Fig. 2: L-Pgds (Ptgds) expression in the SCN in the late subjective night.

a In situ hybridization with a [³⁵S]CTP-labeled antisense probe for L-Pgds in the SCN at CT 21. Representative bright-field photomicrographs are shown for PACAP^−/− and wild-type mice, which were either light stimulated (light+) at CT 21 or kept without light (light−). 3V third ventricle; OC optic chiasma. The dotted line indicates the borders of the SCN area that contain densely aggregated cresyl violet-stained nuclei. Red arrowheads in the right SCN region indicate L-Pgds signals that were merged with counterstained neurons. Bar, 100 μm. b Wild-type mice illuminated with light. Right panel, magnification of the area marked with a black box. Red arrowheads indicate L-Pgds signals that were merged with counterstained neurons. Bars, 10 μm. c The number of L-Pgds-positive cells quantified in the SCN. To quantitatively determine the L-Pgds-expressing neurons in the whole SCN, five coronal SCN sections every four sections per mouse were used for statistical analysis. The values are shown as the mean ± SEM (n = 5–9 per group). *p < 0.05. Statistically significant differences were assessed using two-way ANOVA followed by Tukey–Kramer tests. d Representative dark-field photomicrographs. Bar, 100 μm. Right panel, magnification of the area marked with a white box. Red arrowheads indicate L-Pgds signals on individual cell bodies that were merged with Nissl-stained neurons. Bar, 10 μm. e Intensity of in situ hybridization signals for L-Pgds in each cell in the SCN. The values are shown as the mean ± SEM (n = 10 per group). **p < 0.01. Statistically significant differences were assessed using two-way ANOVA followed by Tukey–Kramer tests. Double immunohistochemical staining for L-PGDS (magenta) and VIP (green, f) or AVP (green, g) in the SCN. Right panels, magnification of the areas marked with white boxes. White arrowheads, cells that were immunoreactive for L-PGDS and VIP or AVP. Bars, 100 μm.