Influence of temperature on mammalian clocks in vitro

Although mammalian circadian clocks use temperature compensation mechanisms to maintain a similar period length over a wide range of constant temperatures, they are also capable of resetting their phase upon temperature pulses and of synchronizing to temperature cycles. The use of in vitro model systems allows to isolate the circadian clocks from in vivo homeostatic processes and thus to study the effects of temperature in the absence of confounding signals produced by other sources in the intact animal.

The application of diverse stimuli, including light, temperature or different inducing substances has been shown to reset the clock in a phase-dependent manner. The analysis of such responses allow to establish so called phase response curves (PRCs) (in which observed phase shifts are plotted as a function of circadian time at which the stimulus is given), and phase transition curves (PTCs) (in which the new phase is plotted against the old phase). The extent of the phase shift depends on the strength (amplitude) and the duration of the stimulus.

If the stimulus is short or weak, the resulting phase shifts will be small and cause a smooth

PRC. This PRC is termed type 1 PRC, since the corresponding PTC results in a wavy curve around a straight line with a slope of 1. By contrast, a stronger and longer stimulus immediately resets all oscillators to the same new phase and causes a strong PRC with an abrupt breakpoint between phase advances and phase delays. This PRC is called type 0 PRC, since in this case, the resulting PTC exhibits a linear curve with a slope of 0 (Figure 18) (Johnson, 1999). Temperature pulse-induced PRCs have been done in diverse cell types of homeotherms, including mice and rat SCN tissue slices, peripheral mouse tissue explants, or chicken pineal cells. For example, Barrett and colleagues have shown that temperature pulses (42°C, 6h) are able to shift the phase of the circadian rhythm of melatonin release by chicken pineal cells in a phase-dependent manner (Barrett and Takahashi, 1995). From this experiment, the authors obtained a PRC type 0,qualitatively very similar to the PRC resulting from 6h light pulses in these cells (Robertson and Takahashi, 1988). This similarity let them propose that light and temperature ultimately may have similar effects on the clock. Likewise, a recent study shows that peripheral tissues explants (pituitary and lung) from Per2::luc knockin mice are reset by temperature pulses (36 to 38.5°C, 6h) as reflected by their bioluminescence profiles (Buhr et al., 2010). The PRCs resulting from these experiments

Figure 18. Schematic representations of PRCs and PTCs type 1 (A and C) or type 0 (B and D). In PRCs, delays (represented as negative values) and advances (positive values) are plotted in function of the time circadian of the stimulus. In PTCs, the new phase (after stimulation) is plotted in function of the old phase (before stimulation). The average slope is represented in grey.

were also of type 0. By contrast, the authors observed that SCN slices were resistant to identical temperature pulses, and suggest that this difference could be explained by the fact that the SCN should not react to the timing cues it emits to synchronize peripheral oscillators.

However, this result is in conflict with two previous studies made with SCN slices from rats.

Using SCN neuron firing rates as a readout, one group observed phase delays and advances in response to heat pulses (34 to 37°C, 2h), with a PRC similar to the ones obtained with light pulses for behavioral rhythms (Ruby et al., 1999). In the other study, bioluminescence recordings of SCN explants from Per1-luc transgenic rats, showed that the cultured SCN entrained to daily ∆1.5°C square temperature cycles and that Per1-luc peak expression occurred just prior to the daily increase in temperature, which is similar to what is seen in vivo (Herzog and Huckfeldt, 2003). Together, these two latter approaches support that temperature alter the SCN pacemaker cells and suggest that normal rhythms in brain temperature may serve to stabilize rhythmicity of the circadian pacemaker system in vivo. The discrepancies between these studies may result from interspecies variations (mice versus rats), and further experiments will therefore be required to settle this somewhat controversial issue.

While temperature pulses or steps immediately reset the clock and result in phase shifts, temperature cycles have been shown to influence both the amplitude and the phase of mammalian circadian clocks. Indeed, quantifying circadian transcripts, Brown and co-workers showed that repeated entrainment to ∆4°C square temperature cycles resulted in a strong increase of the amplitude of circadian gene expression as well as in a phase entrainment of the expression of these genes in cultured rat-1 fibroblasts (Brown et al., 2002). They also reported that smoother temperature cycles that mimicked body temperature fluctuations, while incapable of synchronizing circadian gene expression de novo, could maintained previously entrained rhythms. In contrast, by monitoring bioluminescence of pituitary and lung explants from Per2::luc transgenic mice, Buhr and colleagues shows that Per2 expression can be

entrained by body-like temperature cycles (Buhr et al., 2010). These different conclusions may result from different readout resolutions (RNAs quantification versus bioluminescence profiles), different systems analyzed (rat-1 cell line versus mice explants), or different temperature schedules (duration, phase of temperature cycles). Parallel work recording bioluminescence from Per1-luc transgenic rats, showed that ∆1.5°C square temperature cycles were also able to cause a significant increase in the amplitude of Per1 expression and to entrain the phase of Per1 expression in astrocytes isolated from cortical glia (Prolo et al., 2005).

Alltogether, these in vitro experiments suggest that temperature changes have a noticeable effect on the clock of homeothermic organisms, and suggest that body temperature fluctuations could serve as internal Zeitgeber signals for peripheral (and perhaps, SCN pacemaker) clocks. Conceivably, non-photic Zeitgebers such as locomotor activity and feeding behavior may, at least in part, act on the clock via an increase in body temperature.

2.5.2 Influence of temperature on mammalian clocks in the context of the whole