β-arrestin2 regulates cannabinoid CB1 receptor signaling and adaptation in a central nervous system region-dependent manner

Abstract

Background: Cannabinoid CB(1) receptors (CB(1)Rs) mediate the effects of ▵(9)-tetrahydrocannabinol (THC), the psychoactive component in marijuana. Repeated THC administration produces tolerance and dependence, which limit therapeutic development. Moreover, THC produces motor and psychoactive side effects. β-arrestin2 mediates receptor desensitization, internalization, and signaling, but its role in these CB(1)R effects and receptor regulation is unclear.

Methods: CB(1)R signaling and behaviors (antinociception, hypothermia, catalepsy) were assessed in β-arrestin2-knockout (βarr2-KO) and wild-type mice after THC administration. Cannabinoid-stimulated [(35)S]GTPγS and [(3)H]ligand autoradiography were assessed by statistical parametric mapping and region-of-interest analysis.

Results: β-arrestin2 deletion increased CB(1)R-mediated G-protein activity in subregions of the cortex but did not affect CB(1)R binding, in vehicle-treated mice. βarr2-KO mice exhibited enhanced acute THC-mediated antinociception and hypothermia, with no difference in catalepsy. After repeated THC administration, βarr2-KO mice showed reduced CB(1)R desensitization and/or downregulation in cerebellum, caudal periaqueductal gray, and spinal cord and attenuated tolerance to THC-mediated antinociception. In contrast, greater desensitization was found in hypothalamus, cortex, globus pallidus, and substantia nigra of βarr2-KO compared with wild-type mice. Enhanced tolerance to THC-induced catalepsy was observed in βarr2-KO mice.

Conclusions: β-arrestin2 regulation of CB(1)R signaling following acute and repeated THC administration was region-specific, and results suggest that multiple, overlapping mechanisms regulate CB(1)Rs. The observations that βarr2-KO mice display enhanced antinociceptive responses to acute THC and decreased tolerance to the antinociceptive effects of the drug, yet enhanced tolerance to catalepsy, suggest that development of cannabinoid drugs that minimize CB(1)R interactions with β-arrestin2 might produce improved cannabinoid analgesics with reduced motor suppression.

Figure 1

Cumulative THC dose-response curves in WT and βarr2-KO mice subchronically treated with either vehicle or THC (10 mg/kg twice daily, i.p.). A. Following vehicle treatment, βarr2-KO mice display enhanced responses to THC compared to WT mice in the tail-flick antinociceptive test (for interaction of dose and genotype: F_5,78 = 5.11, p = 0.0004, ***p<0.001 Bonferroni post-hoc analysis). Following THC pretreatment, βarr2-KO mice remain more responsive than WT mice (for genotype: F_1,78 = 12.31, p = 0.0008, *p<0.05 Bonferroni post-hoc analysis). Both genotypes display tolerance following THC pretreatment (vehicle vs. THC: WT: for interaction of dose and pretreatment: F_5,84 = 11.25, p <0.0001, ^{^}p<0.05, ^{^^^}p<0.001 Bonferroni post-hoc analysis. βarr2-KO: for pretreatment: F_1-72 = 22.78, p <0.0001, ^#p<0.05, ^##p<0.01 Bonferroni post-hoc analysis.) B. Following vehicle treatment, βarr2-KO mice display equivalent hypoactivity in response to THC compared to WT mice as assessed by the ring test for catalepsy (for genotype: F_1,78=2.53, p=0.1159). Both genotypes displayed tolerance following the THC pretreatment (vehicle vs. THC: WT: for interaction of dose and pretreatment: F_5-84=2.49, p=0.0376, ^{^^}p<0.01 Bonferroni post-hoc analysis. βarr2-KO: for interaction of dose and pretreatment: F_5-72= 4.91, p=0.0006, ^###p<0.001 Bonferroni post-hoc analysis). Following THC pretreatment, βarr2-KO mice become more tolerant to the hypolocomotor effects of THC than WT mice (for genotype: F_1,78 = 3.97, p=0.0497, ^#p<0.05 Bonferroni post-hoc analysis). C. Following vehicle treatment, βarr2-KO mice display greater hypothermia in response to THC compared to WT mice as determined by changes in rectal body temperature (for genotype: F_1,78=8.15, p=0.0055). Following THC pretreatment, WT and βarr2-KO mice did not differ in their response profiles (for genotype: F_1,78=0.09, p=0.9941). Both genotypes displayed tolerance following the THC pretreatment (vehicle vs. THC: WT: for interaction of dose and pretreatment: F_5-84=13.26, p<0.0001, ^{^^^}p<0.001 Bonferroni post-hoc analysis. βarr2-KO: for interaction of dose and pretreatment: F_5–72=6.94, p<0.0001, ^###p<0.001 Bonferroni post-hoc analysis). Data are presented as the mean ± S.E.M. (n = 8 WT, n = 7 βarr2-KO). The log values of the indicated doses are graphed on the abscissa and nonlinear regression curves are provided.

