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. 2020 Feb 12;6(7):eaay1502.
doi: 10.1126/sciadv.aay1502. eCollection 2020 Feb.

Endocannabinoid Genetic Variation Enhances Vulnerability to THC Reward in Adolescent Female Mice

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Free PMC article

Endocannabinoid Genetic Variation Enhances Vulnerability to THC Reward in Adolescent Female Mice

Caitlin E Burgdorf et al. Sci Adv. .
Free PMC article

Abstract

Adolescence represents a developmental period with the highest risk for initiating cannabis use. Little is known about whether genetic variation in the endocannabinoid system alters mesolimbic reward circuitry to produce vulnerability to the rewarding properties of the exogenous cannabinoid Δ9-tetrahydrocannabinol (THC). Using a genetic knock-in mouse model (FAAHC/A) that biologically recapitulates the human polymorphism associated with problematic drug use, we find that in adolescent female mice, but not male mice, this FAAH polymorphism enhances the mesolimbic dopamine circuitry projecting from the ventral tegmental area (VTA) to the nucleus accumbens (NAc) and alters cannabinoid receptor 1 (CB1R) levels at inhibitory and excitatory terminals in the VTA. These developmental changes collectively increase vulnerability of adolescent female FAAHC/A mice to THC preference that persists into adulthood. Together, these findings suggest that this endocannabinoid genetic variant is a contributing factor for increased susceptibility to cannabis dependence in adolescent females.

