Figure 2

Late-stage zebrafish larvae acquired the conditioned fear response. (a) A typical HB pattern in the classical fear conditioning, showing the heart movement in the 15^th trial of the habituation session, the 16^th trial of the acquisition session, and the 8^th trial of the probe session, and the timing of the CS (gray boxes) and US (yellow line). About 20-dpf wild-type larvae were used. Representative data obtained from a single individual larva are shown. The x-axis shows the time (sec). The y-axis shows the heartbeats as monitored by luminosity (arbitrary unit) at an appropriate region of the heart in IR video. Each peak represents one heartbeat. In the acquisition trial, the US elicited the bradycardic response. Note that in the late acquisition session (middle panel) and in the probe session (right panel), bradycardia, denoted by horizontal bars, occurred after the start of the CS presentation. (b) The relative HB frequency in the habituation and probe sessions. The HB frequencies from 10 trials (6^th–15^th trials of habituation; 1^st–10^th trials of the probe session) were averaged, divided by the control HB frequency (the average HB frequency for 2 s before the CS), and indicated as a relative HB frequency in the graph (y-axis). The relative HB frequency was determined every 11.1 ms. The shaded error region shows the standard deviation (SD); gray boxes show the timing of the CS. Representative data obtained from a single individual larva are shown. We compared the relative HB frequency between the habituation and probe sessions, and identified larva with a significantly reduced relative HB frequency during the first 2 s of the CS period as learners. (c) Percentage of wild-type fish showing CS-dependent bradycardia (learners) in the delayed fear conditioning and the control backward conditioning. 15 of 40 larvae were learners in the delayed conditioning, and none of 10 were learners in the backward conditioning.