Fig. 3

Simultaneous Analysis of Escape Behavior and M-Cell Calcium Signals during ASR Habituation in 6 dpf Zebrafish Larvae

(A) Simultaneous behavior (2.5× objective, 1,000 fps) and GCaMP5 calcium imaging of M-cells (40× objective, 40 fps) with two independent light paths.

(B) Representative image of time-series projection (ventral view) of sound-induced escape behavior.

(C) Tracking of tail movement during response to acoustic stimulus at t = 20 ms.

(D) Representative image (dorsal view) of tg(s1181t:Gal4;UAS:kaede) transgenic zebrafish line. Scale bar, 25 µm.

(E and F) GCaMP5 calcium activity traces from M-cells in response to acoustic stimulus at 400 ms.

(G) Acoustic startle responses of individual larvae ordered along y axis by probability of escape over the stimulus series. Each dot represents a sound-induced escape event: large dot represents escape with two M-cells active; small dot represents escape with one M-cell active.

(H) Decrease in ASR probability during stimulus series (F-statistic versus constant model: 41.5, p < 10^-9, adjusted R-squared = 0.085).

(I) ASR habituation accompanied by significant decrease in the fraction of escapes with two active M-cells (F-statistic versus constant model: 9.15, p < 0.01, adjusted R-squared = 0.028). Statistics: linear regression model analysis. N_larvae = 29. Error bars, SEM.