Acute dosing experiment
Limonene - Open Field Test
Heatmaps created from video recordings show the combined group behaviour for the control and limonene groups over the duration of the open field trials (Fig. 1a).
Time in Zones
We observed a significant difference between groups for time spent in the center zone (Fig. 1b; H(3) = 17.02, P =0.0007). Dunn’s multiple comparison test indicated significant differences compared to controls for treatments 0.5% (P = 0.0004) and 0.75% (P = 0.0228). Significant differences between groups were also observed for time spent in the transition zone (Fig. 1c; H(3) = 23.77, P = <0.0001). Dunn’s multiple comparison test showed that the 0.25% (P =0.0149), 0.5% (P= 0.0008), and 0.75% (P = <0.0001) treatments were significantly different from controls. The time spent in the thigmotaxis zone was also significantly different between groups (Fig. 1d; F(3, 69) = 8.248, P = <0.0001). Dunn’s multiple comparison test showed that the 0.25% (P = 0.0190), 0.5% (P = 0.0002), and 0.75% (P =<0.0001) treatments groups were significantly different from controls.
Locomotion
We observed a significant difference in distanced moved between treatments (Fig. 1e; H(3)= 29.82, P = <0.0001). Post-hoc Dunn’s multiple comparison test indicated that there were significant differences compared to controls in distance moved for 0.25% (P = 0.0004), 0.5% (P = 0.0026), and 0.75% (P = <0.0001). Significant differences in velocity were also observed between treatments (Fig. 1f; H(3)= 29.81, P = <0.0001). Dunn’s multiple comparison test showed significant differences compared to controls for treatments 0.25% (P =0.0004), 0.5% (P =0.0026), and 0.75% (P =<0.0001). No significant differences were observed between groups in meandering (Fig. 1g; H(3) = 3.099, P = 0.3766). Immobility was also observed to be significantly different between groups (H(3) = 13.85, P = 0.0031). Dunn’s multiple comparison test showed that the 0.75% (P = 0.0012) group spent significantly more time immobile than controls. We observed a significant difference between groups in high mobility (H(3) = 17.91, P = 0.0005). Dunn’s multiple comparison test showed that the 0.75% group had decreased high mobility compared to controls (P = <0.0001).
Linalool - Open Field Test
Heatmaps created from video recordings show the combined group behaviour for the control and linalool groups over the duration of the open field trials (Fig. 2a).
Time in zones
We observed a significant difference between groups for time spent in the center zone (Fig. 2b; H(3) = 7.642, P = 0.0540). Dunn’s multiple comparison test indicated that the 0.001% group spent significantly more time in the center zone than controls (P =0.0286). There were no significant differences between groups for time spent in the transition (Fig. 2c; H(3) =4.963, P =0.1745). Moreover, no significant differences were observed for time spent in the thigmotaxis zone (Fig. 2d; H(3) = 5.624, P = 0.1314).
Locomotion
Here, we observed a significant difference in distance moved between groups (Fig. 2e; H(3) = 15.54, P = 0.0014). Dunn’s multiple comparison test indicated that the 0.0001% (P = 0.0311) linalool treatment group had travelled a significantly less than controls. A significant difference was also observed for velocity between groups (Fig. 2f; H(3) = 15.53, P = 0.0014). Dunn’s multiple comparison test indicated that the 0.0001% (P = 0.0320) linalool treatment group had a significantly decreased velocity compared to controls. We observed no significant differences between groups in meandering (Fig. 2g; H(3) = 4.810, P = 0.1862), and immobility (H(3) = 5.165, P = 0.1601). We did observe a significant difference between groups in high mobility (Fig. 2h; H(3) = 10.32, P = 0.0160). However, Dunn’s multiple comparison test did not indicate any significant differences for treatment groups compared to controls.
Myrcene - Open Field Test
Heatmaps created from video recordings show the combined group behaviour for the control and myrcene groups over the duration of the open field trials (Fig. 3a).
