Fig 2: DAT nanodomain localization is regulated by the D2R and involves changes in lateral mobility(A–F) Dispersal of DAT nanodomains is promoted by the D2R antagonist haloperidol and blocked by the agonist quinpirole and by the N-type VGCC blocker ω-conotoxin. Shown are example varicosities from dSTORM of DA neurons subjected to 5-min treatment with haloperidol (10 nM), haloperidol (10 nM) plus quinpirole (50 μM), haloperidol (10 nM) plus ω-conotoxin (1 μM), or haloperidol (10 nM) plus ω-agatoxin (1 μM).(A) DAT clusters with a diameter greater than 75 nm are shown in green and clusters smaller than 75 nm in magenta.(B) Dense DAT clusters are shown in red (>80 localizations; radius, 50 nm).(C) Localizations segmented by Voronoi tessellation.(D and E) Normalized fraction of DAT clusters greater than 75 nm (percent) (D) and normalized fraction of DAT localizations in dense clusters (percent) (E). Means ± S.E.; one-way ANOVA, ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.(F) PDFs (curves are color coded as in the bar diagrams) for the Voronoi tessellated areas averaged by varicosity. Data are from 3 cultures and 153 control, 166 haloperidol, 167 quinpirole, 178 ω-conotoxin, and 176 ω-agatoxin varicosities.(G–R) Single-particle tracking dSTORM (SPT-dSTORM) identifies changes in DAT mobility in response to NMDA and D2R antagonism.(G) Example live dSTORM images of DA neurons labeled with the fluorescent cocaine analog DG3–80.(H) Boxed region in (B).(I and J) Detected tracks (I) and distribution of segment lengths (J) in a varicosity from the control SPT-dSTORM experiment (16-ms frame rate).(K) Example traces of a confined and an unconstrained freely moving particle (DG3–80-labeled DAT).(L) Fraction of unconstrained tracks of the total number of tracks. Data are means of 23 tracks ± S.E.(M) PDF for total mean jump distances (MJD) (i.e., the distance an individual molecule/particle has traveled between each image) for control experiments.(N) PDF when separated into static and freely moving particles. Among the freely moving particles, some moved very fast with MJDs of 200–350 nm.(O) NMDA (20 μM, 5 min) increases the population of DAT molecules with MJDs greater than 200 nm, which is blunted by AP5 (100 μM).(P) Fraction of fast-moving particles (>200 nm). Means ± S.E.; data from 13 control, 4 NMDA, and 5 NMDA plus AP5 experiments.(Q) The D2R antagonists haloperidol (10 nM, 5 min) and UH-232 (1 μM) increase the population of DAT molecules with MJDs greater than 200 nm.(R) fraction of fast-moving particles (>200 nm), Means ± S.E.; data are from 10 control, 6 haloperidol, and 5 UH-232 experiments; one-way ANOVA, *p < 0.05; **p < 0.01.See also Figure S5, Videos S1 and S3.