3.1. Body mass and body temperature
Body mass (g) and body temperature (°C) were taken to ensure animal’s health was not substantially affected by the study procedure (Fig. 1AB). Animals were weighed 24 h after acute administration of THC doses (0–10 mg/kg, Fig. 1A). A three-way ANOVA with drug dose as a within-subjects factor and sex and genotype as between-subjects factors demonstrated a significant main effect of sex, F(1, 34) = 6.6, p = 0.014. Male mice (35.3 ± 0.92, n = 18) weighed more on average than females (31.9 ± 0.95, n = 20), which was not affected by genotype. No significant THC dose effects or interactions were noted.
Animal body temperature was also measured as an indicator of animal’s health 45 min following acute THC injections (Fig. 1B). No significant main effect or interactions were noted, indicating that body temperature was not altered by THC doses, sex, or genotype. Overall, acute administration of THC doses did not lead to significant changes in body mass or body temperature, confirming the continued health of the animals after acute drug exposure.
3.2. Spontaneous heat-evoked nociception
The tail-flick and hot-plate assays were conducted 45 min after drug injections to assess effects of acute THC (0–10 mg/kg) exposure on heat-evoked pain like behaviors in the Tat transgenic mouse model (Fig. 1CD). The tail-flick test was used to assess spinal-related spontaneous nociception (Fig. 1C). A three-way ANOVA with drug dose as a within-subjects factor and sex and genotype as between-subjects factors demonstrated a significant main effect of THC doses, F(1, 34) = 146.7, p < 0.001. Bonferroni’s post hoc tests indicated that all THC doses, including vehicle, significantly differed from each other (p < 0.001) except 1 mg/kg from 3 mg/kg, indicating that THC administration induced dose-dependent antinociception, thus increasing latency to remove the tail. Importantly, a significant main effect of genotype was noted, F(1, 34) = 13.1, p < 0.001, that was altered by drug dose, drug dose x genotype interaction: F(1, 34) = 11.4, p < 0.001, with Tat(+) mice showing higher response latencies, thus antinociception, with increasing THC doses. When conducting individual comparisons for each drug dose, significant genotype effects were noted for 3 mg/kg THC, F(1, 34) = 5.7, p = 0.023, and 10 mg/kg THC, F(1, 34) = 13.5, p < 0.001. No significant sex effect or interactions were noted.
The hot-plate test was used to assess supraspinal-related spontaneous nociception (Fig. 1D). Similar to the tail-flick data, a three-way ANOVA with drug dose as a within-subjects factor and sex and genotype as between-subjects factors demonstrated a significant main effect of THC doses, F(1, 34) = 12.3, p < 0.001, with elevated THC doses increasing time of paw withdrawal or lick, thus inducing antinociception. Bonferroni’s post hoc tests indicated that the 10 mg/kg THC dose significantly differed from all other groups (p < 0.001). No other effect or interaction was significant.
Overall, spontaneous heat-evoked nociception is significantly decreased in a THC dose-dependent manner, thus withdrawal latencies are increased, and Tat induction enhances THC-induced antinociception in the spinal-related tail-flick task at higher THC doses.
3.3. Locomotor activity and rotarod coordination
To understand the effects of acute THC (0–10 mg/kg) exposure on motor function we assessed locomotor activity and rotarod performance 30 min and 1 h after injections, respectively (Fig. 1EF). The locomotor activity task was conducted to assess effects of acute THC administration in Tat transgenic mice on motor activity (Fig. 1E). A three-way ANOVA with drug dose as a within-subjects factor and sex and genotype as between-subjects factors demonstrated a significant main effect of THC doses, F(1, 34) = 3.4, p = 0.021, with Bonferroni’s post hoc tests indicating higher activity after 1 mg/kg THC administration compared to vehicle exposure (p < 0.001). Specifically, female Tat(−) and female Tat(+) mice, but not males, demonstrated a significant drug dose effect [F(1, 27) = 3.0, p = 0.047 and F(1, 27) = 3.4, p = 0.031, respectively], with female Tat(−) mice showing higher activity after 1 mg/kg THC compared to vehicle administration (p = 0.013) and Tat(+) mice showing higher activity after 10 mg/kg THC compared to vehicle (p = 0.012). No other significant effects were noted.
