The genetic basis of circadian behavior

Review

The genetic basis of circadian behavior

H. Oster*

Laboratory for Chronobiology and Signal Transduction, Max Planck

Institute for Experimental Endocrinology, 30625 Hannover,

Germany

*Corresponding author: H. Oster, Laboratory for Chronobiology

and Signal Transduction, Max Planck Institute for Experimental

Endocrinology, 30625 Hannover, Germany. E-mail: henrik.oster@

mpihan.mpg.de

In most species, an endogenous timing system synchro-

nizes physiology and behavior to the rhythmic succession

of day and night. The mammalian circadian pacemaker

residing in the suprachiasmatic nuclei (SCN) of the

hypothalamus controls peripheral clocks throughout the

brain and the body via humoral and neuronal trans-

mission. On the cellular level, these clockworks consist

of a set of interwoven transcriptional/translational feed-

back loops. Recent work emphasizes the tissue specificity

of some components of these molecular clockworks and

the differential regulation of their rhythmicity by the SCN.

Keywords: Behavior, circadian, clock genes, physiology, SCN

Received 20 January 2005, revised 17 May 2005, accepted

for publication 14 June 2005

One of the most prominent characteristic features of life on

earth is the constant repetition of night and day. The 24-h

rhythm of light and darkness, coupled with warm and cold,

exerts a fundamental influence on physiology and behavior of

most species dwelling on this planet. Life has coped with

this rhythm by evolving biological clocks tracking time and

enabling the organism to anticipate and prepare for predict-

able environmental changes (Pittendrigh 1993).

Biological clocks exist not only to deal with daily rhythms

(termed circadian clocks, from the Latin circa dies, meaning

about one day) but are also employed to measure shorter

(ultradian) or longer (infradian) intervals of time (Wollnik

1989). We are currently beginning to unravel the molecular

basis of some of these clocks measuring short and long time

spans such as the circannual timekeeper controlling migra-

tion behavior in birds (Dawson et al. 2001). This review,

however, will focus on the circadian system, the so far

best characterized clockwork constituting a unique show-

case of the molecular basis underlying the regulation of

complex physiological and behavioral processes.

Most aspects of temporal homeostasis, the timed co-

ordination of the physiological status, are under the control

of the internal pacemaker (Perreau-Lenz et al. 2004). The

signaling pathways employed to transmit timing information

from the central clockwork of the hypothalamus to the var-

ious sites controlling metabolism and physiology and the

mechanisms that keep these oscillations in phase are still

poorly understood (Gachon et al. 2004). Even less is known

about the molecules that mediate the circadian control of

complex behavioral systems such as the sleep/wake cycle

(Monk & Welsh 2003; Pace-Schott & Hobson 2002), anxiety

(Jones & King 2001), learning and memory consolidation

(Chaudhury & Colwell 2002), behavioral flexibility and atten-

tion (Aston-Jones et al. 2000), and social behavior (Insel &

Young 2001; Reijmers et al. 2001; Schwartz & Reppert

1985).

In addition to its ability of self-sustained oscillation, the

endogenous clock also receives input from the environment

to synchronize internal and external time. The most promi-

nent timing signal or Zeitgeber (German for time cue) of the

mammalian circadian system is light. Illuminance levels are

measured by special photosensory cells in the retina (Berson

2003) that signal via glutamate and pituitary adenylate

cyclase-activating peptide (PACAP) to light responsive cells

of the central mammalian pacemaker, the suprachiasmatic

nuclei (SCN), localized in the ventral part of the hypothalamus

(Moore 1978; Rusak & Zucker 1979).

Other synchronizing signals have also been identified and

include temperature (Rensing & Ruoff 2002), enforced loco-

motor activity (Wickland & Turek 1991), sleep deprivation

(Mistlberger 1992), injections of melatonin (Korf & Stehle

2002), leptin (Prosser & Bergeron 2003), gastrin-releasing

peptide (GRP) (McArthur et al. 2000), steroids (Pinto &

Golombek 1999) and opioids (Byku & Gannon 2000). Many

of these so-called non-photic effectors are thought to oper-

ate as feedback mechanisms by which the body affects the

central clock. They use signaling pathways to distinct parts of

the SCN employing neuropeptide Y (NPY) and serotonin as

major neurotransmitters (Mrosovsky 1996).

