hrh1 expression in Turku wt fishes and the mutation site of hrh1^−/− fish. a In situ hybridization of hrh1 in Turku wt zebrafish at 3 dpf (lateral and dorsal view), 5 dpf (lateral and dorsal view). Scale bars: 50 µm. b qRT-PCR results of hrh1 mRNA expression at 5 hpf, 10 hpf, 24 hpf, 3 dpf, 6 dpf, and 12 dpf. N = 6. Mean ± SEM. c The mutation site of hrh1^−/− zebrafish and genomic DNA sequencing results. d Predicated 3D modeling of hrh1^+/+ and hrh1^−/− protein by using SWISS-model online. e Brightfield image of larval zebrafish at 6 dpf. Scale bar: 200 µm.

Histaminergic, dopaminergic, and hcrt systems in hrh1^−/− zebrafish. a Representative pictures (ventral view) for immunohistochemistry of histamine and Th1 in hrh1^+/+ and hrh1^−/− 6 dpf zebrafish brains. Anterior to the left. Scale bars: 100 µm. b Histaminergic neuron numbers. N = 6. c Number of Th1-expressing neurons in group 10. N = 6. d–f qPCR results of th1, th2, chata in hrh1^+/+ and hrh1^−/− in 6 dpf whole zebrafish. N (hrh1^+/+) = 5, N (hrh1^−/−) = 6. g–h Representative pictures (ventral view) for in situ hybridization of hcrt in hrh1^+/+ and hrh1^−/− 6 dpf zebrafish brain and cell numbers. Anterior to the top. Scale bar 50 µm. N = 6. i qPCR results of hcrt in hrh1^+/+ and hrh1^−/− in 6 dpf whole zebrafish. N (hrh1^+/+) = 5, N (hrh1^−/.⁻) = 6. All histograms are mean ± SEM, unpaired t-test, two-tailed. *0.01 < P ≤ 0.05; **0.001 < P ≤ 0.01; ***0.0001 < P ≤ 0.001; ****P ≤ 0.0001

Neurogenesis markers in hrh1^−/− zebrafish. a Representative pictures for in situ hybridization of nes in hrh1^+/+ and hrh1^−/− larval brain at 6 dpf in both dorsal and lateral view. Scale bar: 100 µm. Anterior to the left. N (hrh1^+/+) = 5, N (hrh1^−/−) = 6. c Representative pictures (dorsal view) for in situ hybridization of notch1a in hrh1^+/+ and hrh1^−/− larval brain at 6 dpf. Anterior to the left. Scale bar: 100 µm. N (hrh1^+/+) = 8, N (hrh1^−/−) = 8. b, d, e qPCR results of nes, notch1a, sox2 in whole larval zebrafish at 6 dpf. N (hrh1^+/+) = 5, N (hrh1^−/.⁻) = 6. All bar graphs show mean ± SEM, unpaired t-test, two-tailed. **0.001 < P ≤ 0.01

Proliferation and differentiation markers in hrh1^−/− zebrafish. a Representative pictures (ventral view) for immunohistochemistry of HuC/D in hrh1^+/+ and hrh1^−/− larval brain at 6 dpf. Anterior to the left. Scale bar: 100 µm. N = 6. c Representative pictures (dorsal view) for immunohistochemistry of PCNA in hrh1^+/+ and hrh1^−/− larval brain at 6 dpf. Anterior to the left. Scale bar: 100 µm. N = 6. i Representative pictures (ventral view) for in situ hybridization of pax2a and pax6a in hrh1^+/+ and hrh1^−/− larval brain at 6 dpf. Anterior to the left. Scale bar: 100 µm. b, d–h qPCR results of elavl3, pcna, olig2, slc1a3a, gfap, and isl1 in whole larval zebrafish at 6 dpf. N (hrh1^+/+) = 5, N (hrh1^−/.⁻) = 6. All histograms are exhibit as mean ± SEM, unpaired t test, two tailed. **0.001 < P ≤ 0.01

Larval behavior of hrh1^+/+ and hrh1^−/− fish. a Total distances moved of hrh1^+/+ and hrh1^−/− larvae at 6 dpf in an open field for 15 min. b The frequency of zebrafish entering the center zone of an open field. c The duration of zebrafish staying in the center zone of open field. d Dark flash responses of hrh1^+/+ and hrh1^−/− zebrafish. Each dark period lasted 2 min. Distance moved during every 30-s period is indicated on the Y axis. e Distance moved/s of hrh1^+/+ and hrh1^−/− in the first dark response period. f The index of the first dark response (distances moved in the dark (30 s) minus distances moved in the light (30 s)). a–fN (hrh1^+/+) = 17, N (hrh1^−/−) = 27. g Sleep-like behavior from 5 to 6 dpf of hrh1^+/+ and hrh1^−/− zebrafish. Distance covered during every 30-min period is indicated on the Y axis. N (hrh1^+/+) = 16, N (hrh1^−/−) = 14. h–i Mean values for maximum velocity during 10 acoustic/vibrational stimuli with a 20-s interstimulus interval (ISI) (h) and 30 acoustic/vibrational stimuli with 1 s ISI. N (hrh1^+/+) = 14, N (hrh1^−/−) = 15. Mean ± SEM, two‐way ANOVA

Adult behavior of hrh1^+/+ and hrh1^−/− zebrafish. a Total distances moved in an open-field tank in 15 min in total by adult hrh1^+/+ and hrh1^−/− fish. b Duration of hrh1^+/+ and hrh1^−/− fish in the border zone. N = 10. c Total distances moved in the novel tank in 6 min in total. N (hrh1^+/+) = 13, N (hrh1^−/−) = 10. d Representative tracking map of novel tank for hrh1^+/+ and hrh1^−/− adult zebrafish. e Statistics of cumulative durations in the bottom zone for hrh1^+/+ and hrh1^−/− adult zebrafish. f–g Statistics of the distances between subjects and cumulative duration in proximity for hrh1^+/+ and hrh1^−/− adult zebrafish. N = 20. Mean ± SEM, unpaired t test, two tailed. *0.01 < P ≤ 0.05; no stars mean no significant differences