Abstract

The consumption of Δ9-tetrahydrocannabinol (THC)- or cannabis-containing edibles has increased in recent years; however, the behavioral and neural circuit effects of such consumption remain unknown, especially in the context of ingestion of higher doses resulting in cannabis intoxication. We examined the neural and behavioral effects of acute high-dose edible cannabis consumption (AHDECC). Sprague-Dawley rats (six males, seven females) were implanted with electrodes in the prefrontal cortex (PFC), dorsal hippocampus (dHipp), cingulate cortex (Cg), and nucleus accumbens (NAc). Rats were provided access to a mixture of Nutella (6 g/kg) and THC-containing cannabis oil (20 mg/kg) for 10 minutes, during which they voluntarily consumed all of the provided Nutella and THC mixture. Cannabis tetrad and neural oscillations were examined 2, 4, 8, and 24 hours after exposure. In another cohort (16 males, 15 females), we examined the effects of AHDECC on learning and prepulse inhibition and serum and brain THC and 11-hydroxy-THC concentrations. AHDECC resulted in higher brain and serum THC and 11-hydroxy-THC levels in female rats over 24 hours. AHDECC also produced: 1) Cg, dHipp, and NAc gamma power suppression, with the suppression being greater in female rats, in a time-dependent manner; 2) hypolocomotion, hypothermia, and antinociception in a time-dependent manner; and 3) learning and prepulse inhibition impairments. Additionally, most neural activity and behavior changes appear 2 hours after ingestion, suggesting that interventions around this time might be effective in reversing/reducing the effects of AHDECC.

SIGNIFICANCE STATEMENT The effects of high-dose edible cannabis on behavior and neural circuitry are poorly understood. We found that the effects of acute high-dose edible cannabis consumption (AHDECC), which include decreased gamma power, hypothermia, hypolocomotion, analgesia, and learning and information processing impairments, are time and sex dependent. Moreover, these effects begin 2 hours after AHDECC and last for at least 24 hours, suggesting that treatments should target this time window in order to be effective.:

Introduction

Cannabis is consumed mostly through smoking and inhalation (Borodovsky et al., 2016); however, the use of edible cannabis, consumed by oral ingestion, has increased in recent times (Weiss, 2015). Possible reasons for this increase include the fact that there is no exposure to smoke, the convenience and discreteness of edibles, and their longer-lasting effect compared with other forms of intake (Giombi et al., 2018). However, this increase in popularity has raised concerns about accidental overconsumption of edible products. This arises from the delayed onset of subjective effects, which depends on factors such as body weight, metabolism, and eating habits, resulting in difficulty with dose estimation and titration. These factors often lead people to unintentionally consume more edible products than desired, resulting in accidental cannabis intoxication (Giombi et al., 2018). Moreover, the incidence of accidental cannabis intoxication after high-dose edible cannabis has increased among children and pets (Amissah et al., 2022; Tweet et al., 2023), who mistake edible cannabis for other cannabis-free foods (Wang et al., 2016; Lewis et al., 2020). This emphasizes the need to investigate how edible cannabis affects the body after consumption. Most clinical signs of acute high-dose cannabis consumption are neurologic (Janczyk et al., 2004; Claudet et al., 2017), occurring due to the interaction between Δ9-tetrahydrocannabinol (THC) and type 1 cannabinoid receptors (CB1Rs) expressed in brain regions, including the cingulate cortex (Cg), prefrontal cortex (PFC), hippocampus, and nucleus accumbens (NAc). The THC in edible cannabis is metabolized into 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC), which might be more potent than THC (Barrus et al., 2016) and cause stronger and longer lasting effects (Favrat et al., 2005). Oral THC administration also results in high brain THC levels in rats (Hložek et al., 2017), which may contribute to the high incidence of edible cannabis–induced intoxication (Hudak et al., 2015); however, brain THC levels after edible exposure have not been assessed in humans.

