Fig 2: ENZ modulates midbrain dopaminergic release to striatum. Note. (A) Schematic representation of SNpC dopaminergic projections to striatum and consequent striatal neuronal activation. Immunofluorescence of TH+-DA striatal projections of ENZ-, AAP- and vehicle-treated mice (seen on magnification of squared areas). Scale bar: 100 µm. On the right, Whisker boxes represent TH+ fibers intensity and the number of P-DARPP-32+ cells in the striatum 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 Dopamine cAMP-Regulated Neuronal Phosphoprotein P-DARPP-32+—(green), DA receptor 1+—(DR1, cyan), TH+—(red) positive fibers and DAPI (blue) immunoreactivities in striatum of ENZ-, AAP- and Vehicle-treated mice. Scale bar: 25 µm and 100 µm. AAP, abiraterone acetate-prednisone; DA, dopamine; DAPI, 4',6-diamidino-2-phenylindol; ENZ, enzalutamide; P-DARPP-32, Phosphorylated form of Dopamine cAMP-Regulated Neuronal Phosphoprotein; DR1, DA receptor 1; SNpc, substantia nigra pars compacta; TH, Tyrosine hydroxylase; vHp, ventral hippocampus.

Fig 3: 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.