FIGURE 5.

MOR recycling frequency is modulated by the presence of agonist. A, experimental design for MOR recycling experiments by TIR-FM. Cells are incubated with 10 μm DAMGO to induce MOR internalization and sequentially imaged by TIR-FM. Two 1-min periods are recorded for basal and wash to reduce variabilities. B, recycling frequency was measured before and after wash in HEK293 cells. Removal of DAMGO induced a significant decrease in recycling frequency to 53% compared with basal level (n = 11 cells). C, the number of receptors contained in the recycling vesicles did not change significantly between treatments (n = 64 insertions for basal and n = 66 for wash). D, MOR recycling frequency was also tested in striatal neurons. Agonist washout induced a marked reduction in the recycling frequency of MORs to 24% compared with basal level (n = 10 cells). E, the number of receptors concentrated in single recycling vesicles did not significantly change when comparing conditions (n = 69 and n = 59 insertion events for basal and wash, respectively). F, dissociated striatal cultures transfected with SEP-MOR were preincubated with 10 μm morphine. Recycling frequency was measured in the presence of or after removal of morphine. G, striatal cultures transfected with SEP-MOR were incubated with 10 μm morphine for 10 min, and recycling frequency was measured before and after the addition of 25 μm forskolin to the imaging medium in the presence of morphine.