Enhanced Functional Activity of the Cannabinoid Type-1 Receptor Mediates Adolescent Behavior

Abstract

Adolescence is characterized by drastic behavioral adaptations and comprises a particularly vulnerable period for the emergence of various psychiatric disorders. Growing evidence reveals that the pathophysiology of these disorders might derive from aberrations of normal neurodevelopmental changes in the adolescent brain. Understanding the molecular underpinnings of adolescent behavior is therefore critical for understanding the origin of psychopathology, but the molecular mechanisms that trigger adolescent behavior are unknown. Here, we hypothesize that the cannabinoid type-1 receptor (CB1R) may play a critical role in mediating adolescent behavior because enhanced endocannabinoid (eCB) signaling has been suggested to occur transiently during adolescence. To study enhanced CB1R signaling, we introduced a missense mutation (F238L) into the rat Cnr1 gene that encodes for the CB1R. According to our hypothesis, rats with the F238L mutation (Cnr1(F238L)) should sustain features of adolescent behavior into adulthood. Gain of function of the mutated receptor was demonstrated by in silico modeling and was verified functionally in a series of biochemical and electrophysiological experiments. Mutant rats exhibit an adolescent-like phenotype during adulthood compared with wild-type littermates, with typical high risk/novelty seeking, increased peer interaction, enhanced impulsivity, and augmented reward sensitivity for drug and nondrug reward. Partial inhibition of CB1R activity in Cnr1(F238L) mutant rats normalized behavior and led to a wild-type phenotype. We conclude that the activity state and functionality of the CB1R is critical for mediating adolescent behavior. These findings implicate the eCB system as an important research target for the neuropathology of adolescent-onset mental health disorders.

Significance statement: We present the first rodent model with a gain-of-function mutation in the cannabinoid type-1 receptor (CB1R). Adult mutant rats exhibit an adolescent-like phenotype with typical high risk seeking, impulsivity, and augmented drug and nondrug reward sensitivity. Adolescence is a critical period for suboptimal behavioral choices and the emergence of neuropsychiatric disorders. Understanding the basis of these disorders therefore requires a comprehensive knowledge of how adolescent neurodevelopment triggers behavioral reactions. Our behavioral observations in adult mutant rats, together with reports on enhanced adolescent CB1R signaling, suggest a pivotal role for the CB1R in an adolescent brain as an important molecular mediator of adolescent behavior. These findings implicate the endocannabinoid system as a notable research target for adolescent-onset mental health disorders.

Keywords: CB1 receptor; ENU mutagenesis; adolescence; endocannabinoids; reward processing; risk seeking.

Figure 1.

Location of residue 4.46(238) in the CB1R (a, b) and molecular dynamics results for wild-type (WT) and mutant (MT) CB1R (c, d). In a, the transmembrane helix (TMH) bundle is shown in a side view looking from the lipid bilayer toward TMH2/3/4. Here, it is clear that 4.46(238) (green) is located on the lipid face of TMH4, one turn below the highly conserved class A G-protein-coupled receptor residue, W4.50 (orange). In b, the CB1R is shown in an extracellular view, with the location of residue 4.46(238) highlighted in green. Here, it is clear that residue 4.46(238) faces the TMH2–3-4 outside interface. c presents a plot of root mean square fluctuations (RMSF) for the transmembrane portion of rat WT CB1R(black) and the F238L mutant (red) versus residue number for the period before W6.48 undergoes the χ₁ g+ → trans conformational change (7.5–25 ns portion of MD trajectory). The green bars identify the residue spans for each TMH. The RMSF values for the F238L mutant (red) are larger than WT (black) in the following regions: the intracellular (IC) ends of TMH3/TMH4 (the IC-2 loop), the IC end of TMH6 (IC-3 loop), as well as the TM portion of TMH6 itself. Similar results were obtained for the second trajectory. This increase in RMSF is an indicator of increased flexibility of these regions in the MT receptor compared with WT. d presents a plot of RMSF for the transmembrane portion of rat WT CB1R (black) and the F238L MT (red) versus residue number for the period after W6.48 returns to a g+ χ₁ (75–300 ns portion of MD trajectory). The green bars identify the residue spans for each transmembrane helix. The EC ends of TMH4 and TMH5 (ends of EC-2 loop) of the MT undergoes the largest fluctuation, which is consistent with previous studies showing EC-2 loop movement during GPCR activation (Ahn et al., 2009; Ahuja et al., 2009; Bertalovitz et al., 2010). Similar results were obtained for the second trajectory.

Figure 2.

