Fig. 5. Nanoscale visualization of in vivo cariprazine drug interaction sites by PharmacoSTORM.

a Schematic illustration of the generalizable workflow that enables PharmacoSTORM-based quantitative nanoscale analysis of target engagement sites of drugs administered in vivo. In the specific experiment, intraperitoneal injection of 1 mg/kg cariprazine was followed 2 h later by acute brain slice preparation and incubation with 30 nM Fluo-CAR. b Confocal images show that in vivo cariprazine pretreatment efficiently blocks Fluo-CAR binding in the hilus of the Islands of Calleja. c Fluo-CAR binding sites (yellow) are visualized by PharmacoSTORM imaging. Note the striking lack of Fluo-CAR-binding sites on TH-immunopositive nerve terminals. In unlabeled cariprazine-treated animals, the density of STORM LPs representing Fluo-CAR-binding sites was markedly reduced, indicating that Fluo-CAR binding sites are preoccupied with cariprazine administered in vivo. d Scatter dot plot shows PharmacoSTORM density (LPs/µm²) in the Islands of Calleja in cariprazine- or vehicle-treated animal pairs (n = 4). Data are presented as mean ± SEM. Each data point represents the average density of STORM images obtained in the Islands of Calleja from each animal. Statistical evaluation was performed by two-tailed Mann–Whitney U test (n = 4, P = 0.0286). e Statistical analysis of the nanoscale distribution of Fluo-CAR STORM LPs in relation to TH-immunopositive varicosities. The same analysis was applied as in Fig. 4d. The number of Fluo-CAR STORM LPs was significantly lower on TH-containing structures (n = 12 frames, n = 4 animals). Two-tailed Wilcoxon signed-rank test was performed to test if the median of data is different from the hypothetical median of 1 (P = 0.0057 for Fluo-CAR LPs, P = 0.5042 for random LP distribution).