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Fig. 3 A change in optic flow direction is encoded in enhanced responses to probe stimuli. (A) Schematic of the experimental protocol. Trials 2 to 13 began with an acclimatization phase of 8× optic flow pulses at 1 cm/s followed by a probe. (B to E) Average calcium responses in the backward-tuned cells to the different trials [(B), negative probe; (C), random stimulus timing followed by negative probe; (D), positive probe; and (E), no probe)]. The respective optic flow stimuli and average motor activity are shown above. Gray traces are the responses of individual fish (N = 8). Fold change in peak amplitude in response to the three presentations of the negative probe stimuli with respect to (w.r.t.) the baseline amplitude in trial 1 is shown for (B) and (C). *P < 0.05 and **P < 0.01 by Wilcoxon signed-rank test showing an elevation in amplitude w.r.t. baseline (dashed line at 0). There were no large calcium responses in the backward-tuned cells to the positive probe or the zero probe [(D) and (E)]. (F) Average calcium response in backward-tuned cells to the negative probe stimulus when presented in random order in trial 1 (gray) and to the first presentation of the same stimulus after acclimatization (black). (G and H) The peak amplitude in dF/F of the trial-averaged calcium response to the respective stimuli shown on the x axis for the backward-tuned cells (G) and the forward-tuned cells (H). *P < 0.05 w.r.t. the baseline amplitude by post hoc Conover’s test following a significant Friedman’s test (P = 3 × 10−5). n = 98 cells, N = 8 fish in (G); n = 41 cells, N = 7 fish in (H). No large calcium events were seen in forward-tuned cells to any of the probe stimuli. Hence, no statistical analysis was performed to test for systematic changes in amplitude values across all trial types (see fig. S2).