Functional characterization and expression of the mutated CB1R and other components of the eCB system in the striatum of Cnr1 mutant (MT) and WT rats. a, b, Cannabinoid-induced [³⁵S]GTPγS binding (a; interaction effect: F_(9,54) = 2.1, p = 0.048), as well as PPR (b; genotype effect: F_(1,69) = 13.6, p = 0.001) were significantly higher in MT animals compared with WT littermates. Representative traces for PPR: EPSC amplitudes are normalized to the peak of the first EPSC. In each example, there are three trials superposed (for three different interstimulus intervals). Scale bar, 20 ms. No differences could be observed for sEPSC amplitude (p = 0.27), but the interevent interval was enhanced (p = 0.034) in MT rats. Representative traces for sEPSCs are shown. There are 7 consecutive seconds for each condition (two rows per each). Scale bar, 10 pA; 500 ms (c). Effects of the CB1R antagonist/inverse agonist SR141716 (SR, 2 μm) on field EPSP (fEPSP) were significantly stronger (p = 0.01) in MT rats. Representative traces of field EPSPs recorded before (black) and after (gray) application of SR141716. Scale bar, 0.1 mV; 2 ms (d). CB1R expression did not differ between genotypes. No significant differences could be detected between MT animals and WT controls for protein levels (e) (Western blot analysis) (p = 0.50) and uptake of the CB1R ligand [¹⁸F]MK-9470 (assessed in vivo by PET analysis) (p = 0.95) (f). Protein levels of monoacylglycerol lipase (MAGL) were also unaltered (p = 0.73) (g), but FAAH levels were significantly lower in MT (p = 0.043) compared with WT rats (h). No significant differences between the genotypes could be found for expression of AEA (p = 0.62) (i) and 2-AG (p = 0.58) (j). All data are indicated as means ± SEM ([³⁵S]GTPγS: MT/WT n = 4; PPR: MT n = 12; WT n = 13; sEPSC: n = 12; SR on fEPSP: MT: n = 6, WT n = 5; Western blot CB1R: n = 5; PET analysis: n = 6; Western blot FAAH, MAGL: n = 5; AEA, 2-AG: MT: n = 9, WT n = 14; *p ≤ 0.05).

Figure 3.

Expression of CB1R and other components of the eCB system in the hippocampus. CB1R protein levels in the rat hippocampus did not differ between MT animals and WT controls (a) (p = 0.2). Protein levels of MAGL were also unaltered (b) (p = 0.8). As in the striatum, FAAH levels were significantly reduced in MT compared with WT rats (c) (p = 0.03). No significant differences between the genotypes could be found for expression of AEA (d) (p = 0.32) and 2-AG (e) (p = 0.85). Data are indicated as means ± SEM (Western blot analysis: CB1R and MAGL n = 5; FAAH: MT n = 5, WT n = 4; AEA, 2-AG: MT: n = 9, WT n = 14; *p ≤ 0.05).

Figure 4.

Novelty-seeking and risk-taking behavior of adult Cnr1 MT and WT animals. MT rats showed a significantly higher activity in the open-field test than WT when tested under high-anxiety conditions (unhabituated testing and a high light intensity of 150 lx; p = 0.026) (a). Only a trend for increased activity between the genotypes was observed when animals were retested in the open field under low anxiety conditions (b) (p = 0.062). MT rats were found to show increased risk-taking behavior in the EPM (% time: p = 0.002; percentage entries: p = 0.011) compared with WT (c), although the acitivity level did not differ between the genotypes (closed arm entries: p = 0.38). In the light/dark emergence test (d), MT rats showed a decreased emergence latency (p = 0.021) and spent more time in the lit compartment (p = 0.008) than WT controls. MT also showed higher novelty-seeking during exploration of a novel object (p = 0.005) (e) and novelty preference testing (p = 0.013) (f). Risk-based decision making was assessed in a PORT. First, the latency to collect a palatable food reward was assessed in various contexts with different degrees of familiarization (g). No genotype differences were found in the latency to collect the pellet within in the home cage (p = 0.98), whereas WT rats showed initially a significant higher latency in reward collection in the test apparatus (p = 0.048), which was not observable anymore after 5 d of habituation to the test environment (p = 0.41). MT and WT rats differed significantly during PORT testing (interaction effect: F_(2,32) = 7.72, p = 0.002) (h). Presentation of both predator odors increased reward-collection latency significantly in WT (mountain lion: p < 0.001; coyote: p < 0.001), whereas only the coyote odor affected response latency in MT rats (p < 0.001). Latency did not differ between the genotypes in the control condition (p = 0.57), but was significantly enhanced in WT compared with MT animals for both predator odors (mountain lion: p = 0.01; coyote: p = 0.013). Data are indicated as means ± SEM (open-field, EPM, EMT, novel object exploration, novelty preference: MT n = 13, WT n = 10; PORT: MT: n = 12, WT n = 6; *p ≤ 0.05).

Figure 5.

