Fig 1: ENZ modulates midbrain dopaminergic release to ventral hippocampus. Note. (A) Schematic of dopaminergic projections to ventral hippocampus (vHp) from VTA and consequent neuronal activation. Immunofluorescence of TH+-DA projections likely arising from VTA and projecting to vHp of ENZ-, AAP- and vehicle-treated mice (seen on magnification of squared areas). Scale bar: 100 μm. On the right, Whisker boxes represent the intensity of TH+ fibers and number of P-DARPP-32+ cells in the vHp (right panel) of ENZ- and AAP-treated mice compared with respective vehicles. Statistical quantifications were performed using Mann–Whitney test. Data are represented as box and whiskers of mean ±SEM (n = 4 mice), * p < 0.05. Scale bar: 100 μm. (B) Representative images of P-DARPP-32+—(green), DR1+—(cyan), TH+—(red) positive fibers and DAPI (blue) immunoreactivities in vHp of ENZ-, AAP- and Vehicle-treated mice. Scale bar: 25 μm and 100 μm. (C) Representative example of the immediate early gene (c-fos, green) and NeuN (red) immunoreactivities in the DG, CA3 and CA1 regions of vHp of ENZ-, AAP- and vehicle-treated mice. The boxed areas show DAPI (blue) immunoreactivity. On the left, whisker boxes represent the number of c-fos+ cells in DG, CA3 and CA1 hippocampal areas of ENZ- and AAP-treated mice compared with respective vehicles. Statistical quantifications were performed using Mann–Whitney test. Data are represented as box and whiskers and mean ± SEM (n = 4 mice), * p < 0.05, ** p ≤ 0.01. Scale bar: 100 μm. AAP, abiraterone acetate-prednisone; CA1, cornus ammonis 1; CA3, cornus ammonis 3; c-fos, cellular oncogene c-fos; DA, dopamine; DAPI, 4′,6-diamidino-2-phenylindol; DG, dentate gyrus; ENZ, enzalutamide; NeuN, Neuronal Nuclei Antigen; P-DARPP-32, Phosphorylated form of Dopamine cAMP-Regulated Neuronal Phosphoprotein; DR1, DA receptor 1; TH, Tyrosine hydroxylase; vHp, ventral hippocampus; VTA, ventral tegmental area.
Fig 2: Effects of non-UV irradiation on the cortical surface. (A) Representative photomicrographs of non-UV light-irradiated sections subjected to Nissl staining (at bregma −2.92 mm). Top: Magnification, x 8. Scale bar, 1000 µm. Red arrow, site of the optic cannula tip. (1), (2), and (3) indicate corresponding sites on the top and bottom panels. Bottom: Higher-magnification (x 40) photomicrographs of the same brain slice as that shown in the top panel. (1) Non-UV light-irradiated site. (2) Area adjacent to the non-UV light-irradiated site. (3) Area contralateral to the non-UV light-irradiated site. Scale bar, 200 µm. (B) Representative photomicrograph of a non-UV light-irradiated section subjected to HE staining. Magnification, x 200. Scale bar, 200 µm. Note that no clear cortical lesion was observed in Nissl or HE staining. (C) Representative photomicrographs of non-UV light-irradiated sections subjected to immunohistochemical staining. The left and right panels show staining for NeuN (brown, neuronal marker) and GFAP (brown, astrocyte marker), respectively, and counter-staining with haematoxylin (deep blue). Magnification, x 40. Scale bar, 200 µm. (D) Representative photomicrographs of sections subjected to immunohistochemical staining for Iba-1 (brown, microglial marker) and counter-staining with haematoxylin (deep blue). The left panel shows a non-UV light-irradiated section, and the right panel shows a UV-irradiated section. Magnification, x 200. Scale bar, 100 µm. Neuronal degeneration and glial congregation were hardly observed in the sections subjected to non-UV light irradiation.
Fig 3: The expression of various neuronal markers in the STN. The figure represents the expression of various markers in the adult human STN. (A) nNOS, (B) PAX6, (C) Nissl, (D) FOXP2, (E) NeuN, (F) NKX2.1, (G) parvalbumin, and (H) calretinin. Arrows point to positive neurons. Note that STN neurons have diverse cell body morphology. Bar—50 μm. STN, subthalamic nucleus.
Fig 4: Neuronal degeneration process after UV irradiation. Representative photomicrographs of (A) HE-stained and (B) NeuN-immunostained (brown, neuronal marker) sections at each time point (0 h, 2 h, 6 h, 12 h, 24 h, 3 d, and 5 d) after UV irradiation (at bregma −3.84 mm). NeuN-immunostained sections were counterstained with hematoxylin (blue). Scale bar, 200 μm. Black arrowhead, center of the UV-lesioned site. Gray bar, coverage area of the core of the optic cannula (400 μm). These results were replicated at least 3 times with different animals each time. (C) The number of NeuN-positive cells gradually decreased after the irradiation at the UV-irradiated side (blue solid bars) but not at the control side (gray stripe bar, contralateral of the UV-irradiated side). Cells were counted within two ROIs of 200-μm square each and the total area of the ROIs was 0.08 mm2 (see cell counting in Materials and Methods). Each time point included 3 animals, except for 3 d, which included 2 animals. All data are presented as mean ± SEM. *P < 0.05; **P < 0.01.
Fig 5: Layer-dependent neuronal degeneration after UV irradiation. (A) Heatmaps indicating the topological change of the number of NeuN-positive cells in control (left panel) and UV-irradiated (right panel) sides after UV irradiation. UV-induced neuronal degeneration started from the deep cortical layers within the UV-lesioned area. Cells were counted within an ROI of 200-μm width and 800- or 1000-μm depth located at the center of the UV-lesioned area (see cell counting in Materials and Methods). The total cell number was 3781 in the UV side, and 7176 in the control side from 20 animals. (B) Schematic illustration of absolute (top panels) and relative (bottom panels) location hypotheses. The brown areas indicate the assumed width of the superficial remaining neurons at 24 h after UV irradiation in each hypothesis. (C) Representative photomicrographs of NeuN-immunostained sections in small (created by 1.0 mWh UV irradiation, left panels), medium (created by 2.0 mWh UV irradiation, center panels), and large (created by 4.0 mWh UV irradiation, right panels) at time points of 24 h (top panels) and 3 d (bottom panels) after UV irradiation. This result was replicated 3 times with different animals each time. Scale bar, 200 μm. (D) The number of NeuN-positive cells in superficial and deep layers in the control side. The superficial layers (layer II/III) included more NeuN-positive cells than the deep layers (layers IV and V) in the control side. Top (layer II/III) and bottom (layers IV and V) ROIs were 200-μm square each (see cell counting in Materials and Methods). Cells were counted in 20 animals. Data are presented as mean ± SEM. **P < 0.01.
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