Immunopositive signals of CB1 and VGluTs or VGAT. Representative double immunofluorescent staining of CB1 and VGluTs or VGAT is shown in Fig. 2D. We evaluated the colocalization of CB1 and the terminal markers by calculating the CC values in the CB1-positive varicosities. The CC values of CB1 and VGAT were significantly higher than those of CB1 and 
VGluTs in all cortical layersK162 web Effect of Dark Rearing on CB1 ExpressionTo explore the effect of visual inputs on the developmental regulation of CB1 expression, we examined CB1 expression in mice that were dark reared from birth to P30 or P50. The mice reared in 15900046 the dark from birth to P30 had a lesser quantity of CB1 protein than the normal mice reared under normal light/dark conditions. However, the 
mice that were dark reared until P50 had similar amounts of CB1 protein as the normal mice (Fig. 4A, B). In P30 animals, the pattern of layer distribution of CB1 was similar between the dark-reared and normal groups (Fig. 4C, D). To determine the effect of dark rearing on the synaptic localization of CB1, we compared the colocalization of CB1 and VGAT, VGluT1, or VGluT2 between the dark-reared and normal miceRegulation of CB1 Expression in Mouse VFigure 4. Effects of dark rearing on CB1 expression. (A) Representative western blots of CB1 and GAPDH in V1. The blots of normal light/dark condition-reared (NR) and dark-reared (DR) mice at P30 and P50 are shown. (B) Mean and SEM of the blot density of CB1 (P30: n = 16 (NR) and 21 (DR) animals, P50: n = 5 (NR) and 5 (DR) animals; unpaired t-test, **: p,0.01). (C) Layer distribution of CB1 immunoreactivity in V1. Photographs represent immunostained sections of NR and DR animals at P30. Layer boundaries were determined in neighboring Nissl-stained sections. Scale, 100 mm. (D) CB1 immunoreactivity in individual layers of NR and DR animals at P30. Intensities in each layer are represented as the proportion to the all-layer intensities (two-way ANOVA, p.0.05). (E) Double immunofluorescent staining of CB1 (magenta) and VGAT, VGluT1 in the deep layer of V1 of NR (upper) and DR (lower) animals at P30. Scale, 3 mm. (F) Box and whisker plots showing the CC values of CB1 and VGAT, VGluT1 in the deep layer of NR and DR animals at P30 (n = 3 animals each; NR animals: n = 531 ROIs (CB1/VGAT), 244 ROIs (CB1/VGluT1), DR animals: n 23727046 = 594 ROIs (CB1/VGAT), 343 ROIs (CB1/VGluT1), Mann-Whitney U test, *: p,0.05). doi:10.1371/journal.pone.0053082.gat P30. In the deep layer, the CC value of CB1 and VGAT was significantly higher in the dark-reared mice than that in the normal mice. In contrast, the CC value of CB1 and VGluT1 in the dark-reared mice was significantly lower than that of the normal mice (Fig. 4E, F). In the upper and LY-2409021 web middle layers, dark rearing did not affect the CC value of CB1 and VGluTs or VGAT.Effect of Monocular Deprivation on CB1 ExpressionWe examined the effect of monocular deprivation (MD) on CB1 expression and its time course in mice during the critical period of ocular dominance plasticity. MD for 2 days or 7 days did not affect the expression and the layer distribution of CB1 immunoreactivity (Fig. 5A ). However, in the deep layer of V1, which is contralateral to the deprived eye, the CC value of CB1 and VGAT in 2-day MD mice was significantly higher than that of the normal mice (Fig. 5E, F). On the other hand, the CC value of CB1 and VGluTs did not change significantly following 2 days or 7 days of MD. In the upper and middle layers, MD did not affect.Immunopositive signals of CB1 and VGluTs or VGAT. Representative double immunofluorescent staining of CB1 and VGluTs or VGAT is shown in Fig. 2D. We evaluated the colocalization of CB1 and the terminal markers by calculating the CC values in the CB1-positive varicosities. The CC values of CB1 and VGAT were significantly higher than those of CB1 and VGluTs in all cortical layersEffect of Dark Rearing on CB1 ExpressionTo explore the effect of visual inputs on the developmental regulation of CB1 expression, we examined CB1 expression in mice that were dark reared from birth to P30 or P50. The mice reared in 15900046 the dark from birth to P30 had a lesser quantity of CB1 protein than the normal mice reared under normal light/dark conditions. However, the mice that were dark reared until P50 had similar amounts of CB1 protein as the normal mice (Fig. 4A, B). In P30 animals, the pattern of layer distribution of CB1 was similar between the dark-reared and normal groups (Fig. 4C, D). To determine the effect of dark rearing on the synaptic localization of CB1, we compared the colocalization of CB1 and VGAT, VGluT1, or VGluT2 between the dark-reared and normal miceRegulation of CB1 Expression in Mouse VFigure 4. Effects of dark rearing on CB1 expression. (A) Representative western blots of CB1 and GAPDH in V1. The blots of normal light/dark condition-reared (NR) and dark-reared (DR) mice at P30 and P50 are shown. (B) Mean and SEM of the blot density of CB1 (P30: n = 16 (NR) and 21 (DR) animals, P50: n = 5 (NR) and 5 (DR) animals; unpaired t-test, **: p,0.01). (C) Layer distribution of CB1 immunoreactivity in V1. Photographs represent immunostained sections of NR and DR animals at P30. Layer boundaries were determined in neighboring Nissl-stained sections. Scale, 100 mm. (D) CB1 immunoreactivity in individual layers of NR and DR animals at P30. Intensities in each layer are represented as the proportion to the all-layer intensities (two-way ANOVA, p.0.05). (E) Double immunofluorescent staining of CB1 (magenta) and VGAT, VGluT1 in the deep layer of V1 of NR (upper) and DR (lower) animals at P30. Scale, 3 mm. (F) Box and whisker plots showing the CC values of CB1 and VGAT, VGluT1 in the deep layer of NR and DR animals at P30 (n = 3 animals each; NR animals: n = 531 ROIs (CB1/VGAT), 244 ROIs (CB1/VGluT1), DR animals: n 23727046 = 594 ROIs (CB1/VGAT), 343 ROIs (CB1/VGluT1), Mann-Whitney U test, *: p,0.05). doi:10.1371/journal.pone.0053082.gat P30. In the deep layer, the CC value of CB1 and VGAT was significantly higher in the dark-reared mice than that in the normal mice. In contrast, the CC value of CB1 and VGluT1 in the dark-reared mice was significantly lower than that of the normal mice (Fig. 4E, F). In the upper and middle layers, dark rearing did not affect the CC value of CB1 and VGluTs or VGAT.Effect of Monocular Deprivation on CB1 ExpressionWe examined the effect of monocular deprivation (MD) on CB1 expression and its time course in mice during the critical period of ocular dominance plasticity. MD for 2 days or 7 days did not affect the expression and the layer distribution of CB1 immunoreactivity (Fig. 5A ). However, in the deep layer of V1, which is contralateral to the deprived eye, the CC value of CB1 and VGAT in 2-day MD mice was significantly higher than that of the normal mice (Fig. 5E, F). On the other hand, the CC value of CB1 and VGluTs did not change significantly following 2 days or 7 days of MD. In the upper and middle layers, MD did not affect.