Intraperitoneal THC administration decreased gamma power in the rat hippocampus, a brain region critical for learning (Robbe et al., 2006; Jenkins and Khokhar, 2021), possibly through the inhibition of presynaptic neurotransmitter release (Cortes-Briones et al., 2015). Gamma power refers to the magnitude of neural activity within the gamma frequency range (30–150 Hz). Brain oscillations within this range are called gamma oscillations and are necessary for associative learning, and their alteration may therefore lead to learning deficits (Uhlhaas et al., 2008). In our previous work, THC vapor administration decreased gamma power in the rat dorsal striatum, orbitofrontal cortex, and PFC, regions implicated in the cognitive and psychotic effects of THC (Nelong et al., 2019). Although THC inhalation and injection lead to decreased brain oscillations, few studies have investigated similar effects after edible cannabis consumption. Prepulse inhibition (PPI), a behavioral measure of sensory-motor gating (Jones et al., 2014), is impaired in cannabis users (Kedzior and Martin-Iverson, 2007) and in rodents administered with THC (Wegener et al., 2008); deficits in sensory-motor gating have been reported after disruptions in gamma oscillations, suggesting an association between exposure to THC and gamma power alteration (Jones et al., 2014). PPI can be evaluated using the acoustic startle reflex test (ASR) (Swerdlow and Geyer, 1998). Learning deficits can also be evaluated using the active avoidance task (AAT), and in animal models acute intraperitoneal THC administration impairs AAT performance (Mishima et al., 2001). Acute high-dose edible cannabis consumption (AHDECC) may also produce such deficits. Although THC exposure via vapor, injection, and gavage induces specific behavioral effects referred to as the cannabis tetrad, which includes catalepsy, hypolocomotion, hypothermia, and antinociception (Moore and Weerts, 2022), few studies have evaluated similar behavioral effects using edible cannabis (Moore et al., 2021; English et al., 2022). Like humans (Fogel et al., 2017; Sholler et al., 2021; Graves et al., 2023), sex differences in sensitivity to the effects of THC and its metabolism have also been identified in rodents (Craft et al., 2013; Wiley and Burston, 2014); therefore, it may be worth investigating these differences with respect to AHDECC. Based on the above discussion, we hypothesized that AHDECC in rats would lead to sex-dependent disruptions in gamma power, learning, and sensorimotor gating as well as marked cannabinoid tetrad effects.

This is an important topic given that most patients who report to the emergency unit due to cannabis intoxication report consuming edible cannabis (Noble et al., 2019). Understanding the underlying neural mechanisms and the pharmacokinetics of THC via edible consumption could help prevent and/or identify effective treatments for cannabis intoxication. Therefore, the objective of this study was to investigate the effects of AHDECC on neural activity, behavior, and serum and brain THC and 11-OH-THC levels.

Materials and MethodsAnimals.

Forty-four 56-day-old Sprague-Dawley rats (body weight: 150–250 g; males: n = 22; females: n = 22) were purchased from Charles River for the study. These rats were divided into two cohorts: cohort 1 (males: n = 6; females: n = 7) and cohort 2 (males: n = 16; females: n = 15). The rats were singly housed and allowed to habituate for 1 week. Male and female rats were housed in the same room but on separate racks, which were adjacent to each other. To motivate rats to consume a mix of either Nutella (a brand of sweetened hazelnut cocoa spread; Ferrero USA Inc., Somerset, NJ) and medium-chain triglyceride (MCT) oil (N-MCT; vehicle for dissolving and diluting THC-containing cannabis oil; Alpha Supreme MCT Oil; Assured Natural Distribution Inc., British Columbia, Canada) or Nutella and THC-containing cannabis oil (N-THC; edible), rats were maintained on 85%–90% free-feeding body weight on standard rodent chow for the entire study period except the first week after arrival and the week after stereotaxic surgery (for rats that underwent surgery). The MCT oil was derived from coconut oil, whereas the THC oil was diluted in olive oil. Rats were maintained on a 12-hour light/dark cycle with lights on at 07:00 hours. Experiments were performed during the light phase. All procedures were approved by the University of Guelph Institutional Animal Care and Use Committee (approval number: 4789). Additionally, experiments were performed in accordance with guidelines set by the University of Guelph Animal Care Committee and guidelines in the Guide to the Care and Use of Experimental Animals.

Preclinical Model Development.

On the seventh day after arrival, each rat was given 3 ml Nutella (density: 1.2 g/ml) for 3 hours (Fig. 1A) in a small petri dish placed on the floor in the home cage to familiarize the animals to the Nutella in their home environment. For all subsequent N-MCT/N-THC accesses, each rat was placed alone in an empty cage with a lid and only corn cobs as bedding material before N-MCT/N-THC was provided in a petri dish. With the exception of the duration of Nutella access, which was 3 hours for the seventh day, all subsequent accesses to N-MCT or N-THC was for only 10 minutes. During this 10-minute period, all the rats consumed all of the N-MCT or N-THC provided. Food restriction began afterward and lasted until at least the 13th day. On the ninth and 11th days, rats received N-MCT (Nutella: 6 g/kg; MCT oil: 20 mg/kg) for 10 minutes. High-dose edible cannabis (N-THC) was prepared similar to N-MCT; however, THC-containing cannabis oil [Five Founders THC Oil (30 mg THC/g oil); Ontario Cannabis Store] was added at 20 mg/kg (adjusted to animal’s body weight; this dose was calculated using the formula provided in the “Rat Equivalent Dose Calculation” section of the Supplemental Material file). On the 13th day, rats either were given 10-minute access to N-MCT or underwent stereotaxic surgery.

Fig. 1.