Time in zones
We observed significant difference between groups in time spent in the center zone (Fig. 3b; H(3) = 9.587, P = 0.0225). Dunn’s multiple comparison test indicated that the 0.1% treatment group spent significantly more time in the center zone then controls (P = 0.0083). Additionally, we observed significant differences for time spent in the transition zone (Fig. 3c; H(3) = 13.78, P = 0.0032). Dunn’s multiple comparison test indicated that the 0.001% and 0.1% treatment groups spent significantly more time in the transition zone (P = 0.0365 and P = 0.0028 respectively) than controls. Significant differences between groups were also seen for time spent in the thigmotaxis zone (Fig. 3d; H(3) = 13.87, P = 0.0031). Dunn’s multiple comparison test indicated that the 0.001% and 0.1% treatments groups spent significantly less time in the thigmotaxis zone than controls (P = 0.0426 and P = 0.0023 respectively).
Locomotion
We observed a significant difference between groups in distance moved (Fig. 3e; H(3) = 8.307, P = 0.0401). Dunn’s multiple comparison test indicated that the 0.1% treatment travelled significantly less than controls (P = 0.0190). A significant difference was also observed between groups in velocity (Fig. 3f; H(3) = 8.307, P = 0.0401). Dunn’s multiple comparison test indicated that the 0.1% treatment had a significantly decreased velocity compared to controls (P = 0.0190). No significant differences were observed between groups in meandering (Fig. 3g; H(3) = 1.484, P = 0.6860) or immobility (Fig. 3h; H(3) = 3.824, P = 0.2811). However, we did observe a significant difference between groups in high mobility (Fig. 3i; H(3) = 10.47, P = 0.0150). Dunn’s multiple comparison test indicated that the 0.1% treatment group had significantly shorter duration of time spent high mobile than controls (P = 0.0094).
Limonene - Novel object approach test
Heatmaps created from video recordings show the combined group behaviour for the control and limonene groups over the duration of the novel object approach test trials (Fig. Time in zones
We observed a significant difference between groups in time spent in the center zone (Fig. 4b; H(3) = 18.03, P = 0.0004). Dunn’s multiple comparison test showed that the 0.75% treatment spent significantly more time in the center than controls (P = 0.0001). Significant differences were also observed in time spent in the transition zone (Fig. 4c; H(3) = 15.58, P = 0.0014). Dunn’s multiple comparison test showed that the 0.5% (P = 0.0318) and 0.75% (P = 0.0006) treatments spent significantly more time in the transition zone than controls. Furthermore, we observed a significant difference in time spent in the thigmotaxis zone between groups (Fig. 4d; H(3) = 19.37, P = 0.0002). Dunn’s multiple comparison test showed that the 0.5% (P = 0.0369) and 0.75% (P = <0.0001) treatments spent significantly less time in the thigmotaxis zone than controls.
Locomotion
We observed significant differences in distance moved between treatments (Fig. 4e; H(3) = 19.14, P =0.0003). Dunn’s multiple comparison test indicated that the 0.25% (P = 0.0007), 0.5% (P =0.0064), and 0.75% (P = 0.0048) treatments travelled significantly less than controls. A significant difference between groups was also observed in velocity (Fig. 4f; H(3) = 19.05, P = 0.0003). Dunn’s multiple comparison test indicated that the 0.25% (P = 0.0006), 0.5% (P =0.0062), and 0.75% (P = 0.0066) treatments moved at a significantly decreased velocity compared to controls. No significant differences between groups were observed in meandering (Fig. 4g; H(3) = 4.722, P = 0.1933). However, we did observe significant differences between groups in immobility (Fig. 4h; H(3) = 16.48, P = 0.0009). Dunn’s multiple comparison test showed that the 0.25% (P = 0.0144), 0.5% (P = 0.0062), and 0.75% (P = 0.0028) treatments spent significantly more time immobile than controls. Additionally, we observed a significant difference between treatments in high mobility (Fig. 4i; H(3) = 10.61, P = 0.0141). Dunn’s multiple comparison test indicated that the 0.75% treatment spent significantly less time highly mobile than controls (P = 0.0040).
Linalool - Novel object approach test
Heatmaps created from video recordings show the combined group behaviour for the control and linalool groups over the duration of the novel object approach test trials (Fig. 5a).
Time in zones
Here we observed no significant differences between groups in time spent in center zone (Fig. 5b; H(3) = 1.803, P = 0.6144), transition zone (Fig. 5c; F(3,68) = 1.324, P = 0.2736), or thigmotaxis zone (Fig. 5d; F(3,68) = 1.375, P = 0.2578.
