The SCN resets the phase in peripheral clocks

To coordinate overt behavioral and physiological rhythms, the intrinsic timekeeping signals of the SCN have to be transmitted to the oscillators that are running in other brain regions and in peripheral tissues. The pathways by which the SCN synchronizes these clocks are multiple and are still unclear. They include neuronal connections (Cailotto et al., 2009; Vujovic et al., 2008), endocrine signals (Balsalobre et al., 2000a; Oster et al., 2006), body temperature cycles (Brown et al., 2002; Buhr et al., 2010), and indirect cues, provoked by SCN-controlled rest/activity cycles (Figure 16). The phase angle of circadian gene expression is different in the SCN than it is in peripheral tissues. In mice, there is a ~4h delay in peripheral cells as compared to the SCN. This reflects the hierarchical organization of the circadian system, with peripheral tissues being controlled in a delayed manner by signals whose emission is governed by the SCN (Lopez-Molina et al., 1997; Panda et al., 2002; Yoo et al., 2004).

Graft experiments reveal the importance of both neural and endocrine signalizations from the SCN to other brain areas for circadian outputs. Indeed, fetal SCN grafts contained in a capsule preventing neural outgrowth in the host SCN-lesioned brain are able to restore rhythms of locomotor activity, but do not restore rhythms in the neuroendocrine axis (Meyer-Bernstein et al., 1999; Moore and Eichler, 1972; Silver et al., 1996). It appears thus that paracrine factor(s) released from the SCN can act to coordinate the animal’s activity and that SCN efferents control hormone rhythms such as melatonin and corticosterone. Indeed, the SCN secretes different neuro-hormones, such as prokineticin-2 (PK2) (Cheng et al., 2002), transforming growth factorα (TGF-α) (Kramer et al., 2001) or arginine-vasopressin (AVP) (Shinohara et al., 1994) in a circadian fashion. Brain infusion of PK2 suppresses locomotor activity during the night, whereas TGF-α stimulates it. However, mice lacking PK2 or the PK2 receptor still display dampened rhythms in activity and thermoregulation, indicating that factors other than PK2 can sustain circadian locomotor activity (Li et al., 2006). The SCN might also directly influence physiology, for example by regulating blood pressure via the

circadian release of AVP.

The connections of the SCN with other brain areas have been investigated by injections of antero- and retrograde tracers, showing that the SCN efferents terminate in a wide range of brain sites (Watts and Swanson, 1987). Neural projections from the SCN are well placed to drive endocrine and other circadian cycles, albeit via indirect polysynaptic connections. Extensive links therefrom feed into arousal and sleep-regulatory centers of the orexinergic system (control of wakefulness and food intake) and ventrolateral preoptic area (regulation of sleep and temperature), respectively, as well as into a diversity of autonomic centers. Consequently, many peripheral organs may receive SCN-dependent circadian time cues via their parasympathetic and/or sympathetic innervations (Kalsbeek et al., 2006; Morin and Allen, 2006). The SCN also indirectly controls pineal as well as adrenal cortex function, and as a consequence, the circadian release of melatonin and glucocorticoids. Melatonin is secreted in during the dark phase and regulates many physiological processes, including seasonal rhythms in many species and sleep efficiency in humans. The SCN indirectly mediates the inhibitory effects of light on melatonin release and is involved in the circadian control of melatonin production and secretion rhythms. The SCN also controls the daily release of glucocorticoids, which in collaboration with additional circadian cues, coordinate metabolic (glucose metabolism, circadian cholesterol homeostasis, detoxification, etc.), endocrine, immune and neuronal functions (see below) (Hastings et al., 2007).

Both humoral and neural pathways are also employed by the SCN to synchronize peripheral organs and tissues. The circadian expression of Per2 in peripheral mononuclear leukocytes that have no neuronal connections represents a clear evidence for a role of humoral signals to reset peripheral clocks (Oishi et al., 1998). Moreover, parabiosis between intact and SCN-lesioned mice shows that non-neural (behavioral or blood borne) signals are able to maintain circadian rhythms of clock gene expression in liver and kidney in the SCN-lesioned partner (Guo et al., 2005). Furthermore, in vitro studies show that the expression of Per1 and

