Individual differences in frontolimbic circuitry and anxiety emerge with adolescent changes in endocannabinoid signaling across species

Phenotypic differences in frontolimbic circuitry resulting from FAAH polymorphism emerge during adolescence in human and mouse. (A, Left) Posthoc analyses revealed a significant genotypic effect on UF FA in participants 12 y of age and older [n = 509; 249 females; F(1,491) = 14.02; P = 0.0002] but not in those under 12 y of age [n = 541; 259 females; F(1,523) = 0.513; P = 0.474]. (A, Right) Mask in Montreal Neurological Institute standard space where UF ascends from the temporal lobe used as the seed region for probabilistic tractography in humans (upper left); UF tract mask is derived from probabilistic tractography averaged across human participants (n = 1,050). (B, Left) Consistent with the findings in humans, a significant genotype by age group interaction [F(2,36) = 58.72; P < 0.0001] in IL afferent fibers to BLA emerged, such that knock-in mice (AA: n = 7 per age) had higher fiber density than WT mice (CC: n = 7 per age) during adolescence at age P45 (P < 0.0001) and adulthood at P75 (P < 0.0001). (B, Right) Drawing of anatomical boundaries and anterograde tracer targeted to IL and labeling afferents in BLA. CeA, central amygdala; MeA, medial amygdala; PL, prelimbic; *P < 0.05.

FAAH genotype by age interaction for FA in the UF. FA of the UF showed a significant increase with age [F(1,1,031) = 305.985; P < 2.2 × 10¹⁶; plotted as a function of age in A allele carriers (n = 420; 208 females) and C homozygotes (n = 630; 310 females)]. A significant age by genotype interaction [age by genotype interaction: F(1,1,031) = 5.893; P = 0.0154] revealed that the main effect of genotype [F(1,1,031) = 17.858; P = 2.59 × 10⁻⁵] emerged during adolescence.

Trend for dose dependence association between FAAH genotype and frontolimbic circuitry emerging during adolescence. There was a trend toward a linear effect of number of A alleles on UF FA among participants 12 y of age and older [F(2,491) = 2.03; P = 0.154; AA: n = 41; 15 females; AC: n = 182; 90 females; CC: n = 286; 144 females]. However, there was no effect of number of A alleles on UF FA among participants under 12 y of age [F(2,523) = 1.92; P = 0.167; AA: n = 29; 11 females; AC: n = 168; 92 females; CC: n = 344; 166 females]. Data are presented as means ± SEMs.

No FAAH genotype by age group interaction for FA in the corticospinal tract. (A) Mask in Montreal Neurological Institute standard space of bilateral internal capsules to serve as the seed region for probabilistic tractography. (B) Corticospinal tract mask derived from probabilistic tractography averaged across human participants (n = 1,050). (C) There was a significant effect of age on FA in the corticospinal tract [F(1,1,031) = 531.616; P = 2.0 × 10⁻¹⁶]. However, there was no main effect of genotype [F(1,1,031) = 2.699; P = 0.101] or age by genotype interaction [F(1,1,031) = 0.009; P = 0.927], suggesting specificity of the developmental effects of the FAAH genotype on frontolimbic circuitry. Data are presented as means ± SEMs.

Phenotypic differences in anxiety resulting from FAAH polymorphism emerge during adolescence in human and mouse. (A) A significant gene by development interaction [F(1,198) = 6.2269; P = 0.0134] on anxiety revealed that A allele carriers (n = 54; 19 females) had lower anxiety than C homozygotes (n = 63; 30 females) during adolescence [F(1,102) = 8.96; P = 0.00346] but not during childhood [F(1,83) = 3.82; P = 0.054; A allele carriers: n = 32; 21 females; C homozygotes: n = 65; 28 females]. These findings parallel the adolescent emergence of stronger frontolimbic connectivity among A allele carriers. (B) Consistent with the finding in humans, a significant genotype by age group interaction [F(1,45) = 4.922; P = 0.0316] revealed that FAAH^A/A mice (P23: n = 9; P45: n = 9; P65–P75: n = 10) showed less anxiety-like behavior than FAAH^C/C mice (P23: n = 6; P45: n = 8; P65–P75: n = 7) during adolescence and adulthood, but not during childhood, as measured by percentage of time spent in the open arms of the EPM. *P < 0.05.

No FAAH genotype by age group interaction for total distance traveled in the EPM. Despite a genotype by age group interaction for percentage of time spent in the open arms, there was no genotype by age group interaction for total distance traveled in the EPM [F(1,45) = 2.59; P = 0.114]. Data are presented as means ± SEMs.

Developmentally informed vertically integrated translational approach to genetic variation and treatment targets across development. We have used developmentally informed parallel human and mouse studies to identify the relevance and impact of the FAAH C385A polymorphism on brain biochemistry, neurocircuitry, behavior, and symptoms (i.e., vertical integration across multiple levels of analysis) during development and identified a previously unidentified gene by development interaction.

PHA-L injections in IL cortex of mice. (A) Representative site of IL injection of PHA-L; PHA-L–like positive neurons and neuropil deposits are well-defined in IL, and PHA-L–like immunoreactive positive neurons (red) are counterstained with DAPI. This assessment of injection accuracy was carried out for all seven mice per genotype per age, and all of the injections were within the IL and did not overlap with neighboring regions, such as the prelimbic prefrontal cortex. (B) Bar graph showing numbers of PHA-L–positive cell bodies in IL in different genotypes and ages based on stereological estimation of cell density using the fractionator estimation method. There were no main effects of age [F(1,36) = 0.0273; P = 0.789] or genotype [F(2,36) = 0.453; P = 0.639] and no age by genotype interaction [F(2,36) = 0.309; P = 0.736]. Data are presented as means ± SEMs.