Locomotion
Here, we observed no significant differences between groups in distance moved (Fig. 5e; H(3) = 5.052, P = 0.1680), velocity (Fig. 5f; F(3,68) = 1.297, P = 0.2827), meandering (Fig. 5g; H(3) = 5.010, P = 0.1711), or immobility (Fig. 5h; H(3) = 2.977, P = 0.3952). However, we did observe a significant difference between groups in high mobility (Fig. 5i; H(3) = 15.08, P = 0.0017). Dunn’s multiple comparison test showed that the 0.00125% treatment spent significantly less time highly mobile than controls (P = 0.0033).
Myrcene - Novel object approach test
Heatmaps created from video recordings show the combined group behaviour for the control and myrcene groups over the duration of the novel object approach test trials (Fig. 6a).
Time in zones
Significant differences were observed between groups for time spent in the center (Fig. 6b; H(3) = 17.04, P = 0.0007), transition (Fig. 6c; H(3) = 19.33, P = 0.0002) and thigmotaxis zone (Fig. 6d; H(3) = 19.45, P = 0.0002). Dunn’s multiple comparison test indicated that compared to controls, the 0.1% treatment spent significantly more time in the center (P = 0.0460) and transition zone (P = 0.0014) and significantly less time in the thigmotaxis zone (P = 0.0016).
Locomotion
No significant differences were observed between groups in distance moved (Fig. 6e; H(3) = 6.675, P = 0.0830) or velocity (Fig. 6f; H(3) = 6.745, P = 0.0805). No significant differences between treatments were observed in meandering (Fig. 6g; H(3) = 1.462, P = 0.6910). However, significant differences between treatments were observed in immobility (Fig. 6h; H(3) = 8.327, P = 0.0397) and high mobility (Fig. 6i; H(3) = 11.15, P = 0.0109). Dunn’s multiple comparison test indicated that compared to controls, the 0.1% treatment group spent significantly less time immobile (P = 0.0134) as well as significantly less time highly mobile (P = 0.0033).
Repeated dosing experiment
Because of the robust anxiolytic effects of β-myrcene and limonene in our acute administration study, we tested the effect or repeated administration of these compounds. First, we calculated EC50 values of β-myrcene (0.0083%) and limonene (0.39%) and used these concentrations for the repeated dosing experiment.
Open field test
Time in zones
Here, we observed no significant differences between any group in time spent in the center zone (Fig. 7a; F(2,3) = 0.1441, P = 0.8714). Moreover, no significant differences were seen in time spent in the transition zone (Fig. 7b; F(2, 57) = 0.3813, P = 0.6847) or time spent in the thigmotaxis zone (Fig. 7c; F(2, 57), P = 0.3043).
Locomotion
We observed no significant differences between groups in distance moved (Fig. 7d; F(2, 57) = 0.07074, P = 0.9318). Additionally, there were no significant differences between groups in velocity (Fig. 7e; F(2, 57) = 0.07074, P = 0.9318), meandering (Fig. 7f; F(2, 57) = 0.1237, P = 0.8839), immobility (Fig. 7g; F(2, 57) = 0.06465, P = 0.9375), or high mobility (Fig. 7h; F(2,57) = 0.9144, P = 0.4065).
Novel object approach test
Time in zones
There were no significant differences between groups in time spent in the center zone (Fig. 8a; F(2, 3) = 0.07236, P = 0.9318). No significant differences were seen in time spent in the transition zone (Fig. 8b; F(2, 57) = 2.326, P = 0.1069). Furthermore, we observed no significant differences in time spent in the thigmotaxis zone (Fig. 8c; F(2, 57) = 2.326, P = 0.1069).
Locomotion
Here, we observed no significant differences between groups in distance moved (Fig. 8d; F(2, 57) = 0.7433, P = 0.4801) or velocity (Fig. 8e; F(2, 57) = 0.7434, P = 0.4801). Moreover, there were no significant differences between treatments in meandering (Fig. 8f; F(2, 3) = 0.2369, P = 0.8025), or immobility (Fig. 8g; F(2, 57) = 0.5978, P = 0.5534), or high mobility (Fig. 8h; F(2, 3) = 0.4706, P = 0.6641).