DOI: 10.5061/dryad.000000099
title: Behavioral data for: A preclinical model of THC edibles that produces high-dose cannabimimetic responses

description: No preclinical experimental approach enables the study of voluntary oral
 consumption of high-concentration Δ9-tetrahydrocannabinol (THC) and its
 intoxicating effects, mainly owing to the aversive response of rodents to
 THC that limits intake. Here we developed a palatable THC formulation and
 an optimized access paradigm in mice to drive voluntary consumption. THC
 was formulated in chocolate gelatin (THC-E-gel). Adult male and female
 mice were allowed ad libitum access for 1 and 2 h. Cannabimimetic
 responses (hypolocomotion, analgesia, and hypothermia) were measured
 following access. Levels of THC and its metabolites were measured in blood
 and brain tissue. Acute acoustic startle responses were measured to
 investigate THC-induced psychotomimetic behavior. When allowed access for
 2 h to THC-E-gel on the second day of a three-day exposure paradigm, adult
 mice consumed up to ≈30 mg/kg over 2 h which resulted in robust
 cannabimimetic behavioral responses (hypolocomotion, analgesia and
 hypothermia). Consumption of the same gelatin decreased on the following
 3rd day of exposure. Pharmacokinetic analysis show that THC-E-gel
 consumption led to parallel accumulation of THC and its psychoactive
 metabolite, 11-OH-THC, in brain, a profile that contrasts with the known
 rapid decline in brain 11-OH-THC levels following THC intraperitoneal
 (i.p.) injections. THC-E-gel consumption increased the acoustic startle
 response in males but not in females, demonstrating a sex-dependent effect
 of consumption. Thus, while voluntary consumption of THC-E-gel triggered
 equivalent cannabimimetic responses in male and female mice, it
 potentiated acoustic startle responses preferentially in males. We build a
 dose-prediction model that included cannabimimetic behavioral responses
 elicited by i.p. versus THC-E-gel to test the accuracy and
 generalizability of this experimental approach and found that it closely
 predicted the measured acoustic startle results in males and females. In
 summary, THC-E-gel offers a robust preclinical experimental approach to
 study cannabimimetic responses triggered by voluntary consumption in mice,
 including sex-dependent psychotomimetic responses.
description: <strong>Animal Studies</strong>
 Animal studies followed the guidelines established by AAALAC and
 were approved by IACUC of the University of Washington. Male and female
 C57BL/6J mice ranging from 8-14 weeks of age were used. Animals were
 housed with sibling littermates and were provided with standard chow and
 water, <em>ad libitum, </em>and without any additional
 environmental enrichment. Investigators were not blinded to experimental
 exposure conditions throughout assays due to the noticeable behavioral
 effects measured in response to THC. Animal procedures were approved by
 the Institutional Animal Care and Use Committee of the University of
 Washington and conform to the guidelines on the care and use of animals by
 the National Institutes of Health.
 <strong>Pharmacological Agents</strong>
 Animals received THC (0.1, 0.3, 1, 3, 5, 10, and 30 mg/kg) and
 SR141716 (SR1, 1 mg/kg) <em>i.p.</em> or were exposed to THC
 suspended in gelatin. THC and SR141716 (SR1) were provided by the National
 Institute of Drugs abuse Drug Supply Program (Bethesda, MD). THC in
 ethanol (50 mg/ml) was either added to gelatin mixtures (CTR or Ensure®)
 or prepared for <em>i.p.</em> injection. For
 <em>i.p.</em> injection, both THC (0.1, 0.3, 1, 3, 5, 10, and
 30 mg/kg) and SR1 (1mg/kg) were dissolved in 95% ethanol and then vortexed
 thoroughly with equal volume Cremophor and finally dissolved in sterile
 saline to reach a final 1:1:18 solution consisting of
 ethanol:cremophor:saline. <strong>Gelatin
 formulation </strong> Control gelatin
 (CTR-gel): Deionized water (100 mL) was warmed to 40°C and
 stirred at a constant rate. 2.5 g of Polycal™ sugar and 3.85 g of Knox™
 Gelatin were added and the mixture was maintained at a temperature below
 43°C. The mixture was removed from heat, and THC (50 mg/ml in ethanol) was
 added to reach a concentration of 0.3, 1, 2, or 4 mg/15 ml. An equal
 volume of ethanol was added to vehicle gelatin (&lt;1% total volume).
