Treatment administration and behavioural testing. (A) Phase 1: To establish an effective dose of terpenes on zebrafish behaviour, individual zebrafish were placed in a terpene or control solution for 10 mins then transferred to the testing arena where behaviour was tracked and recorded for 10 mins. (B) Phase 2: To test the effects of rimonabant and AM630, zebrafish were placed in an antagonist or control solution for 60 mins, then transferred to a terpene or control solution for 10 mins, then transferred to the testing arena where behaviour was tracked and recorded for 10 mins. (C) The open field testing arena had a 27 cm diameter, 11.5 cm height, and water depth of 5 cm. The annular zones of the arena created in Noldus Ethovision XT consisted of 3 concentric zones each 4.5 cm wide. The outermost circle is the thigmotaxis zone, followed by the transition zone, and the innermost circle is the center zone.

The effects of bisabolol on anxiety-like and locomotor behaviour in the open field test. (A) Bisabolol had no effect on time spent in the center zone (F(3, 28.02) = 1.4, p = .2636) or (B) time spent in the thigmotaxis zone (F(3, 30.56) = 2.094, p = .1216). (C) Bisabolol increased swimming velocity in the 0.002% group (F(3, 56) = 2.431, p = .0746) but (D) had no effect on time spent immobile (H(4) = 6.355, p = .0955). All data are presented as mean ± S.E.M. Significant differences are indicated by * (p < 0.05).

The effects of terpinolene on anxiety-like and locomotor behaviour in the open field test. (A) Terpinolene increased time spent in the center zone in the 0.1% group (F(3, 44.85) = 5.184, p = .0037) and (B) time spent in the thigmotaxis zone in the 0.1% group (F(3, 61.20) = 12.84, p < .0001). (C) Terpinolene decreased swimming velocity in the 0.05% and 0.1% group (F(3, 75) = 13.64, p < .0001). (D) Terpinolene also increased time spent immobile in the 0.05% group (H(4) = 10.96, p = .0120). All data are presented as mean ± S.E.M. Significant differences are indicated by * (p < 0.05) and * ** * (p < .0001).

The effects of rimonabant and AM630 on BCP-reduced anxiety-like and locomotor behaviour in the open field test. (A) BCP increased time spent in the center zone. Rimonabant did not modulate the effect of BCP on time spent in the center zone (F(4, 17.26) = 6.852, p = .0017). (B) BCP decreased time spent in the thigmotaxis zone. Rimonabant did not modulate the effect of BCP on time spent in the thigmotaxis zone (H(5) = 20.9, p = .0003). (C) BCP decreased swimming velocity. Rimonabant did not modulate the effect of BCP on swimming velocity (F(4, 67) = 7.355, p < .0001). (D) BCP nor rimonabant had an effect on immobility (F(4, 46.93) = 1.644, p = .1791). (E) BCP increased time spent in center zone. AM630 eliminated the effect of BCP on time spent in the center zone (F(4, 44.71) = 2.681, p = .0435). (F) BCP decreased time spent in thigmotaxis zone. AM630 eliminated the effect of BCP on time spent in thigmotaxis zone (H(5) = 13.8, p = .0080). (G) BCP decreased swimming velocity. AM630 did not eliminate the effects of BCP on swimming velocity. When administered alone, AM630 decreased swimming velocity (F(4, 50.84) = 14.11, p < .0001). (H) BCP nor AM630 had an effect on immobility (F(4, 48.61) = 3.816, p = .0089). All data are presented as mean ± S.E.M. Significant differences are indicated by * (p < .05), * * (p < .01), and * ** * (p < .0001).

The effects of rimonabant and AM630 on terpinolene-reduced anxiety-like and locomotor behaviour in the open field test. (A) Rimonabant did not modulate the effect of terpinolene on time spent in the center zone (F(3, 21.5) = 12.26, p < .0001) or (B) time spent in the thigmotaxis zone (F(3, 38.3) = 14.32, p < .0001). (C) Rimonabant also did not modulate the effect of terpinolene on swimming velocity (H(4) = 19, p = .0003). (D) There was no effect on immobility across treatment groups (H(4) = 7.178, p = .0664). (E) AM630 eliminated the effect of terpinolene on time spent in the center zone (F(3, 24.55) = 11.68, p < .0001) and (F) time spent in the thigmotaxis zone. AM630 administered alone also had an effect on time spent in the thigmotaxis zone (F(3, 42.74) = 9.376, p < .0001). (G) AM630 did not modulate the effect of terpinolene on swimming velocity and also had an effect when administered alone (F(3, 45.6) = 9.347, p < .0001). (H) While terpinolene administered before and after AM630 had no effect on immobility, AM630 increased immobility when administered alone (H(4) = 6.871, p = .0761). All data are presented as mean ± S.E.M. Significant differences are indicated by * (p < .05), * * (p < .01), * ** (p < .001), and * ** * (p < .0001).