Selection of Appropriate Behavioral Responses to Acoustic Stimuli Is a Dynamic Process

(A) Temporal projections over 40 ms post-stimulus of wild-type 5-dpf larvae performing SLC and LLC behaviors.

(B) Time course of the initial C-bend of wild-type (WIK) fish performing SLC and LLC responses, 16 dpf. Numbers show elapsed time (in milliseconds) after stimulus; red asterisks indicate active pectoral fin usage.

(C) Average behavioral bias (black, left axis) and response frequency (gray, right axis) of 48 larvae (5 dpf) to acoustic stimuli (p < 0.0001, Kruskal-Wallis test). Relative startle bias index was calculated for each larva at each intensity (see STAR Methods).

(D) Average relative startle bias of 5-dpf larvae following identical 26-dB stimuli at 20-s intervals (stimuli 1–10) and then 1-s intervals (stimuli 11–40).

(E) Average relative startle bias of 5-dpf larvae treated for 20 min with serotonin (5-HT) or dopamine D3 receptor agonist R-(+)-7-Hydroxy-DPAT (7OHD). Numbers of larvae tested are given at base of bars.

Error bars indicate SEM. See also Figures S1 and S2, Table S1, and Video S1.

Isolation of Decision-Making Mutants from a Forward Genetic Screen

(A) Average startle biases of decision-making mutants, presenting the average bias of the “bottom 25%” of all tested larvae from heterozygous mutant carrier incrosses (n ⩾ 47 mutant larvae each), with the bottom 25% of representative wild-type larvae (Tüpfel long fin [TLF], indicated in blue; n = 28).

(B) Temporal projection over 40 ms post-stimulus (26 dB) of wild-type and wrong turn mutant 5-dpf larval responses. Percentage indicates average frequency observed (30 WT and 58 wrong turn larvae).

(C) Percentage of short- and long-latency responses using pectoral fins during the initial C-bend. n = 14 sibling (blue, 250 responses), and n = 14 wrong turn (red, 112 responses), Fisher’s exact test.

(D and E) Acoustic stimulus intensity versus average relative startle index (D) or average overall startle responsiveness (E) for wild-type (blue) and wrong turn mutants (red).

(F) Average relative startle bias of 5-dpf wrong turn and wild-type larvae following identical 26-dB stimuli at 20-s intervals (stimuli 1–10) and then 1–s intervals (stimuli 11–40).

(G) Zebrafish CaSR protein showing locations of the wrong turn^p190 and CaSR^p198 mutations, Signal Sequence (S, yellow), extracellular Venus Fly Trap domain (VFT, blue), cysteine-rich domain (CRD, pink), 7-pass transmembrane domain (7TMD, orange), C-terminal domain (CTD, green), 5 key residues of the ligand-binding pocket of the VFT hinge (light blue), and PKC phosphorylation residue (arrowhead).

Error bars indicate SEM. ^∗∗p < 0.01; ^∗∗∗∗p < 0.0001, Bonferroni-corrected t test. NS, not significant. See also Figures S3 and S4.

Mauthner Morphology and Function Are Largely Unperturbed in CaSR Mutants

(A) Representative projection of wild-type Mauthner-neuron-expressing membrane-targeted gap43-citrine, stained with anti-GFP. Non-Mauthner labeling was thresholded, and neurons were reoriented for clarity.

(B) Surface of wild-type Mauthner neuron from (A) segmented into the lateral dendrite (LD, yellow), ventral dendrite (VD, green), initial axon segment (IAS, magenta), and soma (cyan) for morphological quantification.

(C and D) Quantification of the volume (C) and surface area (D) of segmented Mauthner neuron regions (n = 15 sibling and 12 CaSR mutant neurons).

(E–J) Representative GCaMP6s fluorescence in Mauthner neurons of sibling (E, G, and I) and CaSR mutant (F, H, and J) larvae. Baseline fluorescence immediately prior to stimuli (E and F), and peak fluorescence during SLC (G and H) or LLC (I and J).

(K) Peak ΔF/F in Mauthner soma during responses of sibling (blue) and CaSR mutant (red) larvae to 13-dB acoustic stimuli. n = 28 sibling SLC responses, 12 CaSR SLC responses, 8 sibling LLC responses, and 26 CaSR LLC responses.

Error bars indicate SEM. See also Figure S5 and Table S2.