The rotarod task was conducted to investigate effects of THC exposure in Tat transgenic mice on motor coordination and function (Fig. 1F). A three-way ANOVA with drug dose as a within-subjects factor and sex and genotype as between-subjects factors demonstrated no significant effects or interactions.
Overall, a slight increase of locomotor activity was noticed after administration of THC doses, specifically for female mice, but no effects were noted for motor coordination.
3.4. Anxiety-like behavior
The elevated plus maze task was conducted 30 min after injections to assess the effects of acute THC (0 and 10 mg/kg) exposure in Tat transgenic mice on anxiety-like behavior (Fig. 2). The dependent measures included percent time spent in open arms and number of pokes into open arms. A three-way ANOVA with drug, sex, and genotype as between-subjects factors was conducted for each measure. For percent time spent in open arms (Fig. 2A) a significant main effect of sex was noted, F(1, 30) = 4.2, p = 0.050, in which males showed less anxiety compared to females, thus spending more time in the open arms. No significant effects or interactions were noted for drug or genotype.
For number of pokes into open arms (Fig. 2A) a significant drug main effect was noted, F(1, 30) = 5.4, p = 0.028, in which THC decreased pokes into the open arms compared to vehicle exposure. Interestingly, this was specifically noted for Tat(−) females, F(1, 8) = 6.3, p = 0.037, and Tat(−) males, F(1, 7) = 7.6, p = 0.028, but not Tat(+) animals.
Overall, males showed less anxiety compared to females and THC administration appeared to increase anxiety-like behavior in Tat(−) animals but not Tat(+) mice.
3.5. Novel object recognition
The NOR task was conducted 30 min after injections to assess the effects of acute THC (0 and 10 mg/kg) exposure in Tat transgenic mice on novel object recognition memory (Fig. 3). The dependent measures included total time of exploring both the novel and familiar objects and time spent exploring the novel object over the familiar object (discrimination index). Due to the natural tendency to explore new stimuli, the preference for a novel object means that presentation of the familiar object exists in animal’s memory and thus presents successful object recognition memory (Ennaceur, 2010). For total exploration time (Fig. 3A), a three-way ANOVA including drug, sex, and genotype as between-subjects factors, demonstrated a significant main effect of drug, F(1, 30) = 13.4, p < 0.001, with THC administration decreasing object exploration time compared to vehicle exposure. This main effect of drug was specifically noted for Tat(−) females, F(1, 8) = 15.6, p = 0.004, and Tat(−) males, F(1, 7) = 7.4, p = 0.030, but not Tat(+) animals. Further, a significant main effect of sex was noted, F(1, 30) = 4.2, p = 0.049, with female mice showing higher exploration behavior compared to males. Lastly, a sex x genotype interaction was noted, F(1, 30) = 10.70, p = 0.003, with Tat(+) female mice demonstrating decreased object exploration time compared to Tat(−) females, whereas no effect was noted for males More specifically, vehicle exposed Tat(−) females significantly differed from vehicle exposed Tat(+) female, F(1, 8) = 13.0, p = 0.007.
For object recognition memory, the discrimination index was used, with complete preference for the novel object equal 1, no preference equal 0, and complete preference for the familiar object equal − 1 (Fig. 3B). A two-way ANOVA for each sex revealed a significant drug x genotype interaction for females, F(1, 16) = 4.5, p = 0.049, but not males. Specifically, THC administration significantly impacted object recognition memory in Tat(−) females, F(1, 8) = 5.9, p = 0.041, without affecting recognition memory in Tat(+) females. In the presence of THC exposure the natural tendency of Tat(−) females preferring to explore the novel object was reversed to exploring the familiar object over the novel object. No other effects or interactions were noted.
