Temperature as a universal resetting cue for mammalian circadian oscillators

Abstract

Environmental temperature cycles are a universal entraining cue for all circadian systems at the organismal level with the exception of homeothermic vertebrates. We report here that resistance to temperature entrainment is a property of the suprachiasmatic nucleus (SCN) network and is not a cell-autonomous property of mammalian clocks. This differential sensitivity to temperature allows the SCN to drive circadian rhythms in body temperature, which can then act as a universal cue for the entrainment of cell-autonomous oscillators throughout the body. Pharmacological experiments show that network interactions in the SCN are required for temperature resistance and that the heat shock pathway is integral to temperature resetting and temperature compensation in mammalian cells. These results suggest that the evolutionarily ancient temperature resetting response can be used in homeothermic animals to enhance internal circadian synchronization.

Figure 1. Peripheral tissues, but not SCN are sensitive to temperature changes within the physiological temperature range

(A) Phase transition curves for SCN, pituitary, and lung in response to 6-h (blue) or 1-h (red) 38.5°C temperature pulses from 36°C. Pulse times are plotted as the time in circadian hours of the end of the pulse from the previous trough of bioluminescence. (B) SCN, pituitary, and lung cultures exposed to opposing square-wave 12hr:12hr 36°C:38.5°C temperature cycles. Phase graphs show time of peak PER2^LUC bioluminescence the day after the temperature cycle. Colored bars above represent the times of warm temperature for the points of corresponding color below. Data are mean+/− SEM. n=4 for each SCN point and n=5 for each pituitary and lung point.

Figure 2. Tetrodotoxin reveals temperature sensitivity of SCN cultures

(A) Bioluminescence record from a Per2^Luc SCN treated with 1 μM TTX as indicated. Six-hour 38.5°C pulses noted with yellow bars. (B) Phase transition curve of individual SCN cultures containing 5 μM (blue) or 1 μM (red) TTX or without drug (gray). (C) A Per2^Luc SCN was imaged using an intensified CCD camera. Identical regions of interest equal to or smaller than the size of a single cell were measured. Heat maps display voxels measured from dorsal (top) to ventral (bottom) where red corresponds to peak bioluminescence and green to trough with and without TTX for the same SCN. (D) 1μM nimodipine reduces amplitude of bioluminescence rhythms and reveals temperature sensitivity. (E) Phase transition curve of individual SCN cultures receiving a 6-h temperature pulse of 38.5°C in 10 μM nimodipine. (F) Phase shifts in response to 6-h 38.5°C pulses which ended 22 hours after the trough of bioluminescence displayed as mean +/− SEM. Vehicle (n=14), 1 μM TTX (n=11), 1 μM nimodipine (n=13), 1 μ mibefradil (n=11). * indicates p < 0.05, ANOVA, Tukey post-hoc analysis.

Figure 3. SCN resistance to temperature pulses relies on the integrity of the ventral and dorsal regions

(A) Yellow line shows approximate dissection of the dorsomedial and ventrolateral regions of a coronal SCN slice. White bar represents 500 μm. (B) Dorsal (blue) and ventral (red) sections from the same SCN cultured in separate dishes. (C) Phase transition curves of dorsal and ventral SCN sections in response to 6-h pulses of 38.5°C (D) Coronal sections of the SCN were also dissected sagittally so that the left and right SCN were cultured separately. (E) Bioluminescence of right and left SCN from the same animal. (F) Phase transition curves of right (blue) and left (orange) SCN sections in response to 6-h pulses of 38.5°C.

Figure 4. KNK437 phase shifts the clock and blocks temperature-induced phase changes

(A) SCN, lung, or pituitary cultures receiving a 1-h pulse of 100 μM KNK437 indicated by an orange arrow. (B) Phase response curves of lung and pituitary receiving a 1-h 38.5°C “warm” pulse, 33.5°C “cool” pulse, or 100 μM KNK437 pulse. (C) Average phase shifts (mean +/− SEM) from lung or pituitary cultures receiving a 1-h 38.5°C pulse in DMSO (n=6), 1-h 38.5°C pulse in 100 μM KNK437 or 100 μM Quercetin (n=5), or a 1-h pulse of 100 μM KNK437 or 100 μM Quercetin alone (n=4) given 9–11 h (lung) or 4–8 h (pituitary) past peak luminescence. * indicates p < 0.01, ANOVA, Tukey post-hoc analysis. (D) Phase transition curves of SCN, lung, and pituitary receiving 1-h pulses of indicated treatments.

Figure 5. Inhibiting HSF-mediated transcription lengthens circadian period and impairs temperature compensation

(A) SCN in KNK437 where marked by an orange line. Numbers indicate the period of the rhythm underneath the line. (B) Periods of SCN in KNK437 (orange) at 100 μM (n=5), 30 μM (n=5), 10 μM (n=5), 3 μM (n=5), and 1 μM (n=5) displayed as mean +/− SEM. Gray points indicate the periods of the same SCN cultures after KNK437 was removed. * indicates p < 0.05 from paired t-tests corrected for multiple measures. (C) Traces of bioluminescence from pituitary or lung cultured with DMSO (black/gray) or 100 μM KNK437 (orange/red). (D) Periods (mean +/− SEM) of SCN or pituitary in 100 μM KNK437 or DMSO. SCN: 30°C DMSO n=8, KNK n=5, and TTX n=; 36°C DMSO n=9, KNK n=8, and TTX n=8; 38°C DMSO n=8 and KNK n=8. Pituitary: 30°C DMSO n=6 and KNK n=6; 36°C DMSO n=9 and KNK n=7; 38°C DMSO n=7 and KNK n=8. p < 0.05 comparing drug treatment or temperature within KNK groups (ANOVA); N.S. for temperature in vehicle groups (ANOVA). (E) Model representing the communication between dorsal and ventral SCN which confers phase resistance to body temperature changes which are regulated by the SCN. Body temperature then entrains peripheral oscillators by acting through HSF mediated transcription.