The anatomy of the mammalian circadiantiming system

The central clockwork of the SCN controls circadian adapta-

tion of the physiological state via direct humoral and neuronal

Genes, Brain and Behavior (2006), 5 (Suppl. 2), 73–79 # 2006 The Author

Journal compilation # 2006 Blackwell Munksgaard

doi: 10.1111/j.1601-183X.2006.00226.x 73

regulation of the metabolic, the endocrine, the immune

and the nervous system (Perreau-Lenz et al. 2004; Reppert

& Weaver 2002) and indirectly via its influence on the

animal’s activity, coupled to body temperature (Schibler

et al. 2003).

Some peptides secreted from the SCN – such as

Prokineticin 2 (PK2; Cheng et al. 2002) or transforming

growth factor a (TGFa; Kramer et al. 2001) – have been

shown to suppress wheel running activity. The expression

pattern of the corresponding receptors, however, suggests a

primary action on adjacent nuclei of the hypothalamus that

conduct relay signals from the SCN. Glucocorticoids

(Balsalobre et al. 2000), retinoic acid (McNamara et al.

2001) and noradrenaline (Terazono et al. 2003) have the

potential to reset specific body clocks, but so far no general

mechanism has been described for peripheral pacemaker

regulation in vivo (Hirota & Fukada 2004; Schibler et al. 2003).

It is not entirely clear whether these peripheral clocks are

self-sustaining pacemakers or merely hourglass-like time-

keepers that need to be regularly reset by SCN signals to

continue working (Yamazaki et al. 2000; Yoo et al. 2004). A

unique organization of coupling between the single neurons

in the SCN may ensure the rhythm persistence within this

nucleus and distinguish it from the rest of the body (Ohta

et al. 2005). In the periphery, in the absence of exogenous

synchronizing signals from the SCN the phases of single cell

oscillators would gradually drift apart resulting in a dampened

oscillation observed on the whole tissue level (Welsh et al.

2004).

While an animal’s locomotor activity rhythms are con-

trolled at least partially via diffusible molecules (Ralph et al.

1990; Silver et al. 1996), some rhythms such as the release

of corticosteroids from the adrenal gland have been shown

to require neuronal input (Dijkstra et al. 1996; Meyer-

Bernstein et al. 1999) emphasizing the importance of intact

SCN projections to other centers of the CNS (Watts &

Swanson 1987; Watts et al. 1987) and indirectly to various

organs of the body (Fig. 1) (Buijs et al. 2003; de la Iglesia

et al. 2003; Terazono et al. 2003; Warren et al. 1994).

Electrophysiological studies have shown that neurons dis-

play circadian firing rhythms in many brain regions outside

the hypothalamus (Aston-Jones et al. 2001; Granados-

Fuentes et al. 2004; Koolhaas et al. 1980; Ono et al. 1986;

Semm & Vollrath 1980; Watts et al. 1987). The SCN projects

to three different neuronal targets: endocrine and autonomic

neurons of the paraventricular nucleus of the hypothalamus

(PVN) and other hypothalamic structures such as the dor-

somedial nucleus of the hypothalamus (DMH) and the sub-

paraventricular zone (Leak & Moore 2001) that relay timing

signals to other parts of the brain. The PVN secretes

hormones that regulate the activity of the pituitary and con-

trols peripheral targets via autonomic innervation (Buijs &

Kalsbeek 2001). The DMH regulates the circadian firing

pattern of the locus coeruleus (LC) (Aston-Jones et al.

2001) that sends noradrenergic projections to many brain

areas and the spinal cord (Aston-Jones 2004), some of

which may house their own circadian clockworks.

A recent series of publications from the group of Steven

McKnight has described one of these brain oscillators and

characterized its specific contribution to the organism’s

circadian timing system. It was found that the forebrain

contains a neuronal PAS protein 2 (Npas2 )-dependent circa-

dian clock (Reick et al. 2001). Npas2-deficient mice show

changed locomotor activity patterns under normal lighting

conditions as well as an altered adaptability to a rapid shift

in the external light schedule (a simulated jet lag) and

daytime feeding paradigms (Dudley et al. 2003). This pioneer-

ing work highlights the functional importance of extra-

hypothalamic oscillators and complements lesion studies

emphasizing the necessity of correct neuronal wiring of the

SCN within the CNS (e.g. Challet et al. 1996; Fischette et al.

1981; Goodless-Sanchez et al. 1991; Harrington & Rusak

1988; Lissak et al. 1975; Schwartz et al. 1986) for the func-

tionality of the circadian system.

