The clock shop: Coupled circadian oscillators

Abstract

Introduction

What woke you up today? If you ask this question of people in developed countries today, over 80% will credit their alarm clock (Roenneberg et al., 2012). But when they permit themselves a morning without an external timer, they will wake naturally to the call of an internal, daily clock. This chapter is about the cellular organization of this daily clock. We review evidence that this daily clock is comprised of thousands of intrinsically oscillatory cells that must synchronize to environmental cues and to each other. We summarize what is known about the signaling pathways involved in their communication with each other to generate a coherent daily rhythm in behavior.

Life on Earth has evolved in the presence of a daily light-dark, warm-cool cycle. Nearly all organisms alive today anticipate and synchronize (entrain) to these potent cues (Dunlap, 1999). These daily rhythms persist in the absence of these cues, for example in a deep cave or during the dark winter months near the Poles (Cavallari et al., 2011). Under these constant conditions, most organisms will wake and sleep with a period close to 24 h. These circadian (from the Latin, circa meaning approximately and diem, a day) rhythms include cycles of sleep-wakefulness, feeding-fasting, metabolism and hormone release (Honma et al., 2003; Sack et al., 2007). This same circadian clock regulates seasonal changes in many mammals including breeding, fat storage and hibernation (Geiser, 2004).

A simple circadian system: Input-pacemaker-output

It has been useful to evaluate the circadian system conceptually as a pacemaker that regulates a variety of rhythmic outputs and entrains to a variety of environmental timing cues through input pathways. Anatomically, the master circadian pacemaker of mammals has been localized to the suprachiasmatic nucleus (SCN), a group of cells in the base of the anterior hypothalamus situated directly above the optic chiasm (Fig. 1). The bilateral rodent suprachiasmatic nuclei are comprised of about 20,000 neurons packed into an area approximately 1 mm in diameter (Klein et al., 1991).

Figure 1

The cellular and molecular organization of the mammalian circadian system. Neurons within the suprachiasmatic nucleus (SCN) are competent circadian pacemakers (1). They depend on intracellular, transcription-translation negative-feedback (2) mechanisms ...

Many intrinsically circadian cells of the SCN collectively encode the precision, amplitude, waveform and robustness of daily rhythms

In 1995, David Welsh and colleagues demonstrated that individual SCN neurons fire action potentials each day and fall silent each night for many days in vitro (Welsh et al., 1995). Remarkably, they found that when dispersed into a culture dish at relatively low density (~3000 cells/mm²), SCN neurons expressed different circadian periods from each other so that, for example, some neurons started their daily firing every 23 h while others initiated firing every 28 h. These period differences between cells appeared to persist even when all action potentials were blocked with tetrodotoxin. They concluded that single SCN neurons are likely to be competent circadian pacemakers. Subsequently, this has been assumed to be the case in all of the molecular analyses of the mechanisms that generate circadian rhythms (see Chapter by Joseph Takahashi). Over a decade later, Alexis Webb and colleagues isolated single SCN neurons (by killing all but one cell) in the Petri dish and found that individual SCN neurons can indeed express circadian rhythms in firing rate and in gene expression (Webb et al., 2009). She went on to show that this was a property of multiple cell types in the SCN. Notably, when isolated from their network, SCN neurons express unstable circadian rhythms with lower amplitude and more variable cycle-to-cycle periods. This has led to the conclusion that cell-cell interactions within the SCN make the circadian pacemaker more robust (Hogenesch & Herzog, 2011; Hogenesch & Ueda, 2011). Thus, the machinery needed to generate circadian rhythms appears to be intracellular and intrinsic to many cells in the SCN.

Watching the SCN tick in vitro and in vivo is now possible in real-time. Using reporters of gene or protein expression, intracellular Ca⁺² levels or phosphorylated CREB activity, researchers have noted that while the cells in the SCN march to the same beat, they vary in the waveform and phasing of their daily rhythms (Kuhlman et al., 2000; Yamaguchi et al., 2001, 2003; Yoo et al., 2004; Welsh et al., 2005; Cheng et al., 2009). Although it is not yet clear whether all cells at all times are capable of circadian rhythmicity (Silver & Schwartz, 2005), the heterogeneity in the relative phasing of cells appears to underlie adaptation to the long days of summer and short days of winter (Jagota et al., 2000; Schaap et al., 2003; Inagaki et al., 2007; VanderLeest et al., 2007; Ciarleglio et al., 2011; Schwartz et al., 2011). In this way, the population of SCN oscillators encodes photoperiod.

Neuropeptides synchronize SCN cells to each other

To produce a coherent daily output, the cells of the SCN must entrain to each other. Early work showed that nonlinear integration unifies the intrinsic properties of cells to produce circadian rhythms in behavior (Low-Zeddies & Takahashi, 2005; Butler & Silver, 2009; Welsh et al., 2010; Mohawk & Takahashi, 2011). More than 20 transcription factors, kinases, phosphatases and their regulators have been shown to determine circadian cycle length (Shimomura et al., 2001; Reppert & Weaver, 2002; Ueda et al., 2005; Maywood et al., 2011a)(and see Chapter by Joseph Takahashi), yet only a few signaling molecules have been identified as critical for synchrony among SCN cells (Fig. 2).

Figure 2

A model of how SCN neurons synchronize their circadian cycles to each other. By releasing and responding to extracellular factors, SCN cells shift the intracellular rhythm-generating mechanism until all cells oscillate with the same circadian period. ...