Reward-related behavior in Cnr1 MT and WT animals. Limited-access SCM intake was significantly increased in adult MT (p = 0.00002) (a), but not adolescent MT rats (p = 0.083), compared with age-matched WT animals, indicating a ceiling effect in adolescent rats (b). During PR responding, the highest completed ratio (p = 0.0001) and the inactivity ratio (p = 0.000002) were significantly enhanced in MT rats (c). Social play, a highly rewarding social activity most pronounced during mid-adolescence, was strongly enhanced in MT rats compared with WT controls (attacks received: p = 0.0005; attacks initiated: p = 0.0002; pinning: p = 0.014) (d). Morever, impulsive choice was increased in MT compared with WT animals during delay discounting testing (interaction effect: F_(5,100) = 4.9, p < 0.001) (e). MT rats were also found to show increased reward sensitivity toward the rewarding effects of cocaine. CPP for cocaine (p = 0.007) (f), cocaine-induced sensitization (genotype effect: F_(1,24) = 13.1, p = 0.003) (g) and the acute stimulatory effects of cocaine (h) were also increased in MT rats compared with WT controls (interaction effect: F_(2,34) = 5.3, p = 0.01). Data are indicated as means ± SEM (SCM intake, PR: MT: n = 13, WT n = 10; social play: MT: n = 11, WT n = 9; delay discounting: MT: n = 13, WT n = 9; cocaine CPP: MT: n = 6, WT n = 8; cocaine sensitization: MT: n = 6, WT n = 8; cocaine dose–response curve: MT: n = 7, WT n = 12; *p ≤ 0.05).

Figure 6.

Effects of the CB1R antagonist/inverse agonist SR141716 (SR) on palatable food intake in MT/WT animals (a) and adolescent/adult Fischer344 rats (b). A low dose of SR (0.3 mg/kg) was found to significantly attenuate intake of SCM in MT and adolescent Fischer rats to intake levels of WT or adult Fischer rats, respectively, whereas 0.3 mg/kg SR had no effect on SCM intake in WT animals and adult Fischer rats (WT/MT: interaction effect: F_(1,19) = 19.5, p < 0.001; adolescent/adult: interaction effect: F_(1,15) = 11.8, p = 0.004). Effects of SR or vehicle (VEH) on SCM intake were tested in a within-subject design. Data are indicated as means ± SEM (MT: SR/VEH n = 10, WT: SR/VEH n = 11; adolescent: SR/VEH n = 8, adult: SR/VEH = 9; *p ≤ 0.05).

Figure 7.

Expression and functionality of the adolescent CB1R and behavioral characterization of adolescent and adult Fischer 344 rats. Striatal CB1R protein levels were higher in adolescent compared with adult rats (p = 0.036) (a). No differences were found for PPR (age effect: F_(1,83) = 3.2, p = 0.08) Representative traces for PPR: EPSC amplitudes are normalized to the peak of the first EPSC. In each example, there are three trials superposed (for three different interstimulus intervals). Scale bar, 20 ms. (b). The CB1R antagonist/inverse agonist SR141716 (SR) was found to exert a stronger effect in adolescent compared with adult rats on fEPSPs (p = 0.033). Representative traces of fEPSPs recorded before (black) and after (gray) application of SR141716. Scale bar, 0.1 mV; 2 ms (c). The amplitude of sEPSCs did not differ between the two groups (p = 0.08), but, as in MT rats, the sEPSC interevent interval was significantly enhanced in adolescent rats (p = 0.012). Representative traces for sEPSC: There are 7 consecutive seconds for each condition (two rows per each). Scale bar, 10 pA; 500 ms (d). Adolescent animals were further screened for a selection of behavioral characteristics observed before in MT animals. Adolescent rats showed increased risk-taking in the EPM (e) compared with adult Fischer rats (EPM: percentage time: p = 0.009; percentage entries: p = 0.028). Exploration of a novel object (f) was also increased (p = 0.009). Adolescent Fischer rats showed an increase in reward-related behaviors compared with adult rats. Intake of a palatable food reward (g), as well as PR responding (h), was significantly higher in adolescents than adults (intake: p = 0.001; PR testing: p = 0.004). Social play behavior was also strongly increased in adolescent animals (i) compared with adult controls (attacks received, initiated and pinning: p < 0.001). Finally, the acute stimulatory effects of cocaine (j) were significantly enhanced in adolescence compared with adulthood (F_(2,40) = 3.3, p = 0.047). Data are indicated as means ± SEM (CB1R Western blot: n = 5; PPR: adolescent: n = 11, adult n = 12; sEPSC: adolescent: n = 11, adult n = 10; effects of SR on fEPSP: n = 6; EPM, novel object exploration, food reward intake, PR: n = 12; social play: n = 9; cocaine dose–response curve: adolescent: n = 12, adult n = 10).