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. 2021 Nov 11;12:6505. doi: 10.1038/s41467-021-26757-z

Fig. 3. Multiscale anatomical mapping of fluo-cariprazine-binding sites in the brain.

Fig. 3

a Low-magnification epifluorescence image of a mouse brain coronal section after incubation with 300 nM Fluo-CAR (yellow) and nuclear staining with DAPI (blue). The inset highlights the intense Fluo-CAR labeling in the Islands of Calleja. b Although the high-density cell masses of the Islands of Calleja appear distinctly segregated on coronal sections, specific areas that are rich in Fluo-CAR labeling interconnect them. These Fluo-CAR-positive areas represent the hilus of the Islands of Calleja. c–e 3D morphological analysis of the gross anatomical architecture of the Islands of Calleja based on prominent Fluo-CAR labeling. Precise reconstruction of high-density binding sites (c) demonstrates that the hilus represents a large, continuous area. Ventral (d) and lateral (e) views of the reconstruction (yellow), integrated into a 3D mouse brain atlas (dark gray: nucleus accumbens (NAc), light gray: caudoputamen (CP)). White capital letters mark anatomical directions (R rostral, C caudal, V ventral, D dorsal). f, g Representative images of Fluo-CAR labeling in the Islands of Calleja from Drd3+/+ (f) and Drd3−/− (g) animals. Acute slices were treated with 300 nM Fluo-CAR. Fluo-CAR binding is dramatically decreased throughout the entire brain structure in the absence of D3Rs. h Demonstration of the feasibility of PharmacoSTORM super-resolution imaging in brain tissue preparations. Images were taken in the hilus of the Islands of Calleja of Drd3+/+ and Drd3−/− animals after 300 nM Fluo-CAR treatment. i Two-tailed Mann–Whitney U test was performed (P = 0.0357) to compare Fluo-CAR binding site density (STORM LPs/µm2) in the hilus of Drd3+/+ (n = 5) and Drd3−/− mice (n = 3). j Lateral (x-y) localization precision of Fluo-CAR binding sites based on the analysis of STORM LP blinking distribution. The median lateral localization precision was 9.42 nm (n = 98,260 LPs). k Quantitative analysis of the nanoscale distribution of Fluo-CAR-binding sites within the hilus. Nearest-neighbor distance measurements were performed between Fluo-CAR STORM LPs and between the equal numbers of randomly distributed STORM LPs. Cumulative distribution functions were statistically compared with Kolmogorov–Smirnov test (P < 0.001) and revealed non-random tissue distribution.