A, Representative immunohistochemical labeling for control (Saline) and light- (400 lux, 15 min, CT15) evoked p-p70S6K and p-S6 following infusion of physiological saline (Saline+Light) or a cocktail of CNQX (2.5 mM), AP5 (25 mM) and PACAP 6-38 (P6-38,1 mM: CNQX+AP5+P6−38+Light). Compared to the “Saline+Light” condition (middle panel), the infusion of PACAP and glutamate receptor antagonists led to an attenuation of p70S6K and S6 phosphorylation (right panel). Scale bars: 100 μm. B, quantitative analysis of p-S6 expression under three dosages of the inhibitor cocktail. C1: CNQX (150 μM), AP5 (1.6 mM) and PACAP 6-38 (60 μM)+Light, C2: CNQX (600 μM), AP5 (6 mM) and PACAP 6-38 (240 μM)+Light, C3: CNQX (2.5 mM), AP5 (25 mM) and PACAP 6-38 (1 mM)+Light. Light stimulus paradigm is described in (A) C and D, quantitative analysis of p-p70S6K (C) and p-S6 (D) expression under the five treatment conditions indicated below each panel. Mice were infused with PACAP 6-38 (P6-38,1 mM) and/or CNQX (2.5 mM) and AP5 (25 mM) at CT 14.5. Light stimulus paradigm is described in (A). E, Representative immunohistochemical labeling for p-S6 following the ventricular infusion of saline or PACAP (200 μM) and glutamate (2.5 mM) at CT14.5. Animals were sacrificed 30 or 120 min after infusion. Under the control condition, low p-S6 levels were detected in the SCN. PACAP and glutamate infusion increased S6 phosphorylation. Scale bar: 100 μm. F, histogram quantifying p-S6 under the three conditions outlined in (D). S: Saline, G+P30′: sacrificed 30 min after glutamate and PACAP infusion, G+P120′: sacrificed 120 min after glutamate and PACAP infusion. G, Representative confocal images of p70 S6K phosphorylation in cultured SCN neurons. Cultures were stimulated (30 min) with glutamate (10 μM), PACAP (200 nM) or glutamate and PACAP (10 μM and 200 nM, respectively), then fixed and immunolabled. Relative to mock stimulation (ACSF), glutamate or PACAP alone induced a moderate increase in p-p70 S6K expression. However, the coadministration of glutamate and PACAP induced robust p70 S6K activation. H, Quantitative analysis of p-70S6K expression in SCN neurons under the conditions described in G. For the all histograms, data were normalized to the saline (or ACSF) condition, which was set to a value of 1. Please see the Methods section for a detailed description of the quantitation methods.

A and B, Representative double-plotted actographs of wheel-running activity. Mice were dark adapted (DD) and infused with DMSO vehicle (panel A) or rapamycin (100 μM: panel B) 30 min before light (100 lx, 15 min) exposure at CT 22 (red asterisks). Mice were allowed to free-run under DD for 19 days, and then they received a second infusion of rapamycin (panel A) or DMSO (panel B) followed by light exposure. Regression lines approximate the light-evoked phase shifts. C, Representative actograph shows that rapamycin infusion at CT22 did not markedly affect clock timing or phasing. D, Statistical analysis of late night phase shifting. Of interest, the phase advancing effect of light was significantly enhanced by rapamycin infusion. Numbers above the bars denote the number of animals analyzed for each condition.

A and C, Representative immunohistochemical labeling for PER1 (A) and PER2 (C) protein expression in the SCN. Cannulated mice were dark-adapted for two days and then infused (CT 14.5) with rapamycin (100 μM) or DMSO vehicle and exposed to light (400 lx,15 min) at CT 15. Following light exposure, animals were returned to darkness for 4 hours and then killed at CT19. In addition, two “no light” control groups were infused with DMSO or rapamycin at CT 14.5 and killed at CT 19. Immunolabeling revealed that light (DMSO + Light) evoked an increase in PER1 and PER2 expression, relative to the control condition (DMSO). PER1 induction was mainly located in the ventral SCN (A) while the increase in PER2 expression was predominantly located in the lateral and dorsal regions of SCN (C). For each representative section the boxed area is magnified and shown below. Scale bars: 100 μm. B and D, Quantification of light-induced PER1 (B) and PER2 (D) expression in the SCN. Of note, the light-induced increase in PER1 and PER2 expression was significantly attenuated by rapamycin. Under basal conditions, rapamycin led to a modest, but statistically insignificant decrease in PER1 and PER2 protein expression. Numbers in the bars denote the number of animals analyzed for each condition. PER1 and PER2 expression data were normalized to the DMSO infusion conditions, which were set to a value of 1.