Experiment timeline and details. (A) Schematic showing experiments performed each day during the study period. Gray circles, final experiments. C1, cohort 1; C2, cohort 2; Ctrl, control rats for cohort 2; D, day; LFP, 15 minutes local field potential recording; N, Nutella; Test, test rats for cohort 2; Tetrad, tetrad behavior tests. (B) Schematic showing details of final experiments performed over 24 hours. Tetrad + LFP: The cannabis tetrad was performed 15 minutes before LFP recording. Saphenous vein blood draws and brain (for cannabinoid measurements) were collected after AAT/ASR. Empty circles, experiment performed during the 24-hour period; black circles, time after exposure when each experiment was performed during the 24-hour period. X, time that N-THC was administered.

Experiment Paradigm.

The first rat cohort was used for electrophysiology experiments. These rats underwent cannabis exposure as described above for the development of the preclinical model for AHDECC except that on day 13 they underwent stereotaxic surgery for multielectrode array implantation (described below). One week after surgery, rats were rehabituated to the N-MCT and tetrad behavior test, and the results were used as baseline (time point 0) for comparison with the tetrad test results from the AHDECC experiment. Afterward, rats were habituated to the recording chamber, where they were tethered to the headstage for 15 minutes. The data recorded during headstage habituation were used as baseline local field potential (LFP) activity. During the THC test, performed 2 days later, rats were given N-THC for 10 minutes. Subsequently, the cannabis tetrad test followed by LFP activity recording 15 minutes later was performed 2, 4, 8, and 24 hours after AHDECC (Fig. 1B). Rats were subsequently euthanized and their brains retrieved for electrode location verification.

The second rat cohort was used for behavioral [active avoidance test (AAT) and acoustic startle response (ASR)] and pharmacokinetics experiments (Fig. 1A). Upon arrival, these rats received Nutella for 3 hours on day 7, received N-MCT for 10 minutes on day 9, and received N-MCT for 10 minutes as well as underwent the baseline cannabis tetrad on day 11. On days 13 and 15, rats received N-MCT and underwent habituation and baseline measurements for PPI in the ASR operant box. Subsequently, rats in each sex group were divided into the control [(ctrl) male: n = 8; female: n = 8] and the test [(test) male: n = 8; female: n = 7] groups. On day 17, ctrl rats received N-MCT, whereas the test rats received N-THC. Two and a half hours later, both groups (ctrl and test) underwent the AAT (Fig. 1B). For the test groups, saphenous blood was also collected 4, 8, and 24 hours after AHDECC. The brains of the test rats were also retrieved after the 24-hour blood collection (Fig. 1B). After 5 days (day 22), the remaining ctrl rats (males: n = 8; females: n = 8) received N-THC and 3.5 hours later underwent the ASR. Similarly, saphenous blood was collected 4, 8, and 24 hours after AHDECC (Fig. 1B). Test rat brains were retrieved after the 24-hour blood collection. In this study, rats were euthanized through exposure to isoflurane first, leading to unconsciousness, followed by exposure to carbon dioxide. For experiments that required brain retrieval, rats were additionally decapitated.

Tetrad Behavior Evaluation.

The tetrad comprised tests for hypothermia, analgesia, catalepsy, and hypolocomotion, similar to that described in a previous study (Moore and Weerts, 2022). To test for hypothermia, the rectal temperature of rats was measured using a microprobe thermometer (model BAT-4; Physitemp Instruments Inc.), which was inserted into the rectum for 5 seconds. To test for analgesia (thermal pain sensitivity), the tails of rats were placed in the groove on the tail flick analgesia meter (Columbus Instruments, Columbus, OH) containing a radiant heat source and latency to tail flexion was recorded. The tail flick analgesia meter was calibrated to an intensity setting of 10. The tails of rats were removed from the groove after 15 seconds (if the rat did not do so by itself) to prevent tissue damage. Catalepsy was evaluated using an open-source automated catalepsy bar apparatus (Luciani et al., 2020). During the test, the two front paws of the rats were placed on the catalepsy bar with the hindlimbs on the floor of the apparatus. The bar (diameter: 1.27 cm) was set to a height of approximately 12 cm from the floor of the apparatus. In the catalepsy test, a cutoff of 15 seconds was used. Due to the absence of catalepsy, the test lasted for approximately 2 seconds. Hypolocomotion was measured over a 10-minute period in an open field box 45 × 45 cm in size. Rats were placed in the middle of the box at the start of the test, and their total distance moved was recorded using EthoVision XT 16.0 video tracking software (Noldus Information Technology).

Two-Way Active Avoidance Test.

The AAT was conducted using a standard two-way shuttle box (model H10-11R-SC; Coulbourn Instruments, Allentown, PA) placed in a ventilated isolation chamber (height: 51 cm; width: 53 cm; length: 80 cm; model H10-24; Coulbourn Instruments) with a grid floor made of stainless-steel bars. A metallic wall partition with a 9 × 9–cm door separated the shuttle box, which contained signal lights, a house light, and an infrared sensor to detect transitions between chambers, into two identical chambers. Scrambled electrical foot shocks were delivered via the grid floor by a precision animal shocker (model H13-15; Coulbourn Instruments). The Graphic State software version 5.9 (Coulbourn Instruments) was used to program the experimental protocol.