Multiple signals synchronize SCN cells to environmental cycles

How do we synchronize to local time and what makes adjusting to travel across time zone or shift work so difficult? It is clear that we depend on the SCN to both entrain to daily cues and to coordinate with the brain and body to produce internal synchrony and well-being. Here, we briefly review mechanisms by which the SCN entrains to light (Fig. 3).

Figure 3

A model of how SCN neurons synchronize to environmental cycles. For the master circadian pacemaker to be of use to an organism, it must entrain to local time. The SCN responds to pituitary adenylate cyclase activating peptide (PACAP) and glutamate released ...

Photic entrainment likely depends on light-induced release of PACAP (Hannibal et al., 1997; Harrington et al., 1999) and glutamate (Meijer et al., 1988; Ding et al., 1994; Akiyama et al., 1999; Asai et al., 2001) from the terminals of the ipRGCs onto SCN neurons. This leads to a necessary increase in intracellular Ca⁺² and activation of the mitogen activating protein kinase (MAPK) pathway (Pizzio et al., 2003; Golombek et al., 2004).

In addition to this monosynaptic pathway from retina to SCN, light information must follow indirect paths to shifting SCN cells. Normal light-induced shifts of behavior also requires SCN cells to respond to nitric oxide, VIP, GRP and other neuropeptides released by SCN cells and to neuropeptide Y released by cells of the intergeniculate leaflet of the thalamus (Albers et al., 1995; Piggins et al., 1995; McArthur et al., 2000; Watanabe et al., 2000; Reed et al., 2001; Kallingal & Mintz, 2006). These diverse convergent (multiple cells influencing one cell) and divergent (one cell influencing many) pathways could be designed to filter and to encode aspects of the light cycle that change with time of day or time of year. This is supported, for example, by the recent discovery that VIP-deficient mice fail to show the behavioral and SCN adaptations following exposure to short days (Lucassen et al., 2012).

All roads lead through cAMP and Ca⁺²

These extracellular signals must impinge on the intracellular circadian gene network to synchronize rhythms across the population of cells. The second messengers identified to date appear to all converge on intracellular cAMP and calcium (Fig. 3). For example, VIP acts through its G-protein coupled receptor to modulate cAMP and calcium levels (Irwin & Allen, 2010) to upregulate expression of clock genes and, ultimately, adjust the circadian phase of each cell. We envision two mechanisms that cooperate to coordinate the rhythms of the SCN and behavior: circadian synchronization among cells within the SCN and induction of cell-cell signaling by environmental timing cues. For example, VIP release in the SCN is both circadian (Shinohara et al., 1995; Francl et al., 2010) and increased by light (Shinohara et al., 1993, 1995).

Appropriately timed release of these signals appears to lead to entrainment. For example, daily exposure to VIP entrains SCN rhythms in vitro (An et al., 2011). VIP release during the day into the early evening leads to shifts of cellular circadian rhythms through parallel increases in the activities of both adenylate cyclase (AC) and phospholipase C (PLC) and subsequent increases in cAMP and Ca⁺² (An et al., 2011). On a daily basis, this leads to a stable phase relationship between the molecular and physiological rhythms of the SCN and the light cycle.

The precise phase relationship of each cell to its neighbors and to the environment is likely defined by its intrinsic properties and the signals it receives. For example, cells in the ventral SCN are the first to respond and fastest to shift following light-induced release of glutamate from the RHT at night (when increasing cAMP alone does not shift) (Prosser and Gillette 1989; Tischkau et al. 2000). This is strikingly reminiscent of the integrative, state-dependent switching effects of neurotransmitters and neuropeptides in other neural networks like the central pattern generators underlying other oscillatory behaviors like locomotion, respiration and chewing (Marder & Goaillard, 2006).

Significantly, some clock genes, e.g. Period1 and Period2, are immediate early genes, whose promoters also contain functional CREs (cAMP/Ca⁺²-response elements). In the SCN, in vivo and in vitro, appropriate activation of cAMP/Ca⁺² signaling by extracellular stimuli induces Period gene expression, and thereby facilitates clock resetting at night (phase entrainment) (Jenkins et al. 2007; O’Neill et al. 2008; Obrietan et al. 1999).

Circadian sleep disorders

The coordinated molecular and cellular interactions of the SCN generate a coherent daily timer that plays a fundamental role in regulating sleep. Disturbances to the circadian system (e.g. genetic or environmental) can lead to sleep disorders. This is formally distinct from the mechanisms that respond to cumulative hours of wakefulness (sleep homeostasis). As highlighted by the large number of sleep clinics and the dearth of circadian clinics, remarkably little is known about circadian disruptions causing sleep disorders. In principle, those changes which alter the period of the circadian clock can advance or delay the timing of sleep onset. The polymorphisms in the Period2 or Casein Kinase1 ε genes associated with Familial Advanced Phase Sleep Syndrome are examples of genetic perturbations in the rhythm generating mechanism that alter sleep timing (Xu et al., 2005, 2007). Loss of circadian timekeeping could lead to fragmented sleep. No human genetic variations have been linked to a loss of circadian rhythms yet, but aging, irregular work schedules and a variety of neurodegenerative diseases have been associated with a loss of daily rhythms and increasingly fragmented sleep (Nakamura et al., 2011; Kudo et al., 2011; Oliver et al., 2012). Finally, disruption of circadian outputs such as hormonal rhythms could impact one’s ability to sleep soundly (De Leersnyder et al., 2001). Thus, factors which alter the entrainment of the SCN to the environment, synchrony among SCN cells, and the output signals from the SCN are likely to play critical roles in getting a good night’s sleep.

Footnotes

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