A, Representative immunohistochemical labeling for PER1 protein expression. Canulated animals were dark-adapted for two days and then infused at CT 19.5 with rapamycin (100 μM) or DMSO vehicle. Mice were exposed to light (400 lx,15 min) at CT 20, returned to darkness and killed 6 hr later (CT 2). Control mice were infused with DMSO as described above, and killed at CT 2. Immunolabeling revealed that light induced a moderate increase in PER1 expression in the SCN (DMSO+Light vs DMSO). The light-evoked increase in PER1 was attenuated by rapamycin infusion (Rapa + Light). The presentation of high magnification images of a single SCN was necessitated by the relatively modest light-evoked PER1 induction pattern. Scale bar: 100 μm. B, Quantification of PER1 expression. Numbers above the bars denote the number of animals analyzed for each condition. PER1 expression data were normalized to the DMSO infusion condition, which was set to a value of 1. Please see the Methods section for a detailed description of the quantitation methods.

A, Representative immunohistochemical labeling for c-Fos protein. Mice were dark-adapted for two days, and then infused with rapamycin (100 μM) or DMSO vehicle 30 min before light (400 lx,15 min) at CT 15. Animals were killed 0-10 min after termination of the light stimulus. The robust light-evoked increase in c-Fos expression (DMSO+Light) was not affected by rapamycin treatment (Rapa+Light). Framed areas of the SCN are magnified and shown below. Scale bars: 100 μm. B, Quantification of c-Fos protein expression in the SCN. Numbers above the bars denote the number of animals analyzed for each condition. Please see the Methods section for a detailed presentation of the cell counting method.

A, Top: Representative micrographs of tissue labeled for the Thr-2448-phosphorylated form of mTOR. Relative to the control condition (No Light), photic stimulation (400 lx, 15 min, CT 15) triggered an increase in mTOR phosphorylation. Mice were killed 5 min after cessation of the light pulse. Middle panels: Representative confocal images of SCN tissue double labeled for the expression of Thr-389-phosphorylated p70 S6K (p-p70 S6K, green) and Ser-240/244 phosphorylated S6 (p-S6, red). Relative to the control condition (No Light), light triggered a marked increase in the phosphorylated forms of both proteins. Bottom: Merging of the two signals revealed cellular colocalization of the two antigens. The framed area is magnified to the right. 3V, third ventricle; OC, optic chiasm; Scale bars: 100 μm. B, Top and middle rows: Temporal profile of light-induced p70 S6K and S6 phosphorylation. Mice were killed at the noted times following light exposure (400 lx, 15 min) at CT 15. Control mice (No Light) were killed at CT 15. Bottom row: Immunohistochemical profile of total S6 expression following light exposure. Scale bars: 100 μm. C, Quantification of light-induced p-mTOR, p-p70 S6K, p-S6 and S6 expression. * p < 0.05 compared to the “no light” control value, which was normalized to a value of 1. Data were collected from 4-5 animals for each group. D and E, Control and light-pulsed animals (CT15, 400 lux, 15 min) were sacrificed at CT 15.25 and SCN tissue was examined via Western analysis. Probing with a p-p70 S6K antibody (D) revealed that light treatment (L1 and L2: biological replicates) stimulated an increase in the phosphorylation of an ~ 70 kD band relative to the control “no light” condition (NL1, NL2: biological replicates). Relative to the “no light” condition, an increase in p-mTOR and pS6 expression (E) was also detected. The molecular weights of these bands are consistent with the sizes of mTOR (~ 289 kD) and S6 (~ 32 kD). Arrow indicates a non-specific band. As loading controls, blots were probed for total ERK expression.

A and B, Representative double-plotted actographs of wheel-running activity. Initially, mice were entrained on a 12 h light/dark (LD) cycle and then transferred to total darkness (DD). After ~ 10 days under DD, mice were infused with DMSO vehicle (panel A) or the mTOR inhibitor rapamycin (100 μM: panel B) 30 min before light exposure (100 lx, 15 min) at circadian time (CT) 15 (red asterisks). Animals free-ran for 14 days, and then received a second infusion of rapamycin (panel A) or DMSO (panel B) followed by light treatment. Regression lines approximate the phase delaying effects of light. The small horizontal red bars in the activity records denote an “off-line” period when wheel-running activity was not recorded. C, Representative actograph shows that rapamycin (100 μM) infusion at CT15 did not markedly affect clock timing or phasing. D, Statistical representation of the early night phase shifting data. Of note, the light-evoked phase delay was significantly attenuated by rapamycin. Further, the modest phase delay evoked by rapamycin was not significantly different from the effects of DMSO infusion. Numbers above the bars denote the number of animals examined for each condition. E and F, Light-evoked p70 S6K phosphorylation. To test whether the light intensity (100 lx, 15 min) used in the behavioral experiments evokes mTOR activation, mice were exposed to light (100 lx, 15 min) at CT22 and killed immediately thereafter. Immunohistochemical labeling (E) revealed a light-evoked increase in p-p70 S6K, relative to control mice (no light). F, Quantitation of p-p70 S6K immuno-positive cells. Numbers above the bars denote the number of animals analyzed for each condition. Scale bar: 100 μm.