Rats were placed individually into the shuttle box and allowed to habituate for 30 seconds. They underwent 70 signaled avoidance trials, with intertrial intervals ranging from 15 to 60 seconds. The trials were subsequently divided into seven blocks of 10 trials each. Each trial consisted of a conditioned stimulus (CS; asynchronously flashing signal and house lights at a frequency of 2 Hz) and an unconditioned stimulus (US; 0.5 mA foot shock). The CS was presented for 10 seconds, and the US was applied during the last 2 seconds of the CS presentation. Moving to the opposite chamber during the CS prevented the delivery of foot shock (avoidance), whereas moving to the opposite chamber during the US (shock) delivery attenuated the foot shock (escape). The number of avoidance and escape behaviors within each block was detected and expressed as percentages per block.

Prepulse Inhibition of the Acoustic Startle Reflex.

The ASR was performed using Med Associates Acoustic Startle Chambers (MED-ASR-PRO1). Each chamber was soundproof and equipped with a ventilation fan, house light, load cell platform, and two speakers for acoustic stimuli (white noise) delivery. The platform was calibrated by adjusting the load cell amplifier gain, which ranged from −2047 to 2047 arbitrary units to 200 arbitrary units with a standard weight of 300 g. In the chamber, rats are restrained using a plexiglass cylinder mounted on the platform. A 70-dB white noise was used as background noise during the experiment.

During the baseline session, rats were placed in the plexiglass cylinders in the startle reflex chambers for 15 minutes while white noise (background noise) was on. Five acoustic startle sounds were played between 5 and 10 minutes. In the test session, rats were allowed 5 minutes to acclimate to the chamber while the 70-dB background noise was on. Subsequently, five consecutive startle stimuli (120 dB) were presented, followed by 50 trials separated into 10 blocks, and then five more startle stimuli. Each block comprised five trials in a randomized order: 1) startle stimuli alone; 2) a 73-dB prepulse and startle stimuli; 3) a 76-dB prepulse and startle stimuli; 4) an 82-dB prepulse and startle stimuli; and 5) no stimulus (only background noise). These prepulse intensities were selected because they did not elicit significant startle reflex when applied alone. The startle stimulus lasted for 40 milliseconds, whereas the prepulse stimulus lasted for 20 milliseconds. The prepulse stimulus was applied 120 milliseconds prior to the onset of the startle stimulus. The intertrial interval ranged from 15 to 30 seconds. In this study, PPI was defined as a decrease in the magnitude of the startle reflex elicited by a startling stimulus when it is preceded by a nonstartling stimulus (prepulse). The PPI for each prepulse intensity was calculated as follows:

Serum and Brain THC and 11-OH-THC Concentration Quantification.

Quantification of serum and brain levels of THC and 11-OH-THC were performed as previously described (Baglot et al., 2021).

Materials

Reference standards of 11-OH-THC and THC and their deuterated internal standards 11-OH-THC-D3 and THC-D3 were purchased from Sigma-Aldrich Canada (Oakville, ON, Canada). Liquid chromatography mass spectrometry (LC-MS)-grade acetonitrile, methanol, and formic acid were purchased from Fisher Scientific (Fisher Chemical Optima grade). Ammonium formate was purchased from Sigma (St. Louis, MO), and water was obtained from the Milli-Q system (Millipore, Bedford, MA).

Serum Sample Preparation.

To extract 11-OH-THC and THC from rat serum, a Captiva enhanced matrix removal lipid (EMR-Lipid) 96-well plate (Agilent, Santa Clara, CA) was used. Briefly, 250 μl acetonitrile (acidified with 1% formic acid) was added to each well, and then 50 μl rat serum and 20 μl (10 μg/ml) internal standard solution were added. After the sample passed through under positive pressure at 3 psi, the extraction plate was washed with 150 μl of a mixture of water/acetonitrile (1:4; v:v) and passed through under gradually increasing pressure up to 15 psi. The effluent was evaporated under nitrogen at 40°C, and the residual was reconstituted with 100 μl mobile phase for subsequent liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Calibration standards (2–1000 ng/ml) and quality controls (3 ng/ml and 800 ng/ml) were prepared on the day of analysis by spiking standard working solutions into blank rat serum.

Brain Sample Preparation.

The brain tissue samples were removed from a −80°C freezer and immediately placed into a cooled rat brain matrix. A coronal slice between 6 and 7 mm posterior to the frontal tip of brain was cut, weighed, and placed into the ice-cooled glass tubes for manual homogenization with a glass tissue homogenizer. Acetonitrile (2 ml) and 50 ml internal standard solution (10 μg/ml) were added, and the tissue was homogenized completely. Samples were sonicated in an ice bath for 30 minutes and stored overnight at −20°C. The next day, samples were centrifuged at 1800 rpm at 4°C for 4 minutes, and the supernatant were transferred to a new glass tube and evaporated under nitrogen. The tube sidewalls were washed with 250 μl acetonitrile and evaporated again. The samples were reconstituted in 100 μl of the mobile phase and centrifuged for 20 minutes (15,000 rpm, 4°C), and the supernatants were transferred to the vials. Calibration standards (1–4000 ng/ml) and quality controls (10 ng/ml and 2000 ng/ml) were prepared on the day of analysis by spiking standard working solutions into the mobile phase.

