(A) Sequences upstream of the slc5a7a transcriptional start site before (WT) and after integration of Cre (blue indicates donor DNA) at sgRNA target site (red nucleotides and PAM sequences in green). (B) Schematic diagram of intersectional strategy using Cre/lox mediated recombination and the QF2/QUAS binary system. QF2 is driven by lratd2a regulatory sequences and the slc5a7a promoter drives Cre leading to reporter/effector expression in lratd2a neurons in the right dHb. (C) Dorsal view of GFP labeling in only the right dHb after Cre-mediated recombination in a 5 dpf Tg(lratd2a:QF2), Tg(slc5a7a:Cre), Tg(QUAS:loxP-mCherry-loxP-GFP-CAAX) larva. Scale bar, 25 μm. (D) BoTxBLC-GFP-labeled cells (open arrowhead) in the right dHb in Tg(lratd2a:QF2), Tg(slc5a7a:Cre), Tg(QUAS:loxP-mCherry-loxP-BoTxBLC-GFP) 5 dpf, 37 dpf, and 4 mpf zebrafish. Upper images show mCherry-labeled lratd2a Hb neurons, middle images show the subset of right dHb neurons that switched to GFP expression, and the bottom row are merged images. Scale bar, 50 μm. (E) Transverse section of BoTxBLC-GFP labeled axonal endings of dHb neurons that express Cre and lratd2a in a subregion of the vIPN (bracket) in Tg(lratd2a:QF2), Tg(slc5a7a:Cre), Tg(QUAS:loxP-mCherry-loxP-BoTxBLC-GFP) 37 dpf juveniles. Scale bar, 50 μm. (F, G) Preferred tank location prior to and after cadaverine addition of adults genotyped for absence (Cre-) or presence (Cre+) of Tg(slc5a7a:Cre). (F) Representative 1 min traces for single Cre- (blue) and Cre+ (purple) adults recorded over 10 min prior to (min 0–5) and after (min 6–10) addition of cadaverine to one end of the test tank (open arrows). (G) Preference index for all adults for an average of 2 min before (white) and for each of 3 min after (gray) the addition of cadaverine. In Cre- fish, aversive behavior was significantly increased at 2 min (p = 0.0116) and 3 min (p = 0.0344), n = 15 fish for each group. In contrast, Cre+ fish, showed no significant difference in their preferred location over time. Dashed red lines in F and G denote midpoint of test tank. Two-way ANOVA reveals significant effects of time [F(3, 27) = 29, p < 0.0001], but no effect of group [F(1, 14) = 2.381] and interaction [F(3, 33) = 1.813]. Post-hoc analysis by Bonferroni’s multiple comparisons. (H) Swimming speed during 1 min period before and after addition of alarm substance was similar for Cre- [3.68 ± 0.47 and 7.43 ± 1.1 cm/s] and Cre+ [4.02 ± 0.42 s and 7.93 ± 0.92 cm/s] adults. Two-way ANOVA reveals significant effects of time [F(1, 16) = 39.61, p < 0.0001], but no effect of group [F(1,16) = 0.2236] and interaction [F(1,16) = 0.0141]. Post-hoc analysis by Bonferroni’s multiple comparisons. (I) Duration in the upper half of the test tank prior to and after addition of alarm substance for Cre+ adults was 96.6 ± 15.72 s and 13.23 ± 3.34 s and for Cre- adults was 125.53 ± 18.6 s and 8.26 ± 2.5 s. Two-way ANOVA reveals a significant effect of time [F(1, 16) = 63.79, p < 0.0001], but no effect of group [F(1,16) = 1.048] and interaction [F(1,16) = 0.0141]. Post-hoc analysis by Bonferroni’s multiple comparisons. (J) Onset of fast swimming after application of alarm substance was observed at 25 ± 4.05 and at 22.7 ± 3.72 sec for Cre- and Cre+ fish, respectively [p = 0.679, unpaired t-test]. (K) Time interval between increased swimming speed and freezing behavior for Cre- (68.88 ± 23.36 s) and Cre+ (20.88 ± 3.93 s) adults [p = 0.051, unpaired t-test]. For H-K, all numbers represent the mean ± SEM.
