Rapid resetting of the mammalian circadian clock

Abstract

The suprachiasmatic nuclei (SCN) contain the principal circadian clock governing overt daily rhythms of physiology and behavior. The endogenous circadian cycle is entrained to the light/dark via direct glutamatergic retinal afferents to the SCN. To understand the molecular basis of entrainment, it is first necessary to define how rapidly the clock is reset by a light pulse. We used a two-pulse paradigm, in combination with cellular and behavioral analyses of SCN function, to explore the speed of resetting of the circadian oscillator in Syrian hamster and mouse. Analysis of c-fos induction and cAMP response element-binding protein phosphorylation in the retinorecipient SCN demonstrated that the SCN are able to resolve and respond to light pulses presented 1 or 2 hr apart. Analysis of the phase shifts of the circadian wheel-running activity rhythm of hamsters presented with single or double pulses demonstrated that resetting of the oscillator occurred within 2 hr. This was the case for both delaying and advancing phase shifts. Examination of delaying shifts in the mouse showed resetting within 2 hr and in addition showed that resetting is not completed within 1 hr of a light pulse. These results establish the temporal window within which to define the primary molecular mechanisms of circadian resetting in the mammal.

Fig. 1.

Photic induction of c-fos in hamster SCN by single and paired light pulses. Autoradiograms of coronal sections of hypothalamus of hamster SCN produced by in situ hybridization for c-fos mRNA after exposure to darkness (15 min, <1 μW/cm²) or light pulses (15 min, 50 μW/cm²) during early subjective night.a, Thirty minutes after exposure to a control dark pulse at CT13. 3V, Third ventricle; oc, optic chiasm. b, Thirty minutes after start of a light pulse at CT13. Note hybridization signal in bilateral SCN. c, One hour after start of a light pulse at CT13. Note hybridization signal is lost. d, Thirty minutes after start of second pulse in a double-pulse protocol. Note reinduction ofc-fos in SCN. Scale bar, 500 μm. e, Western blots probed for c-fos. Lanes 1,2, Negative and positive (metrazole-treated) controls from extracts of hamster cerebral cortex samples; lanes 3–6, tissue extracts from pooled SCN (5 animals per sample);lane 3, dark pulse control, weak signal; lane 4, 2 hr after light pulse at CT13, strong induction;lane 5, 4 hr after light pulse at CT13, signal lost;lane 6, 2 hr after second pulse, 4 hr after first pulse (note strong reinduction).

Fig. 2.

Photic induction of phospho-CREB in mouse SCN by single and paired light pulses (15 min duration). Coronal sections of SCN reveal low expression in dark-pulsed control animals (a) but extensive induction in retinorecipient SCN of animals 10 min after start of a light pulse (200 μW/cm²) at CT13 (b).c, Time course for phospho-CREB induction in mouse SCN (mean ± SEM) shows rapid and transient response, with reinduction by second pulses presented 1 or 2 hr after initial pulse (arrows).

Fig. 3.

Representative double-plotted actograms of wheel-running activity of Syrian hamsters held in continuous dim red light and exposed to brief pulses of light (15 min,asterisks) at one pulse only at CT13 (a), paired pulses first at CT13 and a second 2 hr later (b), at CT20 and a second 2 hr later (c), and at CT18 and a second 2 hr later (d). Lines on leftside indicate phase shift of activity onset. Pulses ina, b, 15 min, 50 μW/cm²; pulses in c,d, 15 min, 200 μW/cm².

Fig. 4.

a, PRCs (mean ± SEM) of the circadian activity rhythm of Syrian hamsters held in continuous dim red light and exposed to a brief pulse of light (15 min) of 50 (filled symbols; n = 84) or 200 (open symbols; n = 45) μW/cm². The dotted line about the abscissa is the SEM for control dark pulses (mean shift, −0.01 hr;n = 15). b, Predicted and observed resetting to paired pulses in delaying phase of PRC. Shifts to individual pulses (15 or 30 min, 50 μW/cm²) delivered at CT13, CT14, or CT15. NO, Predicted shift if no resetting within 2 hr of first pulse; YES, predicted composite shift if delay arising from pulse at CT13 is completed before second pulse is given; OBS, observed data.n = 11–25; n = 99. **p < 0.01, pairwise comparison. c,d, Predicted and observed resetting to paired pulses in advancing phase of PRC. Shifts to individual pulses (15 min, 200 μW/cm²) delivered at CT18, CT20, CT22, CT23, or CT24. NO, Predicted shift if no resetting within 2 hr of first pulse; YES, predicted composite shift if advance arising from pulse at CT20 or CT18 is completed before second pulse is given; OBS, observed data. n = 6–11; n = 74. **p < 0.01, pairwise comparison.

Fig. 5.

Representative double-plotted actograms of wheel-running activity of ICR(CD-1) mice held in continuous dim red light and exposed to one or a series of four light pulses (200 μW/cm²; asterisks).Lines on left side indicate phase shift of activity onset. a, Single pulse (15 min) at CT13;b, single pulse (1 hr) at CT13; c, series of four pulses of 15 min presented at 2 hr intervals, first at CT13.

Fig. 6.

a, PRC (mean ± SEM) of the circadian activity rhythm of ICR(CD-1) mice held in continuous dim red light and exposed to a brief pulse of light (15 min, 200 μW/cm²) (n = 153). Thedotted line about the abscissa is the SEM for control dark pulses (mean shift, −0.05 hr; n = 15).b, Predicted and observed resetting to single and serial pulses (4 pulses, delivered at intervals of once every 1 hr or once every 2 hr) in delaying phase of PRC. Shifts to individual pulses (15 or 60 min, 200 μW/cm²) delivered at CT13 or CT14 as indicated. NO, Predicted shift after four pulse series if resetting does not occur until final pulse is delivered; YES, predicted composite shift if resetting does occur in the interval of either 1 or 2 hr between pulses;1–2, predicted shift if resetting occurs within 2 hr but not within 1 hr; OBS, observed data.n = 11–17; n = 73. **p < 0.01, pairwise comparison.