Liquid Chromatography Tandem Mass Spectrometry Method.

Serum and brain concentration of 11-OH-THC and THC were determined using an LC-MS/MS method. The LC separation was achieved on a Thermo Scientific Vanquish Flex UHPLC system. Five microliters of sample extracts was injected and separated on an ACQUITY UPLC BEH C18 Column (1.7 μm, 2.1 mm × 50 mm; Waters, Ireland) connected with a VanGuard UPLC BEH C18 Pre-column (Waters, Ireland). The auto sampler was kept at 4°C, and column temperature was at 35°C. The mobile phase containing A: 10 mM ammonium formate with 0.1% formic acid aqueous solution and B: acetonitrile with 0.1% formic acid was at a flow rate of 400 μl/min under a gradient mode. The gradient conditions were: from 0.1 to 4 minutes ramp from 40% to 95% of mobile phase B, maintain 95% B for 2 minutes, and then ramp back to 40% B. 11-OH-THC and THC were eluted at 3.5, 4.0, and 4.6 minutes, respectively, with a total run time of 7 minutes. MS analysis was conducted with a Thermo Q Exactive Focus Orbitrap mass spectrometer equipped with an Ion Max source in positive electrospray ionization (ESI) mode. The source conditions were optimized as spray voltage 3.5 kV, capillary temperature 300°C, and auxiliary gas heater temperature 425°C.

Data were acquired and processed in parallel‐reaction monitoring (PRM) mode using Thermo Scientific TraceFinder software. In this PRM mode, protonated 11-OH-THC ion [mass/charge (m/z) 331.23] and THC ions (m/z 315.23) were selected as precursors and then fragmented in the higher-energy collisional dissociation cell at collision energy of 20 eV for 11-OH-THC and 25 eV for THC. The resulting MS/MS product ions were detected in the Orbitrap at a resolution of 17,500 (full width at half maximum at m/z of 200) with AGC target set at 1e5. The most abundant fragments from the MS/MS spectra (m/z 313.22 for 11-OH-THC and m/z 193.12 for THC) were selected as the quantifying ions. Other specific fragments, m/z 193.12 for 11-OH-THC and m/z 259.17 for THC, were selected as the confirming ions. The resulting chromatograms were extracted and reconstructed with a mass accuracy of 5 ppm for quantification and confirmation. The optimized MS/MS compound parameters are summarized in Table 1.

TABLE 1

Optimized LC-MS/MS compound parameters for quantitation of 11-OH-THC and THC using PRM mode

Stereotaxic Surgery.

After habituation to the N-MCT, rats underwent stereotaxic surgery for electrode implantation, which begins with anesthesia using isoflurane, as previously described (Thériault et al., 2021; Quansah Amissah et al., 2023). A custom-built, 18-channel microelectrode array was constructed using Delrin templates and stainless-steel wires insulated with polyimide tubing. Four regions were targeted bilaterally with eight electrodes: the medial PFC [anterior-posterior (AP): +3.24 mm; mediolateral (ML): ±0.6 mm; (dorsoventral (DV): −3.8 mm], Cg (AP: +1.9 mm; ML: ±0.5 mm; DV: −2.8 mm), NAc (AP: +1.9 mm; ML: ±1.2 mm; DV: −6.6 mm), and dorsal hippocampus [(dHipp) AP: −3.5 mm; ML: ±2.5 mm; DV: −2.6 mm], as per the coordinates found in the Paxinos rat atlas (6th edition) (Paxinos and Watson, 2009). After the surgery, rats were allowed 1 week to fully recover before experimentation began. All electrodes were confirmed to be accurately placed within the brain regions of interest in the rats at the end of the experiment.

Electrophysiological Data Acquisition and Analysis.

LFP data were acquired at a sampling rate of 1 kHz using a TDT RZ10X multichannel acquisition system (Tucker Davis Technologies, Florida). The data were filtered between frequencies of 0.1 to 300 Hz. A notch filter was used to remove any 60-Hz frequency noise present in the signal during recording. The recording lasted for 15 minutes in awake, freely behaving rats in an open field box. LFP analysis was performed using MATLAB (R2020a; The MathWorks) and routines from the open source Chronux package (Mitra and Bokil, 2008). The data were preprocessed using detrending and denoising routines in Chronux. Subsequently, the data were low-pass filtered to eliminate frequencies higher than 100 Hz. Afterward, band pass filters were used to separate the LFP data into its constituent brain rhythms: delta (0.1–4 Hz), theta (>4–12 Hz), beta (>12–30 Hz), low gamma (>30–59 Hz), and high gamma (>60–100 Hz). Continuous multispectral power was calculated for each of the brain rhythms in each brain region, and coherence between regions was determined.