Overall, total object exploration time was significantly higher in females compared to males and altered by genotype; THC administration decreased object exploration time in males and females, specifically for the Tat(−) groups. For object recognition memory, THC administration had no effect on males but differentially affected recognition memory in female mice by reversing the natural tendency of Tat(−) females without affecting the recognition memory in Tat(+) females, which showed no preference.
3.6. CNS levels of endocannabinoids and related lipids
To assess the impact of acute THC (0 and 10 mg/kg) exposure on the endogenous cannabinoid system, changes in levels of 2-AG, AEA, PEA, OEA, and AA were assessed 60 min after injections in various CNS regions of Tat transgenic female and male mice (n = 4–5 per group), including the prefrontal cortex, striatum, cerebellum, and spinal cord (Supplemental Table S2, Fig. 4). Lipid molecule concentrations (nmol/g) differed significantly between CNS region; 2-AG, F(3, 111) = 41.7, p < 0.001, demonstrated differences in expression levels between all CNS regions (p’s < 0.001) with lowest 2-AG levels for the prefrontal cortex, followed by the striatum, cerebellum, and the highest 2-AG levels expressed in the spinal cord. AEA levels, F(3, 111) = 23.7, p < 0.001, were found to be higher in the PFC and striatum compared to the cerebellum and spinal cord (p’s < 0.001). AA, F(3, 111) = 17.6, p < 0.001 and OEA, F(3, 111) = 26.1, p < 0.001, showed highest expression in the striatum compared to the other three CNS regions (p’s < 0.01) and PEA levels, F(3, 111) = 9.6, p < 0.001, were also highest in the striatum but only differed significantly from the cerebellum and spinal cord (p’s < 0.01, Supplemental Table S2, Fig. 4). To assess treatment effects a multivariate analysis was conducted for each lipid molecule with drug, sex, and genotype and as between-subjects factors. No effects or interactions were noted for acute THC administration on any measure indicating that acute exposure of THC did not alter the endocannabinoid system and related lipid molecules. The most prominent findings were noted for AEA and AA levels with some minor effects for 2-AG (Fig. 4) and PEA and OEA (Supplemental Table S2).
For the prefrontal cortex (Fig. 4A), significant sex effects were noted for 2-AG, F(1, 30) = 9.3, p = 0.005, and for AEA, F(1, 30) = 22.3, p < 0.001, with females demonstrating higher 2-AG and AEA levels compared to male mice. For AA, a genotype effect was noted for the prefrontal cortex, F(1, 30) = 6.1, p = 0.019, that was altered by sex, F(1, 30) = 7.5, p = 0.010, with Tat(+) females showing higher AA levels compared to Tat(−) females, F(1, 18) = 12.1, p = 0.003, but no difference was noted for males. Specifically, for females this effect was noted for vehicle-exposed females, F(1, 8) = 6.0, p = 0.040, as well as THC-exposed females, F(1, 8) = 5.5, p = 0.047.
For the striatum (Fig. 4B), significant sex effects were noted for AEA, F(1, 30) = 41.1, p < 0.001, and AA, F(1, 30) = 20.4, p < 0.001, with females demonstrating higher AEA and AA levels compared to male mice. Further, a sex x genotype interaction was noted for AEA in the striatum, F(1, 30) = 11.6, p = 0.002, in which Tat(+) females showed higher AEA levels compared to Tat(−) females, F(1, 18) = 7.7, p = 0.012, but the reversed was true for males, F(1, 16) = 5.5, p = 0.032. Specifically, vehicle-exposed Tat(+) females showed higher AEA levels compared to vehicle-exposed Tat(−) females, F(1, 8) = 5.8, p = 0.042, without any other group showing significance.
