Activation-Dependent Subcellular Distribution Patterns of CB1 Cannabinoid Receptors in the Rat Forebrain

Chronic cannabinoid exposure results in tolerance due to region-specific desensitization and down-regulation of CB1 cannabinoidreceptors (CB1Rs). For most G-protein-coupled receptors, internaliz-ation closely follows rapid desensitization as an important com-ponent of long-term down-regulation. However, in vivo patterns ofCB1R internalization are not known. Here we investigate the subcel-lular redistribution of CB1Rs in the rat forebrain following activationby agonist CP55940 or inhibition by antagonist/inverse agonistAM251. At steadystate, CB1Rs are mainly localized to the cell mem-brane of preterminal axon shafts and, to a lesser degree, to synapticterminals. A high proportion of CB1Rs is also localized to somato-dendritic endosomes. Inhibition of basal activation by acute AM251administration decreases the number of cell bodies containingCB1R-immunoreactive endosomes, suggesting that CB1Rs are per-manently activated and internalized at steady state. On the contrary,acute agonist treatment induces rapid and important increase of en-dosomal CB1R immunolabeling, likely due to internalization and ret-rograde transport of axonal CB1Rs. Repeated agonist treatment isnecessary to significantly reduce initially high levels of axonal CB1Rlabeling, in addition to increasing somatodendritic endosomal CB1Rlabeling in cholecystokinin-positive interneurons. This redistributiondisplays important region-specific differences; it is most pronouncedin the neocortex and hippocampus and absent in basal ganglia.Keywords: axon, basal ganglia, endocytosis, redistribution, toleranceIntroductionRepeated administration of cannabinoids results in attenuatedresponsiveness, or tolerance, essentially through desensitiza-tion and downregulation of type-1 cannabinoid receptors(CB1Rs) (Hoffman et al. 2003, 2007; Sim-Selley, 2003;Howlettet al., 2004; Martin et al., 2004; Gonzalez et al. 2005). Gener-ally, G-protein-coupled receptors (GPCRs) display 3 overlap-ping processes in response to agonists over a time scaleranging from seconds to days: rapid desensitization (secondsto minutes), internalization (minutes to hours) and down-regulation (hours to days) (Ferguson 2001; Tsao et al. 2001).The highly conserved mechanism of rapid desensitization in-volves receptor phosphorylation followed by interaction withcytoplasmic arrestins, which leads to functional uncouplingfrom G-proteins. Next, phosphorylated and arrestin-bound re-ceptors are internalized from the cell membrane to endo-somes, without any notable change in the total number ofreceptors. Subsequently, internalized receptors are either de-phosphorylated and recycled to the cell membrane (resensiti-zation) or degraded in lysosomes. Finally, prolonged agonistexposure leads to a decrease in receptor protein levels,known as down-regulation, caused either by receptor degra-dation, decrease in receptor synthesis or both, ultimately re-sulting in fewer available ligand-binding sites.CB1R desensitization and long-term down-regulation varyin magnitude and time-course across different brain regions,displaying the highest degree of adaptation in the neocortexand hippocampus and the lowest in basal ganglia. Thesedifferences are well correlated with differences in the rate oftolerance development associated with each brain region(Sim-Selley 2003; Martin et al. 2004; Gonzalez et al. 2005).While it is likely that CB1R internalization, which is readilydetectable in cell culture models (Hsieh et al. 1999; Jin et al.1999; Coutts et al. 2001; Leterrier et al. 2004, 2006), closelyfollows rapid desensitization and is an important componentof long-term down-regulation, in vivo internalization patternof CB1R remains unknown. Moreover, because desensitiza-tion and down-regulation are greater in magnitude and moreregionally widespread among CB1Rs than other GPCRs, suchas μ opioid or 5-HT