 Gelatin was poured into plastic cups ranging from 2-10 ml and set into a
 4°C fridge to solidify overnight. Ensure
 gelatin (E-gel): Chocolate-flavored Ensure™ (100 ml) was warmed
 to 40°C and stirred at a constant rate. 3.85 g of Knox™ Gelatin were added
 and the mixture was maintained at a temperature below 43°C. The mixture
 was removed from heat and THC (50 mg/15 ml ethanol) was added to reach a
 concentration of 1, 2, 5, or 10 mg/15 ml. At the 10 mg/15 ml
 concentration, ethanol was evaporated off to 50% volume before being added
 to the mixture to reduce total alcohol concentration below 1%. An equal
 volume of ethanol was added to vehicle gelatin (&lt;1% total volume).
 Gelatin was poured into plastic cups ranging from 2-10 ml and set into a
 4°C fridge to solidify overnight. Mice were always exposed to more gelatin
 than they could consume, smaller volumes were used to conserve
 THC. Acute gelatin access: Animals
 were first habituated to gelatin by receiving an excess of gelatin in
 their home cage the day before the first timed access. On the first day of
 access, mice were placed into a home cage-like environment equipped with a
 vehicle gelatin cup that was stabilized to the cage. Behavior was recorded
 during the consumption window via an overhead camera. On the second day of
 access, animals were placed into the same gelatin access cage with either
 a vehicle or THC gelatin cup. Animals experienced either a triad of
 behaviors (open field, tail flick, and body temperature) measured
 immediately preceding and following the consumption window or an acoustic
 startle trial immediately following consumption. On the third and final
 day of access animals were placed into the same cage with a vehicle
 gelatin cup. For all gelatin access days, gelatin cups and animals were
 weighed before and after the consumption window. Access to gelatin during
 the consumption window was limited to either 1 or 2 h after which the
 animals were removed and returned to their home cage.
 <strong>Triad of Cannabimimetic
 behaviors</strong> Hypolocomotion, hypothermia,
 and analgesia were measured 1 h post-<em>i.p.</em> injection
 or immediately following gelatin exposure. Pre-tests were collected
 immediately prior to injection or gelatin exposure.
 Open Field: A 50 cm x 50 cm chamber (25 LUX)
 was equipped with an overhead camera to record movement. Animals were
 placed in the chamber for 15 minutes and then returned to their home cage.
 Total distance traveled (cm) was measured using Noldus Ethovision
 behavioral tracking software. Locomotion behavior was measured immediately
 before and after gelatin access to calculate a gelatin dependent
 difference score (post-pre). Pre-test measurements for CTR-gel were not
 collected and pre-test values were instead normalized to vehicle
 post-tests to produce a difference score as Post-VEH.
 Tail Flick Analgesia: A hot water bath was set
 to 52.5°C. Mice were securely held upright in the air with their tail
 hanging downward. A timer was started as 75% of their tail was submerged
 into the water. Time was measured once a painful response was presented,
 marked as a latency to flick their tail out of the hot water. Tail flick
 responses were measured immediately before and after gelatin access to
 calculate a gelatin-dependent difference score (post-pre).
 Measuring body temperature: Animals were
 placed on a stable surface with their tails lifted. A rectal thermometer
 probe (RET-3 Kent Scientific) was inserted into the anus for 10-20 s until
 the temperature recording stabilized. This test was always performed prior
 to the Tail Flick test to reduce any potential temperature contamination
 effects. Body temperatures were measured immediately before and after
 gelatin access to calculate a gelatin dependent difference score
 (post-pre). <strong>Blood and brain tissue
 collection and quantification</strong> Animals
 underwent the same gelatin access paradigm for day 1 and 2 described in
 acute gelatin access. After 2 h of gelatin access, blood was collected by
 cardiac puncture with a 23-gauge needle and placed on ice. Immediately
 following, brain tissue was collected and flash frozen in liquid nitrogen.
