Figure 3. Rapid, agonist-induced trafficking of D₁ receptors in striatal neurons.

(A) Representative, epifluorescence images of FD1R-expressing striatal neurons after 10 min. incubation with (bottom) or without (top) of 1μM SKF 81297. (B) The total pool of FD1Rs initially present at the cell surface were labeled with Alexa594-conjugated primary antibody (red). After agonist treatment, neurons were fixed under non-permeabilizing conditions and incubated with Alexa488-conjugated secondary antibody (green) to label receptors that remained at the cell surface or recycled during the incubation period. (B) Quantification of FD1R internalization by change in normalized (surface/total) fluorescence ratio as described in (A). The average surface to total (green/red) fluorescence ratio measured in non-treated cells was defined as 1. Data = mean fluorescence ratios +/-SEM, n = 3, 31-36 cells per condition. (C) Representative images of SpH-D1R surface fluorescence in striatal neurons as visualized by TIRF microscopy prior to (left, inset), and 120 sec. after SKF 81297 addition (right, inset). Arrows indicate clusters of SpH-D1R fluorescence that formed after SKF addition and disappeared shortly thereafter, arrowhead indicates a cluster of that formed after SKF addition and persisted throughout imaging. (D) Kymograph of SpH-D1R fluorescence for endocytic events indicated by arrows in (C). Kymograph depicts surface fluorescence intensity (maximum intensity determination) as a function of time. Arrow indicates bath application of 1μM SKF 81297. (E) Average surface fluorescence of SpH-D1R expressing striatal neurons measured every 3 sec. in the absence of (Bleaching Control) or in response to 1μM SKF 81297. Initial fluorescence values normalized to 100%, data = background-corrected, mean surface fluorescence +/− SEM, n = 5-9 cells/condition. Scale bars = 5μm. See also Figure S3.