Compared with the control mice (No Light), photic stimulation (400 lux, 15 min) at CT 15 increased the number of p-p70 S6K-positive (green) and pCREB-positive (red) cells. Merging of p-p70 S6K and pCREB signals (right) revealed that the expression of the activated kinase and transcription factor were colocalized in a subset of the cells (arrows); arrowhead denotes a cell where colocalization was not detected. The boxed regions are magnified and shown below. Scale bar: 20 µm.

Photic input from the retina induces the release of glutamate and PACAP from RHT terminals. Transmitter binding to postsynaptic receptors on SCN neurons evokes a series of intracellular signal transduction events, including MAPK pathway activation. One downstream target of the MAPK cascade is CREB, which, when phosphorylated on Ser-133, drives clock gene transcription. Light-induced activation of the MAPK cascade also stimulates mTORC1, which in turn targets the translation regulators p70 S6K and 4E-BP1. Activated p70 S6K stimulates S6 phosphorylation, whereas 4E-BP1 phosphorylation has been shown to lead to its dissociation from eIF4E, thereby allowing cap-dependent mRNA translation to occur. U0126 is a specific MEK 1/2 inhibitor and rapamycin inhibits mTORC1.

A, Representative immunohistochemical images of phosphorylated p4E-BP1 (p4E-BP1) expression in the SCN. The treatment conditions are shown above the images. Under control conditions (no light) marked p4E-BP1expression was detected in the SCN at CT15. Pretreatment with rapamycin (100 µM, 2 µl) 30 min prior to sacrificing triggered a significant reduction in p4E-BP1 expression. A 15-minute light treatment (400 lux) at CT 15 stimulated a moderate increase of p4E-BP1 expression in the SCN. Rapamycin (100µM, 2 µl) infusion (30 min before light) blocked light-induced p4E-BP1 expression. Mice not infused with rapamycin were infused with dimethylsulfoxide (DMSO, 2 µl). Scale bars: low magnification 250 µm, high magnification 150 µm. B, Quantitative analysis of 4E-BP1 phosphorylation under the four experimental conditions. The treatment conditions are shown below the histograms. Values are normalized against p4E-BP1 levels in the lateral hypothalamus. Please see the Methods section for a description of the quantitation procedures. Error bars denote SEM. The numbers above the bars indicate the number of animals tested for each condition. ***p < 0.001; ** p < 0.01.

A, Representative confocal images showing colocalization of phosphorylated p70 S6K (p-p70 S6K, green) and mTOR (red) in the SCN. Compared with “no light” control animals (top), a 15-minute light treatment (400 lux) at CT 15 elicited robust p70 S6K activation in the SCN. The merged immunolabeling images (right column) reveal a largely nuclear expression pattern for p-p70 S6K and a cytoplasmic expression pattern for mTOR. OC, optic chiasm. Boxed regions are magnified below each image. Scale bars: 20 µm. B, Quantification of light-induced p-p70 S6K expression and its colocalization with mTOR in the SCN. Cellular coexpression of p-p70 S6K with mTOR was detected in 95% p-p70 S6K positive cells (315 out of 330 cells counted from five mice). Quantitation was performed on 5 control (no light) mice. Error bars denote SEM. C, Representative fluorescent micrographs showing that infusion of the mTOR inhibitor rapamycin blocks light-induced p-p70 S6K expression. Two µl of rapamycin (100 µM) or dimethylsulfoxide (DMSO, vehicle) was infused into the lateral ventricle 30 min before a 15-minute light treatment at CT 15. The boxed regions are shown below. Scale bar, low magnification: 100 µm, high magnification: 75 µm; OC, optic chiasm; 3V, third ventricle. Statistical analysis is presented in figure 5C.