Figures

Fig. 1
Fig. 1. Adolescent female mice, but not adolescent male mice, carrying the FAAH SNP demonstrate increased VTA-NAc connectivity and TH-labeled cells in the VTA.
(A) Schematic of PHA-L injected into the VTA of male and female FAAHC/C and FAAHC/A mice at P25. NAc and mPFC were analyzed for fiber density at P35. (B) Representative images of PHA-L projection labeling in the NAc of adolescent male and female FAAHC/C and FAAHC/A mice injected with the anterograde tracer, PHA-L, in the VTA. Scale bars, 10 μm. (C) Adolescent female FAAHC/A mice show increased density of PHA-L projection labeling in the NAc compared to adolescent female FAAHC/C mice, male FAAHC/C mice, and male FAAHC/A mice [one-way analysis of variance (ANOVA): F3,16 = 22.02, P < 0.0001, n = 5 per group, **P < 0.01, ****P < 0.0001 post hoc Bonferroni tests]. (D) Representative images of PHA-L projection labeling in the mPFC of adolescent male and female FAAHC/C mice and FAAHC/A mice injected with the anterograde tracer, PHA-L, in the VTA. Scale bars, 10 μm. (E) Adolescent male and female FAAHC/C and FAAHC/A mice show similar density of PHA-L projection labeling in the mPFC (one-way ANOVA: F3,16 = 0.2351, P = 0.8706, n = 5 per group). (F) Representative images of the VTA showing cells positive for TH immunoreactivity. (G) Adolescent female FAAHC/A mice show increased density of TH-labeled neurons compared to male FAAHC/C mice, male FAAHC/A mice, and female FAAHC/C mice (one-way ANOVA: F3,20 = 14.51, P < 0.0001, n = 6 per group, ***P < 0.001 post hoc test, ****P < 0.0001 Bonferroni test).
Fig. 2
Fig. 2. Adolescent female mice carrying the FAAH SNP demonstrate increased CB1R-labeled terminals forming symmetric synapses and decreased CB1R-labeled terminals forming asymmetric synapses in the paranigral subregion of the VTA.
(A) Adolescent female FAAHC/A mice (A″) have more CB1R-labeled terminals forming symmetric synapses compared to adolescent female FAAHC/C mice (A′) (unpaired t test, t4 = 2.819, P = 0.0479, N = 3 animals, n = 50 to 87 labels characterized per animal; FAAHC/A: N = 3 animals, n = 39 to 76 labels characterized per animal). (B) Adolescent female FAAHC/A mice (B″) have less CB1R-labeled terminals forming asymmetric synapses compared to adolescent female FAAHC/C mice (B′) (unpaired t test, t4 = 2.819, P = 0.0479; FAAHC/C: N = 3 animals, n = 50 to 87 labels characterized per animal; FAAHC/A: N = 3 animals, n = 39 to 76 labels characterized per animal). (C) Adolescent female FAAHC/A mice have a higher density ratio of membrane-bound CB1R on terminals forming symmetric synapses versus asymmetric synapses compared to adolescent female FAAHC/C mice (unpaired t test, t4 = 6.218, P = 0.0034; FAAHC/C: N = 3 animals, n = 50 to 87 labels characterized per animal; FAAHC/A: N = 3 animals, n = 39 to 76 labels characterized per animal). (D) Schematic of cell-specific CB1R action in the VTA. (E) Representative images of c-Fos immunoreactivity in the NAc of mice euthanized 90 min following acute THC. Scale bars, 120 μm. (F) Adolescent female FAAHC/A mice show increased c-Fos labeling in the NAc compared to adolescent female FAAHC/C mice (unpaired t test, t8 = 7.985, ***P < 0.001, n = 5 per group).
Fig. 3
Fig. 3. Female mice with the FAAH SNP do not display THC-induced hypolocomotion.
(A) Experimental protocol of locomotion testing in FAAHC/C and FAAHC/A mice that consists of 30 min of habituation in the locomotor activity box before an acute systemic vehicle or THC injection. Locomotion data are recorded 10 min after injection for 50 min. (B and C) Adolescent FAAHC/C mice show a decrease in binned locomotion (B) (two-way ANOVA, main effect of time: F4,55 = 3.323, P = 0.0165; main effect of treatment: F1,55 = 26.15, P < 0.0001, *P < 0.05, Bonferroni post hoc test) and cumulative locomotion (C) (unpaired t test, t11 = 3.761, **P = 0.0031, n = 7 vehicle and n = 6 THC) following a systemic THC injection compared to mice receiving a systemic vehicle injection. (D and E) Adolescent FAAHC/A mice show a nonsignificant increase in locomotion in first 10 min of binned data (D) (two-way ANOVA, significant interaction, time × treatment: F4,65 = 4.904, P = 0.0016; main effect of time: F4,65 = 10.82, P < 0.0001; main effect of treatment: F1,65 = 8.138, P = 0.0058) but no change in cumulative locomotion (E) (unpaired t test, t13 = 0.4037, P = 0.6930, n = 9 vehicle and n = 6 THC) following a systemic THC injection compared to mice receiving a systemic vehicle injection.
Fig. 4
Fig. 4. Adolescent female mice carrying the FAAH SNP display a preference for THC.
(A) Experimental and developmental timeline for THC CPP protocol in adolescent and adult mice. (B) Adolescent female FAAHC/C mice show significantly lower preference for the THC-paired chamber on the test day compared to the baseline test (paired t test, t15 = 2.782, *P < 0.05, n = 16), while adolescent female FAAHC/A mice show significantly higher preference for the THC-paired chamber on the test day compared to the baseline test (paired t test, t13 = 4.183, **P < 0.01, n = 14). (C) Adolescent female FAAHC/A mice show a significantly higher difference score than adolescent female FAAHC/C mice (unpaired t test, t28 = 4.82, ***P < 0.001, FAAHC/C: n = 16; FAAHC/A: n = 14). (D) Adult female FAAHC/C mice and adult female FAAHC/A mice show no change in preference for the THC-paired chamber on the test day compared to the baseline test (FAAHC/C: paired t test, t14 = 0.9102, P = 0.3781, n = 15; FAAHC/A: paired t test, t19 = 1.316, P = 0.2037, n = 20). (E) Adult female FAAHC/A mice show similar difference scores compared to adult female FAAHC/C mice (unpaired t test, t33 = 1.55, P = 0.1306, FAAHC/C: n = 15; FAAHC/A: n = 20).
Fig. 5
Fig. 5. Preference for THC as a result of the FAAH SNP persists into adulthood when trained in adolescence.
(A) Experimental timeline for persistence of THC CPP protocol. Mice were trained in THC CPP during adolescence and tested in adulthood. (B) Female FAAHC/A mice, but not female FAAHC/C mice, that had been trained in THC CPP during adolescence and then tested in THC CPP during adulthood continue to show a preference for THC, as shown by a significant increase in preference score on test day compared to baseline day (FAAHC/C mice: paired t test, t10 = 0.76, P = 0.4648, n = 11; FAAHC/A mice: paired t test, t9 = 3.164, *P < 0.05, n = 10). (C) Female FAAHC/A mice showed a trending increase in difference score compared to female FAAHC/C mice when trained in THC CPP during adolescence and retested in adulthood (unpaired t test, t19 = 1.958, P = 0.0650, n = 11 FAAHC/C and n = 11 FAAHC/A). (D) Experimental timeline for home cage exposure of THC. Mice were treated during adolescence and trained and tested in THC CPP later in adulthood. (E) Female FAAHC/C mice, when exposed to THC in their home cage during adolescence, develop an aversion for THC in adulthood (paired t test, t8 = 2.438, P = 0.0407, n = 9). In contrast, female FAAHC/A mice do not show a preference for THC as demonstrated by a lack of change in preference scores on adult test day compared to baseline day (paired t test, t8 = 0.5965, P = 0.5673, n = 9). (F) Female FAAHC/C and FAAHC/A mice show similar difference scores when exposed to home cage THC during adolescence and trained and tested in THC CPP during adulthood (unpaired t test, t16 = 0.6575, P = 0.5202, n = 9 FAAHC/C and n = 9 FAAHC/A).
Fig. 6
Fig. 6. Activity of the VTA-NAc pathway is required for persistence of THC CPP in female mice carrying the FAAH SNP.
(A) Experimental timeline for surgery, chemogenetic manipulation, and THC CPP. FAAHC/A mice underwent THC CPP training during adolescence, followed by injection of mCherry-tagged Cre-dependent AAV expressing the inhibitory DREADD hM4Di (AAV2-hSyn-DIO-hM4Di-Gi-mCherry) into bilateral VTA and a retrograde CAV2 expressing Cre recombinase into bilateral NAc. Control mice either underwent sham surgery without viral infusions or did not receive surgery. All mice were tested in THC CPP in adulthood following a CNO injection. (B) Representative image of injection site in VTA with mCherry-tagged virus-labeled cells. (C) Control FAAHC/A mice that received CNO demonstrated increased preference for the THC-paired side, as demonstrated by a significantly increased preference score on adult test day compared to baseline day (paired t test, t8 = 3.147, *P = 0.0137, n = 9). FAAHC/A mice injected with hM4Di DREADD demonstrated no THC preference, as shown by a similar preference score on adult test day compared to baseline (paired t test, t7 = 0.3208, P = 0.7577, n = 8). (D) FAAHC/A mice injected with hM4Di DREADD that received CNO demonstrated a significantly lower difference score than control FAAHC/A mice that received CNO (unpaired t test, t15 = 2.189, *P < 0.05, n = 9 CNO control and n = 8 hM4Di DREADD).

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