Fig. 3. Oscillation of substance P (SP)-immunoreactivity in the superficial layer of spinal dorsal horn originated from Tac1 gene circadian expression in DRG. (A1 ) Immunohistochemistry stain showed the temporal profiles of SP expression (distribution area of immunoreactions outlined in white dotted line ) in either control or formalin-treated wild-type. Scale bar: 100 μm. (A2 ) Quantification of SP expression patterns in control state (open circles ) and formalin treatment (filled squares ) in wild-type mice (mean ± SE). Dashed curves are circadian (24-h) cosine regressions; the values of mesor (M), amplitude (Amp), acrophase (acro), and 95% CI for acrophase are given in table S5. Data at ZT0 is plotted twice (ZT0 and ZT24). (B1 ) Immunohistochemistry staining shows IB4-immunoreactivity expressed in noncircadian fashion in inner part of lamina II of the spinal cord. Scale bar: 100 μm. (B2 ) Quantification of area measurement for IB4-immunoreactivity expression (three mice at each time-point and 10–12 nonadjacent sections per mouse) (mean ± SE), with circadian (24 h, dashed line ) or circasemidian (12 h, dotted line ) cosine regression, neither of which is significant. (C ) After dorsal rhizotomy on the right side, the oscillation of SP expression in the superficial layer of spinal dorsal horn at ZT4 (trough) and ZT20 (peak) abolished on the cut side, but intact on the uncut side. Quantification of SP expression pattern on the two sides (three mice at each time-point and 10–12 nonadjacent sections per mouse), ***P < 0.001, F = 55.85, using one-way ANOVA followed by Tukey post hoc test. Scale bar: 100 μm. DRG = dorsal root ganglia; ir = immunoreactivity; SP = substance P; ZT = zeitgeber time.