Statistical Analysis.

The results are presented as means ± standard error of means. The Shapiro-Wilk test was performed to evaluate normality before subsequent statistical tests were performed. The power spectral density for each brain region and the coherence between pairs of brain regions after AHDECC were compared in male and female rats using the two-way repeated measures (RM) analysis of variance (ANOVA). When sex differences were significant, subsequent one-way RM ANOVA was used to analyze data within each sex. The two-way RM ANOVA was also performed to compare the means of results obtained during the tetrad behavioral tests, ASR, and serum THC and 11-OH-THC concentrations between male and female rats. Comparison of the brain THC and 11-OH-THC concentrations in male and female rats was performed using the two-tailed unpaired Student’s t test. The three-way RM ANOVA was performed to compare the means of the percentage avoidance in rats during the AAT, with sex, trial block, and treatment as independent variables and percentage avoidance as the dependent variable. This was followed by a two-way RM ANOVA with treatment and trial block as independent variables based on the lack of sex differences. All ANOVA tests were followed by a post hoc Bonferroni test to correct for multiple comparisons. Statistical analyses were performed using GraphPad version 6.01 (GraphPad Software Inc., La Jolla CA), and statistical significance was set at P < 0.05.

ResultsSerum and Brain THC and 11-OH-THC Levels after AHDECC.

There was a significant main effect of sex on serum THC concentration (F(1,29)=21.05, P < 0.0001) after AHDECC. In males serum THC concentrations did not differ at any time point, whereas in females AHDECC increased serum THC concentration at the 4-hour time point compared with the 24-hour time point (P = 0.0463). Moreover, AHDECC increased the serum THC concentration at the 4-hour time point in females compared with males (P = 0.0019) (Fig. 2A). The main effects of sex, time, and their interaction on serum 11-OH-THC levels were significant (F(1,29)=86.00, F(2,58)=23.86, and F(2,58)=21.25, respectively; all P < 0.0001). In males there were no time-related differences in serum 11-OH-THC concentration after AHDECC, whereas in females the 11-OH-THC concentration at the 4-hour time point was higher than at the 8-hour (P = 0.0220) and 24-hour (P < 0.0001) time points (Fig. 2B) after AHDECC. Moreover, females had higher serum 11-OH-THC concentration than males at the 4-hour (P < 0.0001) and 8-hour (P < 0.0001) time points after AHDECC. Comparison using t tests showed that AHDECC increased brain THC and 11-OH-THC levels in females (P = 0.0159 and P = 0.0085, respectively) compared with males at the 24-hour time point (Fig. 2, C and D).

Fig. 2.

Concentrations of THC and 11-OH-THC in the serum and brain of rats after acute high-dose edible cannabis consumption. (A and B) Serum THC and 11-OH-THC concentrations in males and females at the 4-hour, 8-hour, and 24-hour time points. (C and D) Brain THC and 11-OH-THC levels in males and females at the 24-hour time point. Filled black circles, male THC/11-OH-THC; filled red squares, female THC/11-OH-THC; $, comparison between males and females at different time points with P < 0.05; #, comparison of concentration at other time points with that at the 4-hour time point in females with P < 0.05; *, comparison of concentration of THC/11-OH-THC in the brain between males and females with P < 0.05.

Effects of AHDECC on Gamma Oscillations.

Although AHDECC had some effect on delta, theta, and beta oscillations, the low and high gamma oscillations were the most consistently affected, consistent with previous findings with THC and cannabis (Nelong et al., 2019; Jenkins et al., 2022). Therefore, only the results for the effects of AHDECC on PFC, dHipp, Cg, and NAc gamma oscillations at the different time points (Fig. 3) will be described. The same data have been plotted as percentage change from baseline and presented in Supplemental Fig. 2 to allow for comparison despite the baseline differences.

Fig. 3.

Power spectral density (PSD) plot for male and female rats after acute high-dose edible cannabis consumption. Representative log-transformed prefrontal cortex (PFC) PSD in male (A) and female rats (B) at each time point. Orange plot, baseline; cyan plot, 2-hour time point; blue plot, 4-hour time point; green plot, 8-hour time point; and pink plot, 24-hour time point. (C and D) Low and high gamma power, respectively, in the PFC between males and females at different time points. (E and F) Low and high gamma power, respectively, in the dorsal hippocampus (dHipp) of males and female rats at different time points. (G and H) Low and high gamma power, respectively, in the nucleus accumbens (NAc) of males and female rats at different time points. (I and J) Low and high gamma power, respectively, in the cingulate cortex (Cg) between males and females at different time points. $, comparison of time points between sexes with P < 0.05; *, comparison of power at other time points with that at baseline in males with P < 0.05; #, comparison of power at other time points with that at baseline in females with P < 0.05.