SCN

PVNDMH

LC

IML

Pineal

Pituitary

Adrenal

Cortex

Thalamus Cerebellum

Melatonin

CRH

ACTH

Adrenaline/noradrenaline Corticoids

?

via SCG

Figure 1: Circadian regulation of the arousal system. As

shown here in the rodent brain, pacemaker neurons of the SCN

rhythmically innervate a web of nuclei in and outside the

hypothalamus which relay timing information to the brain and

the periphery (exemplified here by the adrenal gland). The bimo-

dal regulatory action of endocrine (dotted lines) and neuronal

signals (solid lines) ensures a co-ordinated control of the organ-

ism’s attention state. Noradrenergic and serotonergic projections

from nuclei of the brainstem activate neurons in various areas of

the brain (including the cerebellum and the cortex), thereby

promoting vigilance and arousal. In addition, these systems

have descending projections by which they can enhance or

modulate muscle tonus and activity in the periphery via endo-

crine pathways (e.g. the hypothalamus–pituitary–adrenal (HPA)

axis and melatonin synthesis by the pineal gland) and the auto-

nomic nervous system (ACTH, adrenocorticotropic hormone;

CRH, corticotropin-releasing hormone; DMH, dorsomedial

nucleus of the hypothalamus; IML, intermediolateral column of

the spinal cord; LC, locus coeruleus; PVN, paraventricular

nucleus of the hypothalamus; SCG, superior cervical ganglion).

Oster

74 Genes, Brain and Behavior (2006), 5 (Suppl. 2), 73–79

The molecular clockwork

Circadian clocks have been shown to function in a cell-

autonomous fashion (Welsh et al. 1995), and our current

view is that most if not all cells of the body contain their

own circadian clockwork (Balsalobre et al. 2000). Genetic

tools were first applied to circadian biology by the seminal

work of Konopka and Benzer studying rhythm mutants in

Drosophila (Konopka & Benzer 1971). Yet, it was not until

1994 that the first mammalian clock gene – circadian

locomotor output cycles kaput or Clock – was discovered

in a mutagenesis screen in mice (Vitaterna et al. 1994).

From the late 90’s onward, an increasing number of other

clock genes have been identified and characterized in the

animal model including the Clock partner Bmal1 (or Mop3;

Bunger et al. 2000), Per1, Per2 and Per3 (Bae et al. 2001;

Cermakian et al. 2001; Shearman et al. 2000; Zheng et al.

1999; Zheng et al. 2001), Cry1 and Cry2 (van der Horst

et al. 1999; Vitaterna et al. 1999), Casein kinase1e (CK1e)(Lowrey et al. 2000; Ralph & Menaker 1988), Rev-Erba(Nr1d1) (Preitner et al. 2002) and RORa (Sato et al.

2004). Other genes like the mammalian homolog of

Drosophila timeless, mTim (Barnes et al. 2003) and the

basic helix-loop-helix (bhlh)-PAS (Period-Arnt-Single

minded) proteins Dec1 and Dec2 (Honma et al. 2002)

constitute bona fide candidates but still await thorough

testing in an appropriate animal model.

The molecular clockwork is based on interconnected posi-

tive and negative transcriptional/translational feedback loops

(TTLs; Fig. 2). The transcriptional activators CLOCK and

BMAL1 form heterodimers that activate the expression of

genes containing E-Box cis-regulatory enhancers (including

the Pers, Crys, Rev-Erba and RORa) during the morning

(Gekakis et al. 1998; Hogenesch et al. 1998). PER and CRY

proteins are translated and accumulate in the cytoplasm.

There they form heteromeric complexes together with

CK1e (and maybe d) that eventually translocate into the

nucleus where they interfere with CLOCK/BMAL1-driven

transcription (Griffin et al. 1999; Jin et al. 1999; Kume et al.

1999). In addition, PER and CRY proteins form complexes

which prevent those proteins from being phosphorylated by

CK1 marking them for ubiquitination and degradation (Yagita

et al. 2000). This equilibrium between translation, further

posttranslational modification, translocation, and degradation

postpones the highest inhibitory potential of PER/CRY com-

plexes to the night phase when E-box-driven transcription

declines. The suppressed transcription and subsequent

decrease of PER/CRY protein levels first in the cytoplasm

and later in the nucleus re-activates CLOCK/BMAL1-driven

expression in the early morning and thereby re-initiates the

next circadian cycle.