Figure 2

Agonist-stimulated [³⁵S]GTPγS concentration-effect curves using CP55,940 and CB₁R binding using [³H]SR141716A in WT and βarr2-KO mice subchronically treated with either vehicle or THC (10 mg/kg twice daily, i.p.). A. Desensitization of CB₁R-mediated G-protein activation was attenuated in spinal cords of βarr2-KO mice following subchronic treatment with THC. B. Subchronic administration of THC produced downregulation of CB₁Rs in spinal cords of WT animals, but was attenuated in βarr2-KO mice. EC₅₀ values for CP55,940- or WIN55,212-2-stimulated [³⁵S]GTPγS binding did not significantly differ between treatment groups.

Figure 3

Net CP55,940-stimulated [³⁵S]GTPγS binding reconstructions derived from the image average of all subjects for vehicle- and THC-treated mice of each genotype (n = 8). Original autoradiographic images are shown in grayscale and correspond to the scale (bottom right). AMYG, amygdala; A,V, auditory & visual cortex; CBLM, cerebellum; Cg, cingulate cortex; CPu, caudate-putamen; GP, globus pallidus; HIPP, hippocampus; HYPO, hypothalamus; PAG, periaqueductal gray; Pir, piriform cortex; SS, somatosensory cortex; SN, substantia nigra.

Figure 4

Genotype-specific differences in CP55,940-stimulated [³⁵S]GTPγS binding were distributed primarily in the cortex and caudal hippocampus. Statistical parametric maps (significance is shown in red to yellow color and corresponds to the significance scale bar) show regions where significantly greater CP55,940-stimulated [³⁵S]GTPγS binding was found in βarr2-KO compared to WT mice following vehicle treatment (n = 8 per group). Cg, cingulated cortex; CPu, caudate-putamen; GP, globus pallidus; Pir, piriform cortex; HIPP, hippocampus; SN, substantia nigra; PAG, periaqueductal gray; CBLM, cerebellum.

Figure 5

Net CP55,940-stimulated [³⁵S]GTPγS binding (mean ± SEM) in sampled brain regions (n = 8 per group) of WT and βarr2-KO mice following 6.5 day subchronic administration of either vehicle or 10 pmg/kg THC twice daily. ^*** p < 0.001, ^** p < 0.01, ^* p < 0.05 versus respective vehicle; ^### p < 0.001, ^# p < 0.05 versus WT vehicle; ^$ versus KO THC (Two way ANOVA, Student-Newman Keuls Post-hoc). PAG, periaqueductal gray; POA, preoptic area; SS, somatosensory cortex.

Figure 6

SPM analysis revealed both region- and genotype-specific differences in desensitization of CB₁R-mediated G-protein activation in the reconstructed mouse brain of WT and βarr2-KO mice following 6.5 day subchronic administration of either vehicle or 10 mg/kg THC (n = 8 per group) twice daily. Brain regions within each genotype demonstrating significant desensitization (p < 0.05, 2-way ANOVA, n = 8) are colored in blue/green and correspond to the significance scale (bottom). AMYG, amygdala; A,V, auditory & visual cortex; CBLM, cerebellum; Cg, cingulate cortex; CPu, caudate-putamen; GP, globus pallidus; HIPP, hippocampus; Hypo, hypothalamus; LEnt, lateral entorhinal cortex; PAG, periaqueductal gray; Pir, piriform cortex; cPAG, caudal periaqueductal gray; rPAG, rostral periaqueductal gray; SN, substantia nigra; SS, somatosensory cortex.

Figure 7

Relative efficacy of CP55,940-stimulated [³⁵S]GTPγS binding is positively correlated with the relative magnitude of CB₁R desensitization in βarr2-KO mice following subchronic THC administration. Each point in the graph represents a brain area (see Table 5) corresponding to its mean Desensitization Ratio and mean Relative efficacy. The Desensitization Ratio was calculated by normalizing the magnitudes of desensitization in βarr2-KO mice to the mean magnitude of desensitization in WT mice. Relative efficacy was calculated by normalizing the magnitudes of CP55,940-stimulated [³⁵S]GTPγS binding in vehicle treated βarr2-KO mice by the mean magnitude of CP55,940-stimulated [³⁵S]GTPγS binding in vehicle treated WT mice. Calculated ratios in the graph represent the mean ± SEM (n = 6–8). Linear regression analysis was performed in Graphpad Prism 5.