Time-lapse confocal experiment showing SNC80 (100 nM; a selective agonist)-induced receptor internalization in primary caudate putamen neurons isolated from Oprd1-EGFP/EGFP knockin mouse. All agonists triggered the clustering of delta opioid receptor-EGFP (DOR-EGFP; green)(bright spots) along the plasma membrane both in cell bodies and processes. DOR-EGFP clusters then progressively internalized, producing a typical vesicular punctate pattern. After 20 min, the fluorescent spots finally converged into bigger vesicles.
Representative experiment is shown. Cells were seeded in glass-bottom, 32-mm diameter plastic dishes (MatTek) coated with polyL-lysine (Sigma). Fully matured primary neurons (10–14 days in vitro) were used, and receptor internalization studies were performed in the presence of various drugs. Samples were observed under a Leica confocal microscope (SP2 AOBS MP) with objective 63X at 37°C. Images were automatically recorded during 20 min, with increasing time intervals to avoid bleaching effects because of repetitive scanning. Specifically, 20 frames every 10 s followed by 10 frames every 30 s, and then 12 frames every minute were recorded. Reconstituted videos (TIMT; in-house software) contain 42 images and last 2 s.
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