Agonist-induced internalisation of G-protein-coupled receptors is an important mechanism underlying receptor desensitisation. Recently, agonist-induced internalisation of cannabinoid CB1 receptors has been demonstrated in cultured hippocampal neurons, where the rate of down-regulation was unusually slow (5Ð16 h for maximal loss of labelling; Coutts et al. 2001). Here CB1 receptors are expressed on a subset of inhibitory synaptic terminals; however, in cultures derived from the cerebellum, they are primarily associated with excitatory synaptic terminals (Irving et al. 2002). In the present study, we have used an antibody directed against the N-terminal 77 amino acid residues of the cloned rat CB1 receptor (Coutts et al. 2001) to investigate the down-regulation of surface CB1 receptors in rat cultured cerebellar granule cells.
For cell surface labelling, cerebellar cultures were incubated with rabbit CB1 receptor antibody for 60 min (room temperature) in a standard Hepes-buffered saline; cells were then fixed with 4 % paraformaldehyde for 10 min. The CB1 receptor immunostaining was then visualised by addition of Cy3-conjugated secondary antibody. A laser-scanning confocal imaging system (Zeiss LSM 510) was used for image acquisition and processing. A minimum of nine randomly selected areas (1000 mm2), from three experiments were used to assess total labelling (mean intensity X area of immunoreactivity). Donor animals were humanely killed.
Cell surface CB1 receptor labelling was highly punctate, with clusters decorating a network of fine neurites. Prolonged exposure (18 h, 37 °C) to the cannabinoid receptor agonist WIN55212-2 (1 mM) produced a dramatic decrease in cell surface CB1 receptor labelling (12 ± 5 % control; mean ± S.E.M.; P < 0.01; ANOVA), which was reversed by the selective CB1 receptor antagonist SR141617A (400 nM; to 63 ± 5 %). The time taken for CB1 receptor labelling to decrease by 50 % in the presence of agonist was 0.49 ± 0.12 h (n = 6). Pretreatment of the cerebellar neurons with the endocannabinoid uptake inhibitor AM404 (10 mM) also caused a marked decrease in cell surface labelling (14 ± 2% P < 0.01) and this effect was partially reversed by SR141716A (400 nM; to 57 ± 11 %), demonstrating the presence of endogenous cannabinoids in the cerebellar cultures. These findings also suggest that the rate of agonist-induced down-regulation of neuronal CB1 receptors can vary quite profoundly between different preparations. Ultimately, this could be an important factor in the development of tolerance towards cannabinoids within the brain.
This work was supported by the MRC.
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