A, Compared with “no light” control animals, a 15-minute light treatment (400 lux) at CT15 elicited robust phospho-p70 S6K (p-p70 S6K: green) and phospho-ERK (pERK: red) expression. Merged confocal images revealed that light-induced p70 S6K and ERK activation occurred in the same subset of cells (arrows). Boxed regions are magnified below each image. Scale bars: 20 µm. B, Quantification of colocalized, light-induced, p-p70 S6K expression and ERK activation in the SCN. Of note, after the light flash 81% p-p70 S6K positive cells were also pERK-postive (338 out of 416 cells counted from six mice); quantitation was performed on 7 control (no light) mice. Error bars denote SEM. C, Representative fluorescent micrographs showing that light-induced p-p70 S6K is dependent on the MAPK pathway. Mice received a ventricular infusion of U0126 (10 mM, 2 µl) or dimethylsulfoxide (DMSO, 2 µl) vehicle 30 min before a 15-minute light treatment at CT 15. SCN sections were double labeled for pERK (red) and p-p70S6K (green) expression. The boxed regions are magnified and shown below. Scale bars, high magnification: 75 µm, low magnification: 100 µm; OC, optic chiasm; 3V, third ventricle. Statistical analysis is presented in figure 5C

A, Confocal images showing immunolabeling for S6 phosphorylation (pS6, red) and p-p70 S6K (green) in the SCN. Compared with control animals (no light), a 15-minute light treatment (400 lux) at CT 15 elicited an increase in pS6 and p-p70 S6K. Merging the two signals revealed colocalization of light-induced p-p70 S6K and pS6 expression in a subset of the cells. OC, optic chiasm. Boxed regions are magnified below each image. Scale bars: 20 µm. B, Infusion of U0126 and rapamycin blocked light-induced p-p70 S6K expression in the SCN. U0126 (10 mM, 2 µl), rapamycin (100 µM, 2 µl) or dimethylsulfoxide (DMSO, 2 µl) was infused into the lateral ventricle 30 min before a 15-minute light treatment at CT 15. Also, note that infusion of U0126 and rapamycin led to a decrease in pS6 expression within the brain regions surrounding the third ventricle. Scale bar: 750 µm; 3V, third ventricle. C and D, Quantitative analysis of light-induced p-p70 S6K (C) and pS6 (D) expression in the SCN. For (D), the Y-axis denotes normalized fluorescent intensity (0-255 scale). Please see the Methods section for a description of the quantitation procedure. The treatment conditions are shown below the histograms. Error bars denote SEM. The numbers above the bars indicate the number of animals used for each condition. ***p < 0.001

Animals were initially dark-adapted for two day and then exposed to light (400 lux, 15 min) during the mid-subjective day [circadian time (CT) 6], early night (CT 15), or late night (CT 22). A, Representative immunohistochemical images of p-p70 S6K in the SCN. Relative to control animals (No Light, top row), light exposure (middle and bottom rows) triggered a marked increase in p-p70 S6K expression at the two night time points. The highest level of antigenicity was observed in the ventral region of the SCN. Photic stimulation during the subjective day did not increase p70 S6K phosphorylation, indicating that the capacity of light to couple to p70 S6K is phase-restricted. Boxed regions are magnified below. Scale bars: 100 µm; 3V, third ventricle; OC, optic chiasm. B, Quantification of p-p70 S6K-positive cells per SCN. The number of animals used for each condition is shown above each histogram. Error bars denote the SEM. ***p < 0.001. C, Western blotting analysis of p-p70 S6K expression in the SCN. Mice received a single light pulse (15 min, 400 lux) at CT 15, and SCN tissue was dissected, pooled (n=3 for each condition) and probed for p-p70 S6K expression. Compared with the “no light” control, a modest increase in the density of the 70 kD band was detected in the light-treated condition. As a protein loading control, the blot was also probed for total ERK (erk 1 and erk 2) expression. D, Distribution of light-induced p-p70 S6K expression within the rostrocaudal axis of the SCN. A fifteen-minute light treatment (400 lux) at CT 15 triggered limited p-p70 S6K expression in the rostral and caudal regions of the SCN; the highest level of expression was observed within the central SCN. Scale bars: 100 µm