There was a significant main effect of time on PFC (low gamma) LG power (F(4,96)=17.59, P = 0.0001). AHDECC decreased PFC LG power at all time points compared with baseline in males (P < 0.05) but only at the 2-hour, 4-hour, and 8-hour time points in females (P < 0.0001). At the 24-hour time point, the effect of AHDECC had worn off in female rats, with the PFC LG power returning to baseline levels (Fig. 3, A–D). The main effects of time and its interaction with sex on PFC high gamma (HG) power were significant (F(4,96)=23.42 and F(4,96)=3.169, respectively; all P < 0.05). AHDECC similarly decreased PFC HG power at all time points compared with baseline in males (all P < 0.001) but only at the 2-hour, 4-hour, and 8-hour time points in females (P < 0.0001). Once again, at the 24-hour time point, the effect of AHDECC had worn off in females, causing the PFC HG power to return to baseline levels.

Sex, time, and their interaction had significant main effects on the dHipp LG power (F(1,24)=24.40, F(4, 96)=5.540, and F(4,96)=4.897, respectively; all P < 0.05). dHipp LG power was higher in females at baseline and at the 24-hour time point and similar between sexes at other time points (Fig. 3E). In males, according to the two-way RM ANOVA, AHDECC did not affect dHipp LG power at any time point compared with baseline (Fig. 3E); however, the one-way RM ANOVA revealed that AHDECC decreased dHipp LG power at the 4-hour and 8-hour time points (all P < 0.05; Supplemental Fig. 1). In females, AHDECC decreased dHipp LG power at the 2-hour, 4-hour, and 8-hour time points (P < 0.0001) compared with baseline: however, at the 24-hour time point, the effect of AHDECC had decreased, causing the dHipp LG power to return to baseline levels. The dHipp LG power in females at baseline and the 24-hour time point was higher than that in males. Similarly, the main effects of sex, time, and their interaction on dHipp HG power were significant (F(1,24)=14.38, F(4,96)=5.5, F(4,96)=4.294, respectively; all P < 0.05). In males, according to the two-way RM ANOVA, AHDECC did not affect dHipp HG power at all time points compared with baseline; however, the one-way RM ANOVA revealed that AHDECC decreased dHipp HG power at all time points (P < 0.01; Supplemental Fig. 1). In females, AHDECC decreased dHipp HG power at the 2-hour, 4-hour, and 8-hour time points (P < 0.001) compared with baseline. dHipp HG power was higher in females at baseline and the 24-hour time point (Fig. 3F) compared with males.

Only the main effects of sex and time on the NAc LG power were significant (F(1,24)=8.994 and F(4,96)=4.122, respectively; all P < 0.01). Females had higher NAc LG power than males at baseline (P = 0.0109) and the 24-hour (P = 0.0071) time point (Fig. 3G) after AHDECC. In males, according to the two-way RM ANOVA, AHDECC did not affect the NAc LG power (Fig. 3G); however, the one-way RM ANOVA revealed that AHDECC decreased NAc LG power at the 2-hour, 4-hour, and 8-hour (all P = 0.05) time points compared with baseline (Supplemental Fig. 1C). In females, AHDECC decreased NAc LG power at the 2-hour (P = 0.0130) and 8-hour (P = 0.0009) time points. Sex and time had significant main effects on the NAc HG power (F(1,24)=5.544 and F(4,96)=7.510, respectively; all P < 0.05). Females had higher HG power than males at baseline (P = 0.0357) and the 24-hour (P = 0.0384) time points (Fig. 3H). Similarly, according to the two-way RM ANOVA, AHDECC did not affect HG power at any time point in males (Fig. 3H); however, the one-way RM ANOVA revealed that AHDECC decreased NAc LG power at the 4-hour, 8-hour, and 24-hour (P < 0.01) time points (Supplemental Fig. 1D). In females, AHDECC decreased the HG power at the 2-hour, 4-hour, and 8-hour (all P < 0.001) time points but not the 24-hour time point.

The main effects of sex, time, and their interaction on Cg LG power were significant (F(1,24)=14.69, F(4,96)=8.707, and F(4,96)=4.506, respectively; all P < 0.005). In males, according to both the one-way and two-way RM ANOVA, AHDECC did not affect Cg LG power at any time point (Fig. 3I and Supplemental Fig. 1E, respectively). AHDECC decreased Cg LG power in females at the 2-hour, 4-hour, and 8-hour (all P < 0.0001) time points but not the 24-hour time point. Females had higher Cg LG power than males at baseline (P < 0.0001) and the 24-hour time point (P = 0.0002). No differences were found at the other time points (Fig. 3I). There were significant main effects of sex, time, and their interaction on Cg HG power (F(1,24)=14.92, F(4,96)=7.109, F(4,96)=3.107, respectively; all P < 0.0190). In males, according to the two-way RM ANOVA, AHDECC did not affect Cg HG power at any time point (Fig. 3J); however, the one-way RM ANOVA revealed that AHDECC decreased HG power at the 4-hour, 8-hour, and 24-hour (all P < 0.05) time points (Supplemental Fig. 1F). AHDECC decreased Cg HG power in females at the 2-hour, 4-hour, and 8-hour (all P < 0.05) time points but not the 24-hour time point. Males had lower Cg HG power than females at baseline and the 4-hour and 24-hour (all P = 0.01) time points (Fig. 3J).