 Blood samples were spun in a 4°C centrifuge at 1450 x
 <em>g</em> for 15 min. Plasma was transferred to another tube
 and stored alongside brain samples in -80°C until being shipped on dry ice
 to the Piomelli Lab at UCI for sample analysis. Samples were collected
 immediately following 1 and 2 h gelatin access and 30 min and 24 h after 2
 h gelatin access. THC and its first-pass metabolites 11-OH-THC and
 11-COOH-THC were quantified in plasma and brain tissue using a selective
 isotope-dilution liquid chromatography/tandem mass spectrometry assay
 (26). Concentration data after E-gel consumption were compared to blood
 and brain tissue concentrations after <em>i.p.</em>
 administration from previous publications from the Piomelli Lab
 [35].  <strong>Acoustic
 startle</strong> Acoustic Startle behaviors were
 measured after 1 or 2 h THC-E-gel exposure (10 mg/15 ml) and
 THC-<em>i.p.</em> (0.1, 1, 5, 10 mg/kg) injection.
 Sound-buffered startle chambers (SR-Lab, San Diego Instruments) were used
 to measure acoustic startle responses, equipped with a holding tube and an
 accelerometer. Background sound was maintained at 65 dB from a
 high-frequency speaker producing white noise. Startle tests were conducted
 1 h post-THC-<em>i.p.</em> injection or immediately following
 THC-E-gel exposure. Animals were set in the holding tube for 5 min to
 habituate prior to a series of seven trials presenting escalating sound
 levels of null, 80, 90, 100, 105, 110, and 120 dB. Tones were presented
 for 40 ms with an inter-trial interval of 30 s. Animals were only ever
 exposed to the acoustic startle paradigm once, immediately after gelatin
 access, to avoid auditory habituation.
 <strong>Data/Statistical analysis</strong>
 All data were analyzed using GraphPad Prism 10-11. For all
 statistical analyses (unpaired <em>t</em> test, one- and
 two-way ANOVA, and post hoc analyses), alpha level was set to 0.05. For
 all ANOVA analyses, Sidak’s post-test was performed for increased power
 and repeated measures analysis was performed for time-dependent
 consumption data. All behavioral locomotor tracking was analyzed using
 Noldus Ethovision software and statistical analyses performed through
 GraphPad Prism 10-11. Nonlinear regressions (Figure 4B-d and Figure
 1-figure supplemental 3B-G) were performed with a 3-parameter nonlinear,
 least squares, regression.
description: # Behavioral data for "A preclinical model of THC edibles that
 produces high-dose cannabimimetic responses.
 Data for the manuscript is
 organized here by their respective figures and panel labels. ##
 Description of the data and file structure For ease of access of data when
 referencing the manuscript figures, all data is organized in excel files
 according to the figures they represent. Each figure shows individual data
 points plotted for interpretability and those respective data points are
 listed in the excel files for the figures. Main figures and supplementary
 data are separated into respective excel plots for the ease of access. All
 statistical analyses were performed in GraphPad Prism where data was
 plotted. Supplemental data is included in main figure data excel sheet.
 All data is included in the following outline: Figure_1-dataset : **E-gel
 promotes heightened voluntary oral consumption of THC and induces
 cannabimimetic behaviors by adult mice.** Tab 1: Panel c Tab 2: Panel d
 Tab 3: Panel e Tab 4: Panel g Tab 5: Panel h Tab 6: Panel i Tab 7: Panel k
 Tab 8: Panel l Tab 9: Panel m Tab 10: Supplementary 1 Panels a-b Tab 11:
 Supplementary 2 Panels b-d Tab 12: Supplementary 2 Panels e-g Tab 13:
 Supplementary 3 Panels b+e Tab 14: Supplementary 3 Panels c+f Tab 15:
 Supplementary 3 Panels d+g Figure_2-dataset: **THC-E-Gel reduces
 locomotion during the exposure period.** Tab 1: Panels b-d Tab 2: Panels
 g-i Tab 3: Supplementary data Figure_3-dataset: **Consumption of THC-E-gel
 results in concomitant increases in the levels of THC and its metabolites
 in brain tissue.** Tab 1: Panel b Figure_4-dataset: **Correlating *i.p.*
 THC and THC-E-gel triad cannabimimetic responses predicts
 THC-E-gel-dependent behaviors.** Tab 1: Panel e Figure_5-dataset:
 **THC-E-gel consumption and THC *i.p.* injections induce sex-dependent
 changes in acoustic startle responses.** Tab1: Panels b-c Tab2: Panels e-f
 Tab3: Panel g ## Sharing/Access information All PK results are listed in
 Figure 3-figure supplemental 1-2 tables for brain tissue and plasma
 concentrations.