(A,B) Novel tank assay. Area in each color shows percentage time spent in either half of a novel tank in one minute by five Control fish and five TeTXlc expressing fish. Arrows indicate median swim time in the bottom half for controls (20.4 ± 3.7%) and TeTXlc fish (48.6 ± 9%). (C–F) Response to dilute alarm substance and an overhead shadow. (C) Time spent in the bottom quarter of a tank, and median number of (D) slow swimming, (E) pauses, and (F) darts. Non-parametric, Mann–Whitney U-test was used to compare behavior of control and TeTXlc fish following exposure to the stimuli. (Mann–Whitney U-test, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001). Zebrafish expressing TeTXlc-CFP in the habenula were generated by crossing GAL4^s1019t with UAS:TeTXlc-CFP, and selected on the basis of CFP fluorescence in the brain. For the novel tank assay, fish were transferred into a glass tank measuring 20 × 12 × 5 cm—L × H × W containing 1 L of water and observed for 1 min. Digital videos were recorded as described before (Mathuru et al., 2012). Fish position in the tank was tracked and analyzed automatically using “track objects” algorithm in MetaMorph. 6.3 and custom written macros for Excel. Alarm substance was prepared as described before (Mathuru et al., 2012). This was introduced into the tank and a shadow, created by passing a hand under the overhead light, was presented after 2 min. Darting was defined as episodes during which the swimming speed exceeded baseline speed by more than 8 SD, slow swim episodes were defined as episodes greater than one second in duration during which the swim speed never exceeded half the mean baseline speed, while one second of immobility (speed less than 3 mm/s) was considered as a pause episode.