateral depolarization of DNg13 by current injection (right). Vertical lines mark the onset and offset of stimulation. See also Figure S4.

Figure 4.. Different descending neurons drive distinctive leg gestures

(A) Difference in rotational and forward velocity during unilateral stimulation of DNa02 with CsChrimson (CsCh). Dots are flies, and lines are means. Ipsiversive (positive) and contraversive (negative) are relative to the side of the soma of the CsCh+ cell and randomly assigned for no CsCh controls. Differences are significant for rotational but not forward.n=8 (CsCh+) and 7 (no CsCh) flies. Box marks a significant change. (B) Unilateral stimulation of DNa02 produces leg-specific changes in stride length. Boxes around ipsi/contra mark significant effects ±CsCh in post-hoc tests considering all three legs on one side, while boxes around individual panels mark significant effects for that particular leg. (C) Unilateral stimulation of DNa02 shortens the return stroke. (D) Difference in rotational and forward velocity during unilateral hyperpolarization of DNa02 with GtACR1. n=17 (GtACR1+) and 7 (no GtACR1) flies. (E) Unilateral hyperpolarization of DNa02 increases ipsilateral stride length. (F) Unilateral hyperpolarization of DNa02 lengthens the return and power strokes. (G) The effects of DNg13 current injection are significant for rotational but not forward velocity. n=6 flies. Lines connect data from the same fly. (H) DNg13 current injection produces leg-specific changes in stride length. (I) DNg13 current injection affects stride length during both phases of the stride cycle in the contralateral legs. See also Figures S5 and S6. See Table S1 for statistics.

Figure 5.. Descending neuron activity is correlated with specific leg gestures

(A) Example recordings. DNa02 spiking increases just before a decrease in ipsilateral stride length and ipsiversive turning. DNg13 spiking increases just before an increase in contralateral stride length and ipsiversive turning. Shading highlights the change in behavior. (B) Rotational velocity is related to DNa02 and DNg13 spike rate (normalized per cell). Positive rotational velocity is ipsiversive. Thin lines are mean values for individual flies; thick lines are averages across flies (DNa02: n=4 cells in 4 flies; DNg13: n=9 cells in 9 flies). Mean ± SEM across flies of the correlation between rotational velocity and spike rate is also shown. (C) Same but relating DNa02 spike rate with ipsilateral (i.e. the side affected by optogenetic manipulation) step parameters, contralateral stride length, and step direction. (D) Same but for DNg13 and contralateral (i.e. the side affected by current injection) step parameters, ipsilateral stride length, and step direction. (E) Left: DNa02 and DNg13 spike rate when steering ipsilaterally while also decelerating or accelerating in the forward direction. Dots are means for each fly, lines are means across flies, and gray lines connect points from the same fly. Right: difference in spike rate for acceleration - deceleration. Asterisks mark significant differences. See also Figure S7. See Table S1 for statistics.

Figure 6.. Different descending neurons are recruited at different moments within a steering maneuver

(A) Left: Filters show mean change in rotational velocity around an impulse change in DN firing rate. Thin lines are individual flies, thick lines are means across flies (DNa02: n=4 cells in 4 flies; DNg13: 5 cells in 5 flies). Stripchart shows time of the filter peak; dots are individual flies, horizontal lines are means across flies. Asterisks mark significant differences. Right: Filters show mean change in stride length around an impulse change in DN firing rate (in ipsilateral legs for DNa02 and contralateral legs for DNg13). For ease of comparison, the filter for DNa02 was multiplied by −1. Stripchart shows time of the filter peak. (B) Autocorrelation of DNa02 and DNg13 spike rate during walking (DNa02: n=4 cells in 4 flies; DNg13: 9 cells in 9 flies). Stripchart shows the full-width at half-maximum of the autocorrelation function. (C) Left: Changes in stride length over time for each pair of legs during turning bouts in freely walking flies. Bouts are aligned to the peak in rotational velocity (n=818 bouts in 73 flies). Right: The full width at half-maximum for the change in stride length over time.. Violin plots are Gaussian kernel estimates fit to all data points. (D) Top: example recordings of DNa02 and DNg13, overlaid with the stride cycle phase. The stride cycle is expressed relative to the phase of the ipsilateral (DNa02)/contralateral (DNg13) middle leg. Bottom: Timing of DNa02 and DNg13 spikes relative to the stride cycle 100 ms prior. DNa02: n=4 cells in 4 flies; DNg13: 9 cells in 9 flies. See Table S1 for statistics.

Figure 7.. Steering descending neurons have specialized outputs

(A) Schematic: morphology of the left DNa02 and the right DNg13 axons in the ventral nerve cord. Here, left and right refer to soma locations in the brain; both of these cells send axons to the left side of the cord. Stacked bar charts summarize the cells postsynaptic to each DN in the leg neuromeres of the cord. Note that DNa02 also targets neurons in the neuropils associated with the wings, halteres, and neck, and those are excluded from this plot. (B) The distribution across the three leg neuromeres (T1, T2, T3) of the postsynaptic neurons. Intersegmental neurons are assigned based on their cell body locations. (C) DNa02 and DNg13 axons projecting to the left side of the cord share almost no postsynaptic cells (numbers are cell counts). (D) Monosynaptic and disynaptic connections between DNa02/DNg13 axons projecting to the left side of the cord and motor neurons controlling the front legs. DNa02 and DNg13 graphs are plotted separately, and thus some motor neurons are repeated. Motor neurons are labeled according to their inferred effect based on published anatomical work, with unlabeled motor neurons having unknown effects. Line thickness is proportional to the number of synapses. Connections between premotor neurons are not shown. (E) Left: limb kinematics during the power stroke. Right: cells downstream from DNg13 that can potentially explain its ability to increase stride length during the power stroke. (F) Same but for return stroke. (G) Schematic summarizing the effects of each DN type on stride length, body velocity, and path shape. See also Figure S4.