Extended Data Fig. 10: Transient asymmetries in PFL3 GCaMP activity track the flies’ heading-relative-to-goal with a lag and the turning behaviour induced by unilaterally stimulating subsets of PFL3 neurons in the lateral accessory lobes is probabilistic.

a, Instead of plotting the flies’ turning velocity (that is, the derivative of the flies' heading) during transient increases (top) or decreases (bottom) in PFL3 Right-Left (R-L) activity (black), as we did in Fig. 5c, we plotted the flies’ mean heading-relative-to-goal (teal) during these moments. Mean ± s.e.m. across transients is shown (from 10 flies). Inset shows that the maximum deviation in the R-L PFL3 GCaMP signal occurs ~200 ms after the peak in flies’ heading relative to goal deviation. This delay is in agreement with previous measurements of the lag between the fly’s turning velocity and the change in the EPG bump position in the bridge, measured with GCaMP³⁴ (Extended Data Fig. 3b). This latency is consistent with the transients in LAL activity reflecting a change in the PFL3 heading inputs from the bridge. b, Further analyzing the PFL3 CsChrimson data from Fig. 5, we show the mean ipsilateral turning velocity as a function of the ipsilateral LAL asymmetry (z-scored) during the 2 s stimulation period. The ipsilateral LAL asymmetry is taken as the right–left ΔF/F₀ signal, with the sign of the values flipped for left LAL stimulation trials. Each dot is a trial and all trials from PFL3 CsChrimson flies are shown. In a minority (8%) of all trials, the average turning velocity was in the contralateral (i.e., the unpredicted) direction, despite measuring an LAL asymmetry consistent with the simulation side (trials below the dotted line). An important caveat, when interpreting this result, is that the driver line does not label all PFL3 neurons (see Extended Data Fig. 1g). The measured asymmetry, therefore, does not necessarily reflect the true population-level activity. As such, it is formally possible that during these anomalous trials, the true left/right asymmetry in PFL3 activity was in agreement with the fly’s turning direction. c, Same stimulation trials shown in panel b but here we plotted the mean ipsilateral turning velocity as a function of flies’ mean forward walking velocity 1 s before the onset of the stimulation. Note that “incorrect” trials are not always preceded by moments when the fly is not walking forward (i.e., the points below the dotted line are not all clustered around zero on the x-axis). d, Same stimulation trials shown in panel b but here we plotted the mean ipsilateral turning velocity as a function of flies’ mean ipsilateral turning velocity 1 s before the onset of the stimulation. Note that “incorrect” trials are not always preceded by a contralateral turn (i.e., the points below the dotted line are not all to the left of zero on the x-axis).

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