E-gel promotes heightened voluntary oral consumption of THC and induces cannabimimetic behaviors by adult mice.

a) Mice were given free access to vehicle (VEH), or THC formulated in either CTR-gel or E-gel for 2 h on Day 1 and Day 2. b) Consumption was determined by weighing gelatin at the end of each session. c) Consumption of CTR-gel on Day 2 is decreased after addition of THC. d) Consumption of E-gel on Day 2 is maintained after addition of THC. e) Dose of THC consumed, in mg/kg, when formulated in either CTR-gel or E-gel on Day 2. Results are mean ± S.E.M. Consumption compared ANOVA and Sidak’s, *p<0.05, **p<0.01, and ***p<0.001, N=8-40. f) Diagram of behavioral paradigm before and after i.p. or gelatin administration. g-i) Dose-dependent behavioral responses for hypolocomotion (g), analgesia (h), and hypothermia (i) after THC exposure. Administration by i.p. (grey) is plotted on x-axis by single bolus injection while CTR-gel (green) and E-gel (purple) are plotted based on average THC consumed after 2 h exposure window shown in e. j) Diagram of THC-E-gel exposure, behavioral measurements, and SR1 injection (by i.p.) at 1 h into exposure window. k-m) Individual behavioral responses for hypolocomotion (k), analgesia (l), and hypothermia (m) for each animal. Individual points are plotted based on individual THC consumption with a linear regression to show correlation between consumed THC and behavioral output (p-values: k=0.003, l<0.001, m<0.001). SR1 treated mice are plotted (red) based on consumed THC after exposure to 10 mg/15 ml THC-E-gel with a linear regression to show no correlation across three behaviors (p-values: k=0.09, l=0.44, m=0.45).

THC-E-gel consumption triggers CB1R-dependent behaviors

a) Over a 3-day exposure paradigm, mice received 3 days of E-gel with either VEH or THC (10mg/15ml) E-gel on day 2. b-d) Cumulative gelatin consumption recorded every 10 minutes throughout the 2 h exposure window over the 3-day paradigm. VEH (black) and THC (purple) groups received access to VEH on day 1 (b), VEH or THC on day 2 (c), and VEH on day 3 (d). e) Total gelatin consumption after 2 h of access to gelatin was plotted comparing VEH and THC treatment groups. f) Animal consummatory and locomotor behavior was tracked during gelatin exposure window. g-i) Distance traveled recorded every 5 min over the 3-day paradigm, similar as to b-d. j) Total distance traveled (cm) after 2 h of gelatin access was plotted comparing VEH and THC groups. Main effect over 2 h exposure period (b-d, g-i) measured using Two-way ANOVA with repeated measures and Sidak’s, main effect on total response (e, h) measured by One-way ANOVA and Sidak’s (*p<0.05, **p<0.01, ***p<0.001) N=8-16.

Consumption of THC-E-gel results in concomitant increases in the levels of THC and its metabolites in brain tissue

a) Diagram outlining gelatin exposure paradigm where blood and brain samples were collected immediately following 1 h and at 2, 2.5, and 26 hours from the beginning of 2 h access to 10mg/15ml THC-E-gel. b) Brain concentration of THC, 11-OH-THC, and COOH-THC after E-gel exposure, 1 h access is separated due to a reduced total access time to THC-E-gel compared to the other time points. c) Plasma concentrations for the three compounds plotted similarly to b. d-e) PK concentrations in brain (d) and plasma (e) normalized to the 1 h access period. Statistical comparison to 1 h Two-way ANOVA, Sidak’s, *p<0.05, **p<0.01, and ***p<0.001, N=8-15.

Correlating i.p. THC and THC-E-gel triad cannabimimetic responses predicts THC-E-gel-dependent behaviors.

a) Diagram of 1 h and 2 h THC-E-gel exposure and i.p. administration with behavioral tests. b-d) Cannabimimetic responses after THC administration by i.p. and subsequent dose-response curve in grey. Responses after 1 h or 2 h exposure to 10 mg THC-E-gel are plotted with dotted lines tracking to relative THC-i.p. dose response. e) Predicted i.p. dose after 1 h and 2 h THC-E-gel exposure window from all three triad behaviors.

Sex-dependent acoustic startle responses after i.p. injection of THC and high concentration THC-E-gel consumption

a) Diagram of THC-E-gel exposure or i.p. administration followed by acoustic startle response behavioral testing. b-c) Male and female acoustic startle responses after i.p. administration of THC in response to escalating tones (80, 90, 100, 105, 110, and 120dB) following i.p. administration of THC in males (b) and females (c). d) Male and female acoustic startle dose-responses to a 120dB tone after i.p. THC administration. Results are mean ± S.E.M. One-way ANOVA, Sidak’s comparing VEH and i.p. THC dose between males and females **p<0.01, and ***p<0.001, N=6-11. e-f) Male and female acoustic startle responses after 1 h or 2 h THC E-gel exposure in response to escalating tones (80, 90, 100, 105, 110, and 120dB. g) THC dose consumption based on grams consumed and individual body weight correlated with individual acoustic startle response after 2 h exposure. h-i) Startle response to a 120dB tone for males (h) and females (i) after 1 h or 2 h access to THC E-gel. Predicted doses calculated from a second order polynomial of i.p. dose responses are plotted to show the consistency in predicted dose response after E-gel exposure.