A and B, Representative double-plotted actographs of core body temperature recording. Initially, cannulated mice were entrained on a 12 h light/dark (LD) cycle and then transferred to total darkness (DD). After ~ 10 days under DD, mice were infused with DMSO vehicle or the mTOR inhibitor rapamycin (Rapa: 100 μM) 30 min before light exposure (100 lx, 15 min) at CT 15 (A) or CT 22 (red asterisks). Animals free-ran for 7-14 days, and then received a second infusion of rapamycin or DMSO followed by light treatment. Regression lines approximate the phase delaying (panel A) or advancing (panel B) effect of light. C, Statistical representation of the early night (CT15) and late night (CT22) data sets. Of note, the light-evoked phase delay was significantly attenuated by rapamycin. However, the light-evoked phase advance was significantly enhanced by rapamycin. Numbers above the bars denote the number of animals examined for each condition.

A, Cannulated mice were dark-adapted for two days and then infused (CT 14.5) with actinomycin D (Acti D, 2 μg/μL, 1 μL) and rapamycin (Rapa, 100 μM, 2 μL) or DMSO vehicle and exposed to light (400 lx,15 min) at CT 15. Following light exposure, animals were returned to darkness for 4 hours and then killed (CT19). In addition, two “no light” control groups were infused with actinomycin D and DMSO or rapamycin at CT 14.5 and killed at CT 19. Immunolabeling revealed that in the presence of actinomycin D, light (Acti D + Light) evoked a moderate increase of PER1 expression, relative to the control condition (Acti D). Infusion of rapamycin attenuated the light- evoked increase (Acti D+Rapa+Light) in PER1 expression. For each representative section the boxed area is magnified and shown below. Scale bars: 100 μm. B, Quantification of the data set depicted in (A). Of note, the light-evoked, actinomycin D-insensitive, increase in PER1 expression was significantly attenuated by rapamycin. Numbers on the bars denote the number of animals analyzed for each condition. The PER1 expression data were normalized to the actinomycin-no light infusion condition, which were set to a value of 1. Please see the Methods section for a detailed description of the quantitation methods.

A, Representative immunohistochemical images of eEF1A expression in the SCN. Mice were dark-adapted for two days and then exposed to light (400 lx, 15 min) at CT 15. Animals were killed at CT17. Control animals (No Light) were also killed at CT17. Relative to control animals, light exposure triggered a moderate increase of eEF1A expression in the SCN; the boxed regions of the SCN are magnified and shown to the right. In contrast to the SCN, light did not affect eEF1A expression in the piriform cortex (Cortex). Scale bars: low magnification 100 μm, high magnification 50 μm. B, Quantification of light-induced eEF1A expression in the SCN and piriform cortex (Cortex). Animals were killed 5, 30, 60,120 and 240 min after light exposure at CT15. Control animals (No Light) were killed at CT15. The eEF1A expression in the SCN increased as a function of time following light exposure (up to 2 hrs after light). Numbers on the bars denote the number of animals analyzed for each condition. *p<0.05 vs “no light” control. eEF1A expression data were normalized to the no light conditions, which were set to a value of 1. C, Rapamycin infusion abolished light-induced eEF1A expression in the SCN. Rapamycin (100 μM) was infused 30 min before light treatment at CT15. Animals were sacrificed two hours after light exposure. Data are presented as described in B. D, Representative immunohistochemical labeling for Jun B protein expression in the SCN. Mice were dark-adapted for two days and then infused with rapamycin (100 μM) or DMSO vehicle 30 min before light (400 lx,15 min) exposure at CT 15. Animals were killed 0-10 min after termination of the light stimulus. Immunolabeling revealed that the light-induced increase in Jun B expression (DMSO+Light) was attenuated by rapamycin (Rapa+Light). Scale bars: 100 μm. E, Quantification of light-induced Jun B expression. Numbers above the bars denote the number of animals analyzed for each condition. Please see the Methods section for a detailed presentation of the cell counting method.

Photic input from the RHT drives the release of the excitatory amino acid glutamate and the neuropeptide PACAP. Postsynaptic receptors trigger actuation of the MAPK signaling cassette, ultimately leading to the phospho-activation of the effector kinase ERK. Two principal ERK-mediated signaling events are depicted: activation of MSK and the mTORC1 signaling complex. A potential signaling pathway from ERK to CLOCK (CLK) is also depicted (See Weber, et al., 2006). Signaling through MSK leads to activation of transcription factors such as CREB (see Vermeulen et al., 2009) which in turn drives the induction of early response genes, including c-fos, and the clock gene Period1, and, potentially, Period2 (See Travnickova-Bendova et al., 2002). CREB also stimulates the expression of the microRNA miR-132, which works through as yet unidentified mechanism to limit the resetting effects of light (See Cheng et al., 2007). ERK-dependent activation of mTORC1 causes phosphorylation-dependent activation of a p70 S6K/S6 signaling cassette, which stimulates TOP mRNA translation, and a 4E-BP1 and eEF1a signaling cassette, which increases CAP-dependent translation. These two arms of the mTOR pathway work in conjunction to enhance the rate of mRNA processivity. With respect to clock entrainment, we posit that the ERK-dependent transcription (via MSK/CREB) and ERK-dependent translation facilitation (via the mTOR pathway) lead to a robust induction of the PERIOD protein expression. As a state variable of the clock, (Reppert and Weaver, 2002), the induction of PERIOD leads to a rapid resetting of the molecular oscillator.