For the cerebellum (Fig. 4C), a significant sex effect was noted for AA, F(1, 30) = 11.3, p = 0.002, with females demonstrating higher AA levels compared to male mice. No other significant effects or interactions were noted.
For the spinal cord (Fig. 4D), significant sex effects were noted for AEA, F(1, 30) = 6.6, p = 0.015, and AA, F(1, 30) = 26.6, p < 0.001, with females demonstrating higher AEA but lower AA levels compared to male mice. Further, AEA levels were significantly altered by genotype in the spinal cord, F(1, 30) = 4.9, p = 0.034, with upregulated AEA expression for Tat(+) mice compared to Tat(−) mice, with individually testing genotype effect for each sex not showing significance. In contrast, the sex x genotype interaction for AA levels in the spinal cord, F(1, 30) = 6.1, p = 0.020, showed lower AA levels in Tat(+) females compared to Tat(−) females, F(1, 18) = 11.2, p = 0.004, but no difference was noted for males. Specifically, for females this effect was noted for THC-exposed females, F(1, 8) = 6.6, p = 0.033, but not vehicle-exposed females. Lastly, for 2-AG, a significant genotype effect was found for the spinal cord, F(1, 30) = 4.8, p = 0.036, with higher 2-AG expression levels in Tat(+) mice compared to Tat(−) mice. Specifically, THC-exposed Tat(+) females showed higher 2-AG levels compared to THC-exposed Tat(−) females, F(1, 8) = 7.0, p = 0.029, without any other group showing significance. No other significant effects or interactions were noted.
Results for PEA and OEA mirrored a few of the findings for AEA, specifically some of the noted sex effects in the prefrontal cortex, even though opposite sex effects were noted for the striatum and spinal cord (Supplemental Table S2).
Overall, acute THC administration did not alter endocannabinoids or related lipid molecules in any CNS region. Interestingly, female mice showed higher AEA and/or AA expression levels in almost all CNS regions compared to males, except for AA levels in the spinal cord, with 2-AG being upregulated for females in the prefrontal cortex only. Further, Tat induction seemed to affect 2-AG, AEA and AA levels differently for males and females in some CNS regions, including the striatum, prefrontal cortex, and/or spinal cord.
3.7. Relationships between AEA, AA, and object recognition memory
As the most prominent findings were noted for AEA and AA levels, we assessed the relationship between levels of AEA and AA within each of the four CNS regions. Pearson correlations were conducted between both lipid molecules separately for females and males within each CNS region (Table 1). Data indicate that specifically in the prefrontal cortex significant positive relationships were noted with low AA levels being associated with low AEA levels and high AA levels being associated with high AEA levels. Note that correlations for AA and AEA across CNS regions revealed only selected significant relations (Supplemental Results). For the prefrontal cortex, significant correlations were further assessed by simple regression analyses. Results indicate predictability of AEA levels by AA accounting for 44% − 73% of total variance in the data. Specifically, for females AA predicted 44% of total variance in AEA data, F(1, 18) = 14.07, p = 0.001, which was carried by Tat(+) females only [61%, F(1, 8) = 12.34, p = 0.008]. For males, the AEA variance accounted for by AA was 46%, F(1, 16) = 13.34, p = 0.002, which was found to be significant for both genotypes [Tat(−) males: 73%, F(1, 7) = 18.79, p = 0.003; Tat(+) males: 64%, F(1, 7) = 12.22, p = 0.010].
