Zebrafish NTT experimental paradigm (A) The chemical structures of the test compounds–harmane, norharmane, TMN, TIQ, clorgyline, and pargyline–are shown (B) Zebrafish are placed in a treatment beaker with respective compounds for 20 min, briefly rinsed in system water, and then monitored in a novel tank for 5 min. The time spent at the top and bottom one third of the tank were then analyzed (C) A representative trace and heatmap of the swimming activity is presented for a vehicle control. The heatmap shows the minimum (dark blue) to maximum (dark red) amount of time a fish spent in each pixel. Abbreviations: NTT = novel tank test.

NTT results for harmane, norharmane, and TIQ. Representative heatmaps for (A) harmane (3 mg/L) (B) norharmane (3 mg/L), and (C) TIQ (100 mg/L) during the NTT for one zebrafish per compound are shown. The heatmaps show the minimum (dark blue) to maximum (dark red) amount of time a fish spent in each pixel. Time spent at the top and the bottom of the tank are quantified for (D and G) harmane (1, 3, and 10 mg/L), (E and H) norharmane (0.3, 1, and 3 mg/L), and (F and I) TIQ (10, 30, and 100 mg/L). All compounds show anxiolytic-like effects at at least one of the concentrations and time point. Black dashed line = vehicle control; light blue = lowest concentration; blue = middle concentration; purple = highest concentration. The sample size was 12 except for 10 mg/L harmane, control group for norharmane, 3 mg/L norharmane, and 100 mg/L TIQ, which were n = 11. Data are expressed as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; Abbreviations: NTT = novel tank test; TIQ = 1.2,3,4-tetrahydroisoquinoline.

NTT results for TMN, clorgyline, and pargyline. Representative heatmaps for (A) TMN (30 mg/L) (B) clorgyline (100 mg/L), and (C) pargyline (10 mg/L) during the NTT for one zebrafish per compound are shown. The heatmaps show the minimum (dark blue) to maximum (dark red) amount of time a fish spent in each pixel. Time spent at the top and the bottom of the tank are quantified for (D and G) TMN (3, 10, and 30 mg/L), (E and H) clorgyline (10, 30, and 100 mg/L), and (F and I) pargyline (10, 30, and 100 mg/L). All compounds show anxiolytic-like effects at at least one of the concentrations and time point. Black dashed line = vehicle control; light blue = lowest concentration; blue = middle concentration; purple = highest concentration. The sample size was 12 except for control group for TMN (n = 11), 3 mg/L TMN (n = 9), 10 mg/L and 30 mg/L TMN (n = 7), 30 mg/L Clorgyline (n = 8), and 100 mg/L Clorgyline (n = 6). Data are expressed as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; Abbreviations: NTT = novel tank test; TMN = 2,3,6-trimethyl-1,4-naphthoquinone.

Effects of MAO inhibitors on total distance traveled. Total distance traveled during the NTT is presented for (A) harmane (1, 3, and 10 mg/L) (B) norharmane (0.3, 1, and 3 mg/L) (C) TIQ (10, 30, and 100 mg/L) (D) TMN (3, 10, and 30 mg/L) (E) clorgyline (10, 30, and 100 mg/L), and (F) pargyline (10, 30, and 100 mg/L). Black dashed line = vehicle control; light blue = lowest concentration; blue = middle concentration; purple = highest concentration. The sample size is detailed in the legends for Figures 3, 4. Data are expressed as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001. Abbreviations: NTT = novel tank test; TIQ = 1.2,3,4-tetrahydroisoquinoline; TMN = 2,3,6-trimethyl-1,4-naphthoquinone.

Effects of MAO inhibitors on freezing time. Total freezing time during the NTT is presented for (A) harmane (1, 3, and 10 mg/L) (B) norharmane (0.3, 1, and 3 mg/L) (C) TIQ (10, 30, and 100 mg/L) (D) TMN (3, 10, and 30 mg/L) (E) clorgyline (10, 30, and 100 mg/L), and (F) pargyline (10, 30, and 100 mg/L). Black dashed line = vehicle control; light blue = lowest concentration; blue = middle concentration; purple = highest concentration. The sample size is detailed in the legends for Figures 3, 4. Data are expressed as mean ± SEM. **p < 0.01; ***p < 0.001. Abbreviations: NTT = novel tank test; TIQ = 1.2,3,4-tetrahydroisoquinoline; TMN = 2,3,6-trimethyl-1,4-naphthoquinone.