Per2 is immediately induced by serum in cultured fibroblasts and is followed by the circadian expression of various clock genes for few days (Balsalobre et al., 1998). This indicates not only that signals present in the serum can synchronize fibroblasts oscillators, but also that the signal transduction cascades operative in the SCN upon exposure of animals to light seems to be conserved in the periphery. Given the plethora of potential signals present in the serum, it is difficult to determine which of these plays a role in vivo. In fact, multiple distinct signaling pathways, including cAMP, protein kinase C, glucocorticoid hormones and Ca2+, have been shown to trigger a transient surge of Per1 and/or Per2 expression and elicit rhythmic gene expression in cultured rat-1 cells (Balsalobre et al., 2000b). This suggests that the SCN may exploit multiple chemical cues to synchronize peripheral oscillators in vivo. This renders the task of characterizing the players involved in peripheral tissues resetting even more challenging. Among the humoral cues, glucocorticoids are plausible candidates of hormones that reset peripheral clocks. Indeed, plasma glucocorticoid levels exhibit robust circadian oscillations, and these daily cycles are driven by the SCN via the hypothalamus/pituitary/adrenal axis (Oster et al., 2006). Dexamethasone, a glucocorticoid analog, can reset clocks of rat-1 fibroblasts and phase shift peripheral oscillators in vivo via glucocorticoid receptor (GR) and, presumably, glucocorticiod response elements (GRE) in the promoter and first introns of Per. However, glucocorticoids do not affect SCN rhythms, most likely because the the GR is not expressed at significant levels in SCN neurons (Balsalobre et al., 2000a; Yamamoto et al., 2005). Thus, the SCN influences the periphery by controlling the daily release of glucocorticoids by the adrenal gland via SCN-sympathetic adrenal nerve route (Ishida et al., 2005). Although glucocorticoids are clearly phase resetting signals controlled by the SCN, the steady-state phase of peripheral tissues, such as the liver, remains unchanged in the absence of GR (Le Minh et al., 2001), emphasizing again the redundancy of synchronization pathways.

Neuronal connections may constitute additional synchronization pathways. A recent

study shows that nocturnal light-induced changes in the liver mRNA levels of clock genes and output genes are clearly affected by selective surgical disruption of liver autonomic innervation (Cailotto et al., 2009). This suggests that the autonomic nervous system can be a gateway for the SCN to cause an immediate resetting of peripheral physiology after phase-shift inducing light exposures. In the same perspective, disruption of sympathetic innervation of the submaxillary glands affects the response of clock genes to light cycle information.

While the expression of Per1 in the submaxillary glands stays entrained to the light cycle (or becomes arrhythmic) and fails to entrain to daytime restricted feeding (see below) in intact rats, its phase angle is shifted by 180° and entrained to daytime feeding after denervation (Vujovic et al., 2008). Thus elimination of the light signal by surgical sympathectomy may allow a secondary signal (i.e. feeding) to control the phase. Again, these results support the notion that peripheral oscillators may receive multiple signals contributing to their phase entrainment.

SCN-controlled rest/activity cycles generate feeding-fasting rhythms, which appear to be dominant Zeitgebers for many peripheral organs, including liver, pancreas, kidney, heart, and skeletal muscles. Feeding–fasting cycles can overcome the hierarchical dominance of the central pacemaker, and daytime feeding thus can uncouple peripheral clocks from the SCN (Damiola et al., 2000; Stokkan et al., 2001). Feeding might reset peripheral oscillators via nutrient-sensing hormones (e.g., cholecystokinin, peptide YY, oxyntomodulin, ghrelin, leptin) (Konturek et al., 2004) or food metabolites (e.g., fatty acids, cholesterol, glucose, heme) (Schibler, 2009). Feeding-induced changes in the redox state of the cell may also influence the circadian clock. Indeed, biochemical experiments have already demonstrated that the NAD(P)H/NAD(P)+ ratio can affect the binding of CLOCK/NPAS2-BMAL1 heterodimers.

Reduced NAD(P)H forms stimulate the occupancy of E-box motifs by the heterodimers, whereas oxidized NAD(P)+ inhibit these interactions (Rutter et al., 2001). In addition, as mentioned previously, SIRT1, an NAD⁺-dependent deacetylase binds CLOCK/BMAL1 in a

circadian manner and promotes the deacetylation and degradation of PER2 and BMAL1.

Hence, SIRT1 may affect circadian function depending on the cells’ metabolic states (Asher et al., 2008; Nakahata et al., 2008). Feeding also influences body temperature. Thus, upon daytime-feeding the phase, shape, and amplitude of body temperature rhythms is dramatically altered. A strong depression is observed during the night when food is not available, while zenith temperatures are reached during the dark-light and light-dark transitions (Damiola et al., 2000). In addition to a rise in core body temperature, feeding probably leads to increased levels of reactive oxygen species (ROS), which can elicit protein denaturation. Reinke and co-workers found that heat shock factor 1 (HSF1) exhibits highly circadian DNA binding activity, peaking at the onset of night (corresponding to the onset of feeding and to the increase of body temperature). They thus suggest that HSF1 senses temperature- or food-induced proteotoxic stress and counteracts it by the up-regulation of cytoprotective proteins (Reinke et al., 2008). In addition, HSF1 might also assist in the temperature-dependent synchronization of peripheral circadian oscillators. Indeed, temperature pulses or cycles are able to synchronize circadian gene expression in fibroblasts or peripheral tissue explants in culture (Brown et al., 2002; Buhr et al., 2010). Thus, feeding-dependent changes in body temperature, as well as environmental temperature changes may participate in the entrainment of peripheral clocks (see section 2.3 for a more detailed view of the interplay between temperature and mammalian circadian clocks).

Interestingly, it seems that in the SCN there is no distinction between nocturnal and diurnal rodents with regard to the phase of Per1 and Per2 transcript accumulation, likely because the light-dependent synchronization is similar in all species. However, the phase of these circadian transcripts appears to vary greatly in peripheral tissues (Vosko et al., 2009).

The authors of this study propose therefore that the signals emanating from the SCN must be interpreted in an opposite manner in the periphery of diurnal compared with nocturnal mammals.