L immersion objective lens (NA = 1.4, HCX PL APO, Leica Microsystems) and stored in 8-bit TIFF file format (2,04862,048 pixels; pixel size, 116.25 nm). The focus was set at a depth of 1? mm from the surface of sections. The pinhole size was set at 1.0 Airy unit, and scanning was averaged 8 times. For Alexa 488-labeled samples, the samples were excited by a 488 nm Ar laser, and the beam splitter was set to 505?30 nm. For Alexa 568-labeled samples, the samples were excited by a 543 nm He/Ne laser, and the beam splitter was set to 580?25 nm. The laser power and the gain of the photomultiplier were set to exclude pixels with 0 or 255 498-02-2 site intensity in the image. In the figures, the contrast of the images was adjusted for clearer demonstration. The colocalization of immunofluorescent signals between CB1 and each of synaptophysin, VGAT, VGluT1, and VGluT2 was evaluated by calculating Pearson’s correlation coefficient (CC).Regulation of CB1 Expression in Mouse VFigure 2. Synaptic localization of CB1 in V1. (A) Double immunofluorescent staining of CB1 (magenta) and MAP2 (green) in the upper layer of V1. CB1-positive 307538-42-7 varicosities presumably contact MAP2-positive dendrites (white arrowheads) and soma (asterisk, yellow arrowheads). Scale, 3 mm. (B) Double immunofluorescent staining of CB1 (magenta) and synaptophysin (green) in the upper layer of V1. Rectangles indicate the ROIs for the correlation coefficient (CC) analysis set on varicosities (orange) and shafts (blue) of CB1-positive structures. Scale, 1 mm. (C) Box and whisker plots showing the CC values of CB1 and synaptophysin in varicosities (var, n = 154 ROIs) and shafts (shaft, n = 140 ROIs). The horizontal lines show the 25th, 50th, and 75th percentiles, and the whiskers show the max and minimum values. Mann-Whitney U test, **: p,0.01. (D) Double immunofluorescent staining of CB1 (magenta) and VGAT, VGluT1, VGluT2 (green). Representative photographs of the upper layer (top row), middle layer (middle row), and deep layer (bottom row) of V1. Scale, 3 
mm. (E) Box and whisker plots showing the CC values of CB1 and VGAT, VGluT1, or VGluT2 in each layer of V1 (n = 6 animals each; in the upper layer, n = 1226 ROIs (CB1/VGAT), 1203 ROIs (CB1/VGluT1), 1212 ROIs (CB1/VGluT2); in the middle layer, n = 492 ROIs (CB1/VGAT), 435 ROIs (CB1/VGluT1), 498 ROIs (CB1/VGluT2); 23727046 in the deep layer, n = 1556 ROIs (CB1/VGAT), 1712 ROIs (CB1/VGluT1), 1492 ROIs (CB1/VGluT2)). The small circles indicate the outliers of the distribution of the CC values. In the box and whisker plots containing the outliers, the bottom of the whisker shows the value of the 25th percentile-1.5IQR. Statistical comparison among layers was performed by Bonferronicorrected Mann-Whitney U test (***: p,0.00033). doi:10.1371/journal.pone.0053082.gEach image was smoothed over 363 pixels to remove high frequency noise on the image. We manually set the ROIs (969 pixels, approximately 1 mm2) at varicosity-like structures and shaft structures in CB1 images. The shaft structure of CB1 was defined as the structure that contains thin fibers with low signal intensity and the varicosity-like structure was defined as the structure that has a large immunopositive area with high signal 
intensity connected by thin fibers. CC value was calculated as follows: ? ?i 1 Xi{X Yi{Y CC Pn ?? ?? Yi{Y i 1 Xi{X Pn where Xi and Yi indicate the individual pixel intensities of CB1 and each of synaptophysin, VGAT, VGluT1, VGluT2 in a ROI,respectively. X and Y indicate the mean.L immersion objective lens (NA = 1.4, HCX PL APO, Leica Microsystems) and stored in 8-bit TIFF file format (2,04862,048 pixels; pixel size, 116.25 nm). The focus was set at a depth of 1? mm from the surface of sections. The pinhole size was set at 1.0 Airy unit, and scanning was averaged 8 times. For Alexa 488-labeled samples, the samples were excited by a 488 nm Ar laser, and the beam splitter was set to 505?30 nm. For Alexa 568-labeled samples, the samples were excited by a 543 nm He/Ne laser, and the beam splitter was set to 580?25 nm. The laser power and the gain of the photomultiplier were set to exclude pixels with 0 or 255 intensity in the image. In the figures, the contrast of the images was adjusted for clearer demonstration. The colocalization of immunofluorescent signals between CB1 and each of synaptophysin, VGAT, VGluT1, and VGluT2 was evaluated by calculating Pearson’s correlation coefficient (CC).Regulation of CB1 Expression in Mouse VFigure 2. Synaptic localization of CB1 in V1. (A) Double immunofluorescent staining of CB1 (magenta) and MAP2 (green) in the upper layer of V1. CB1-positive varicosities presumably contact MAP2-positive dendrites (white arrowheads) and soma (asterisk, yellow arrowheads). Scale, 3 mm. (B) Double immunofluorescent staining of CB1 (magenta) and synaptophysin (green) in the upper layer of V1. Rectangles indicate the ROIs for the correlation coefficient (CC) analysis set on varicosities (orange) and shafts (blue) of CB1-positive structures. Scale, 1 mm. (C) Box and whisker plots showing the CC values of CB1 and synaptophysin in varicosities (var, n = 154 ROIs) and shafts (shaft, n = 140 ROIs). The horizontal lines show the 25th, 50th, and 75th percentiles, and the whiskers show the max and minimum values. Mann-Whitney U test, **: p,0.01. (D) Double immunofluorescent staining of CB1 (magenta) and VGAT, VGluT1, VGluT2 (green). Representative photographs of the upper layer (top row), middle layer (middle row), and deep layer (bottom row) of V1. Scale, 3 mm. (E) Box and whisker plots showing the CC values of CB1 and VGAT, VGluT1, or VGluT2 in each layer of V1 (n = 6 animals each; in the upper layer, n = 1226 ROIs (CB1/VGAT), 1203 ROIs (CB1/VGluT1), 1212 ROIs (CB1/VGluT2); in the middle layer, n = 492 ROIs (CB1/VGAT), 435 ROIs (CB1/VGluT1), 498 ROIs (CB1/VGluT2); 23727046 in the deep layer, n = 1556 ROIs (CB1/VGAT), 1712 ROIs (CB1/VGluT1), 1492 ROIs (CB1/VGluT2)). The small circles indicate the outliers of the distribution of the CC values. In the box and whisker plots containing the outliers, the bottom of the whisker shows the value of the 25th percentile-1.5IQR. Statistical comparison among layers was performed by Bonferronicorrected Mann-Whitney U test (***: p,0.00033). doi:10.1371/journal.pone.0053082.gEach image was smoothed over 363 pixels to remove high frequency noise on the image. We manually set the ROIs (969 pixels, approximately 1 mm2) at varicosity-like structures and shaft structures in CB1 images. The shaft structure of CB1 was defined as the structure that contains thin fibers with low signal intensity and the varicosity-like structure was defined as the structure that has a large immunopositive area with high signal intensity connected by thin fibers. CC value was calculated as follows: ? ?i 1 Xi{X Yi{Y CC Pn ?? ?? Yi{Y i 1 Xi{X Pn where Xi and Yi indicate the individual pixel intensities of CB1 and each of synaptophysin, VGAT, VGluT1, VGluT2 in a ROI,respectively. X and Y indicate the mean.