The coherence between pairs of brain regions within the gamma frequency ranges was also evaluated. However, unlike the power spectral density analysis, the results (Supplemental Fig. 3) were inconsistent and will not be described.

Effects of AHDECC on Tetrad Behavior.

Although we evaluated the four cannabis tetrad behaviors (Moore and Weerts, 2022), there were no observable cataleptic effects of AHDECC in either sex; therefore, results will only be presented for hypolocomotion, hypothermia, and antinociception.

The main effects of time and its interaction with sex on rectal temperature were significant (F(4,44)=26.91 and F(4,44)=3.295, respectively; all P < 0.05). In both males and females, AHDECC decreased rectal temperatures at the 2-hour, 4-hour, and 8-hour time points compared with baseline (all P < 0.05). However, at the 24-hour time point, although AHDECC decreased rectal temperature in males (P < 0.05), it did not affect the temperature in females (Fig. 4A). Therefore, females had higher 24-hour temperatures than males (P = 0.014).

Fig. 4.

Acute edible cannabis-induced effects on body temperature, antinociception, and locomotion in male and female rats. (A) Rectal temperatures of males and female rats measured at the time points of interest after acute high-dose edible cannabis consumption. (B) Latency to tail flick of males and female rats at the time points of interest after acute high-dose edible cannabis consumption. (C) Total distance traveled by males and females in the open field box at the time points of interest after acute high-dose edible cannabis consumption. $, comparison of outcome at different time points between sexes with P < 0.05; *, comparison of outcome at other time points with that at baseline in males with P < 0.05; #, comparison of outcome at other time points with that at baseline in females with P < 0.05.

Time and its interaction with sex had significant main effects on tail flick latency (F(4,44)=11.92 and F(4,44)=5.517, respectively; all P < 0.01). AHDECC increased tail flick latency in males at all time points (all P < 0.01) compared with baseline. In females, AHDECC increased the tail flick latency at the 2-hour (P = 0.0209) and 8-hour (P < 0.0001) time points but not at the 4-hour and 24-hour time points (Fig. 4B). After AHDECC, the tail flick latency was longer in females than males at the 8-hour time point (P = 0.0091) but was not different at the 24-hour time point (P = 0.0743).

The main effects of time and its interaction with sex on distance were significant (F(4,44)=32.90 and F(4,44)=2.453, respectively; all P < 0.05). AHDECC decreased the distance moved compared with baseline at all time points in either sex (all P < 0.01) (Fig. 4C).

The data for body temperature, antinociception, and distance moved have been plotted as percentage change from baseline and presented in Supplemental Fig. 4 to allow for comparison despite the baseline differences between the sexes.

Effects of AHDECC on Active Avoidance Learning and Prepulse Inhibition.

Three-way ANOVA in the AAT revealed significant effects of trial block (F(4.322,109.5)=30.14, P < 0.0001), treatment (F(1,26)=15.94, P = 0.0005), trial block × treatment interaction (F(6,152)=10.29, P < 0.0001), and trial block × sex interaction (F(6,152)=4.318, P = 0.0005) on percentage avoidance. No significant effects were found for sex, sex × treatment interaction, or trial block × treatment × sex interaction on percent avoidance. The two-way ANOVA revealed significant main effects of trial block, treatment, and their interaction on percentage avoidance in male rats (F(6,78)=7.037, F(1,13)=4.701, and F(6,78)=3.396, respectively; all P < 0.050). Male THC rats had lower percentage avoidance than control rats during trial blocks 5 (P = 0.0474) and 7 (P = 0.0098) (Fig. 5A). Similarly, male THC rats had higher percentage escape than male control rats during trial blocks 3 (P < 0.0275), 5 (P < 0.0477), 6 (P < 0.0470), and 7 (P < 0.0077) (Supplemental Fig. 5A). There were significant main effects of trial block, treatment, and their interaction on percentage avoidance in female rats (F(6,74)=27.47, F(1,13)=12.79, and F(6,74)=7.552; all P < 0.05). Female THC rats had lower percentage avoidance than control rats during trial blocks 5 (P < 0.0001), 6 (P = 0.0004), and 7 (P = 0.0001) (Fig. 5B). Similarly, female THC rats had higher percentage escape than female control rats during trial blocks 5 (P < 0.0297), 6 (P < 0.0190), and 7 (P < 0.0390) (Supplemental Fig. 5B).