Table 1
Pearson correlation matrix between AEA and AA levels within each of the four CNS regions separate for females and males. As 10 mg/kg THC administration had no effects on any of the assessed lipid molecules drug groups were combined.a
CNS region | Sex | Geno-type | Pearson Correlations within each CNS region: AEA vs. AA |
| | | r | p | n |
Prefrontal cortex | Female | Tat(−) | -0.224 | 0.534 | 10 |
Tat(+) | 0.779 | 0.008 | 10 |
(−) & (+) | 0.662 | 0.001 | 20 |
Male | Tat(−) | 0.854 | 0.003 | 9 |
Tat(+) | 0.797 | 0.010 | 9 |
(−) & (+) | 0.674 | 0.002 | 18 |
Striatum | Female | Tat(−) | 0.474 | 0.166 | 10 |
Tat(+) | 0.324 | 0.362 | 10 |
(−) & (+) | 0.427 | 0.060 | 20 |
Male | Tat(−) | 0.353 | 0.352 | 9 |
Tat(+) | 0.086 | 0.826 | 9 |
(−) & (+) | 0.336 | 0.172 | 18 |
Cerebellum | Female | Tat(−) | 0.634 | 0.049 | 10 |
Tat(+) | -0.431 | 0.213 | 10 |
(−) & (+) | 0.283 | 0.227 | 20 |
Male | Tat(−) | 0.154 | 0.692 | 9 |
Tat(+) | -0.179 | 0.644 | 9 |
(−) & (+) | -0.039 | 0.879 | 18 |
Spinal cord | Female | Tat(−) | -0.510 | 0.132 | 10 |
Tat(+) | -0.395 | 0.259 | 10 |
(−) & (+) | -0.504 | 0.023 | 20 |
Male | Tat(−) | 0.322 | 0.398 | 9 |
Tat(+) | 0.395 | 0.293 | 9 |
(−) & (+) | 0.431 | 0.074 | 18 |
a No effects or interactions were noted for acute THC administration on any measure and are thus not shown in this table. Bolded values denote significant differences at p ≤ 0.05. CNS, central nervous system; n, sample size. |
Next, we were interested in exploring the relationship between the two lipid molecules in the prefrontal cortex and the observed behavioral outcome in the novel object recognition task. Analyses were conducted separately for sex and genotype as both factors differentially affected object recognition memory and/or AEA and AA levels. As no effects were noted for males, only female correlation and simple regression data are shown (Fig. 5). Pearson correlations revealed a significant negative relationship between AEA levels and object recognition memory for Tat(+) females (exposed to vehicle and THC combined), with low AEA levels being associated with better object recognition memory, thus the preference to explore the novel object over the familiar object (Fig. 5A). A simple linear regression demonstrated predictability of object recognition memory (discrimination index) by AEA levels in the prefrontal cortex of Tat(+) females accounting for 44% of total variance in the data (F(1, 8) = 6.31, p = 0.036). No significant effect was noted for female Tat(−) mice (Fig. 5A). No significant effects were noted for AA levels and object recognition memory (Fig. 5B).
3.8. Expression levels of cannabinoid receptors and endocannabinoid degradative enzymes in the prefrontal cortex
To assess the impact of acute THC (0 and 10 mg/kg) exposure on cannabinoid receptors and cannabinoid catabolic enzymes, changes in protein expression levels of CB1R, CB2R, MAGL, and FAAH were assessed 60 min after injections in the prefrontal cortex of Tat transgenic female and male mice (n = 4–5 per group; Fig. 6). Data were normalized to the housekeeping protein GAPDH and fold-change was calculated using a control sample represented on all blots. Note that Pearson correlation analyses for object recognition memory and expression levels of CB1R, CB2R, MAGL, or FAAH revealed no significant relations (Supplemental Table S3). For CB1R protein expression (Fig. 6A), a three-way ANOVA including drug, sex, and genotype as between-subjects factors, demonstrated a significant main effect of genotype, F(1, 30) = 8.8, p = 0.006, with Tat induction increasing CB1R expression levels. Interestingly, the genotype effect was altered by sex with a significant sex x genotype interaction, F(1, 30) = 26.1, p < 0.001, with Tat(+) female mice demonstrating increased CB1R expression compared to Tat(−) females, F(1, 16) = 24.9, p < 0.001, whereas no effect was noted for males. The upregulation of CB1R expression in Tat(+) female mice compared to Tat(−) females was note din the absence and presence of THC exposure (F(1, 8) = 15.2, p = 0.005 and F(1, 8) = 10.2, p = 0.013, respectively).