Specificities for complex formation seem to exist between

different PER and CRY paralogs in vivo (Oster et al. 2002b;

Oster et al. 2003). Together with the opposite regulation of

Per1 and Per2 expression under different lighting conditions

(Steinlechner et al. 2002), this may adjust the duration of

CLOCK/BMAL1 suppression to the photoperiod and may

therefore constitute a mechanism of adaptation of the clock-

work to winter and summer (Daan et al. 2001; Oster et al.

2002a).

Rev-Erba and RORa form additional feedbacks that stabil-

ize the clock rhythm via transcriptional regulation of Bmal1.

These supporting TTLs seem to be less critical for normal

rhythm generation but add to the precision of the clockwork

and its insensitivity to external and internal noise (Preitner

et al. 2002; Sato et al. 2004).

In addition, CLOCK and BMAL1 directly (via E-boxes) or

indirectly control the rhythmic transcription of a set of clock-

controlled genes (CCGs). These CCGs are tissue-specific and

in the SCN include Vasopressin (Avp; Jin et al. 1999) and

CLOCK/BMAL1

E-box

Pers/Crys/Rev-Erbα/Rorα/CCGs

RORE

Bmal1

PERs

PER/CRY/CK1complex

CRYs

PERs CRYs CK1ε(δ)

PP

PP

RORαBMAL1

REV-ERBα

Figure 2: Multiple transcriptional/translational feedback

loops stabilize the cellular core oscillator of the circadian

clock in the SCN. E-box containing clock and first-order

clock-controlled genes (CCGs) are rhythmically activated by

CLOCK/BMAL1. The translated proteins form positive and nega-

tive feedbacks on their own synthesis via regulation of Bmal1

transcription and direct inhibition of the CLOCK/BMAL1 enhan-

cer complex. Complexation, posttranslational modification and

subcellular localization of clock proteins ensure the delayed tim-

ing of this feedback essential for the oscillation of the molecular

clockwork (for details see the text; RORE, retinoic acid-related

orphan receptor response element).

Genetic basis of circadian behavior

Genes, Brain and Behavior (2006), 5 (Suppl. 2), 73–79 75

Prokineticin2 (Pk2) (Cheng et al. 2002) encoding neuro-

peptides that mediate clock rhythms to other areas in the

brain.

Peripheral clocks

Genomic approaches using microarray technology have

revealed that in most tissues, about 8–10% of all expressed

genes are oscillating in a circadian fashion (Akhtar et al. 2002;

Panda et al. 2002; Storch et al. 2002; Ueda et al. 2002). In the

liver, many transcripts encoding rate-limiting enzymes of

essential metabolic pathways such as glycolysis, fatty acid

metabolism and gluconeogenesis are under circadian regula-

tion (Storch et al. 2002) offering a mechanism for the control

of organ physiology by the circadian clock (Rutter et al. 2002).

Not all of these CCGs are directly controlled by CLOCK/

BMAL1 or their tissue-specific paralogs (like NPAS2 in the

forebrain and in the vasculature; McNamara et al. 2001).

Second-order CCGs may be indirectly regulated via CLOCK/

BMAL1-controlled mediators such as D-element-binding pro-

tein (DBP) that has been shown to control the rhythmic

expression of some metabolic enzymes in the liver (Lavery

et al. 1999). DBP acts together with the basic leucine zipper

transcription factor E4BP4, whose rhythm is opposite to that

of DBP, via inverse effects on D-element cis-regulatory

enhancers of responsive genes (Mitsui et al. 2001).

The cell autonomy of the circadian clockwork is the basis

of tissue and even cell-specific modulation and interpretation

of endocrine or neuronal timing signals from the SCN. This

specificity enables the organism to spatially and temporally

fine tune its body functions in an efficient and economic

manner. The SCN as the central pacemaker synchronizes

the internal rhythm to the environment. It acts like the

conductor of an orchestra that gives the pace he reads

from the score (e.g. the sun). The music, however, is played

by the instruments, the peripheral oscillators controlling the

physiological and the behavioral state of the organism.

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Acknowledgments

The author thanks Ms. Diya Abraham and Drs Gregor Eichele and

Erik Maronde for their critical comments on the manuscript. This

work was supported by the Max Planck Society and the EC

BrainTime grant (QLG3-CT-2002-01829).



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The genetic basis of circadian behavior