For CB2R protein expression (Fig. 6B), a three-way ANOVA demonstrated a significant main effect of sex, F(1, 30) = 4.0, p = 0.054, with females showing higher CB2R expression levels compared to males. No other significant effects were noted.
For MAGL enzyme expression (Fig. 6C), a three-way ANOVA demonstrated a similar effect as noted for CB2R expression levels, with a significant main effect of sex, F(1, 30) = 4.9, p = 0.035, with females showing higher MAGL enzyme protein expression levels compared to males. No other significant effects were noted.
For FAAH enzyme expression (Fig. 6D), a three-way ANOVA demonstrated a significant main effect of sex, F(1, 30) = 9.8, p = 0.004, with females showing higher FAAH enzyme protein expression levels compared to males. Further, a significant main effect of genotype, F(1, 30) = 25.3, p < 0.001, with Tat induction increasing FAAH levels. This genotype effect was found in female mice, F(1, 16) = 11.2, p = 0.004, and male mice, F(1, 14) = 16.4, p = 0.001, specifically in the THC-treated groups (THC-exposed females: F(1, 8) = 7.9, p = 0.023; THC-exposed males: F(1, 8) = 14.1, p = 0.006) but not vehicle-exposed mice.
Overall, females showed higher expression levels in the prefrontal cortex for CB2Rs, MAGL, and FAAH compared to males. Further, Tat induction increased CB1R expression in Tat(+) female mice compared to Tat(−) females, as well as FAAH levels in THC-exposed Tat(+) females and THC-exposed Tat(+) male mice compared to their THC-exposed Tat(−) counterparts.
3.9. Plasma and cortex levels of THC and its metabolites
Levels of THC and its metabolites were assessed 60 min after acute THC (10 mg/kg) exposure using a different cohort of animals. Female and male Tat(−) mice (n = 7 per group) were subcutaneously injected with 10 mg/kg THC and sacrificed 1 h later, around the time when behavior was assessed (Fig. 7). THC levels and its metabolites, THC-COOH and THC-OH, were detected in all plasma (ng/mL) and cortex (ng/mg) samples. A two-way mixed ANOVA was conducted with sample as a within-subjects factor and sex as a between-subjects factor. For THC levels (Fig. 7A), a significant sample effect was noted, F(1, 12) = 18.33, p < 0.001, with higher concentrations in plasma compared to cortex samples. This effect was noted for females, F(1, 6) = 75.32, p < 0.001, and males, F(1, 6) = 11.84, p = 0.014. No sex effect or interaction was found. For THC-COOH levels (Fig. 7B), a significant sample effect was noted, F(1, 12) = 95.28, p < 0.001, with higher concentrations in plasma compared to cortex samples. This effect was found to be present in females, F(1, 6) = 67.77, p < 0.001, and males, F(1, 6) = 28.38, p = 0.002. Further, a significant main effect of sex was noted, F(1, 12) = 9.84, p = 0.009, that was altered by sample, sample x sex interaction: F(1, 12) = 9.91, p = 0.008, with higher THC-COOH levels found for female mice in plasma compared to males, F(1, 12) = 9.87, p = 0.009, but no differences was noted for cortex samples. For THC-OH levels (Fig. 7C), a significant sample effect was noted, F(1, 12) = 50.70, p < 0.001, with higher concentrations in plasma compared to cortex samples. This effect was noted for females, F(1, 6) = 16.89, p = 0.006, and males, F(1, 6) = 5.97, p = 0.050. No sex effect or interaction was found.
Overall, levels of THC and its metabolites were detected in plasma and cortex after 1 h acute 10 mg/kg THC administration and found to be higher concentrated in plasma compared to cortex samples. THC levels did not differ based on sex, but its metabolite THC-COOH demonstrated a selected sex effect in which females had higher THC-COOH plasma levels compared to male mice.