Time in the brain: rhythms of intercellular and intracellular processes

Giorgio RACAGNI, PhD
Center of Neuropsychopharmacology,
of Pharmacological Sciences
University of Milan
Milan, ITALY
and Genetic Unit, IRCCS San
Giovanni di Dio, Fatebenefratelli
Brescia, ITALY
Daniela TARDITO, PhD
Maurizio POPOLI, PhD
Center of Neuropsychopharmacology,
of Pharmacological Sciences
University of Milan
Milan, ITALY

Time in the brain:
rhythms of intercellular and
intracellular processes

by D. Tardi to, G. Racagni ,
and M. Popoli ,I taly

Most biological functions are expressed in an oscillating manner within a 24-hour circadian period, regulated by endogenous biological clocks. The rhythms are generated in the suprachiasmatic nuclei, groups of neurons in the anteroventral hypothalamus, and are synchronized by regularly recurring environmental stimuli or “zeitgebers” (light, social stimuli, physical activity, etc). The different stimuli are conveyed to the suprachiasmatic nuclei through afferent pathways that utilize different neurochemical and neuroendocrine signals, such as glutamate, serotonin, and melatonin. In turn, the suprachiasmatic nuclei communicate with other brain regions and peripheral systems to impart or entrain circadian rhythms in behavioral and physiological processes. It is now known that other brain regions (ie, the hypothalamic nuclei, hippocampus, frontal cortex, etc) contain autonomous oscillators and are capable of generating circadian rhythms. From a molecular point of view, circadian clockmechanisms comprise a core set of genes whose expression oscillates in an autonomous manner generated by a transcriptiontranslation negative feedback loop with a crucial delay between stimulus and response. Posttranslational modifications (ie, phosphorylation events) play a key role in this feedback loop. Recent evidence demonstrates that circadian autonomous oscillations are also evident in components of signaling cascades with a key role in memory formation, neuroplasticity, and depression; for instance, the mitogen activated protein kinases, brain-derived neurotrophic factor, and cAMP response element binding protein. This is thus opening up new lines of research in the field of psychiatry.

Medicographia. 2010;32:152-158 (see French abstract on page 158)

Circadian rhythms in the brain: the role of the suprachiasmatic nuclei

Time-linked modifications are identifiable at all levels of biological functioning, from biochemical processes to whole organism behavior, and these changes are regulated by a system of endogenous regulatory biological clocks. Biological activities follow cycles of various lengths, from very short rhythms (ultradian) to rhythms with a period of nearly 24 hours (circadian), and rhythms with longer cycles, froma week, to a season, or even longer.Most biological functions are expressed in an oscillating manner within a 24-hour period: the rest/activity cycle and sleep phases, body temperature, blood pressure and heart rate, hormone concentrations in the blood (melatonin, cortisol, thyroid-stimulating hormone, insulin, growth hormone, and other hormones), hepatic metabolism and detoxification (cytochrome P450 en- zymes), renal elimination, and gene transcription and translation. These biochemical, physiological, or behavioral rhythms are generated by endogenous biological clocks, ie, internal systems capable of indicating the passage of time, as well as a response to fluctuations in the environment.1

In the absence of time cues, the endogenous rhythms are self-sustained and have a period of approximately that of the earth’s rotation (hence the adjective circadian, meaning about 1 day). In mammals, the master pacemaker controlling the generation and coordination of circadian rhythms is sited in the suprachiasmatic nuclei (SCN), which are two bilaterally paired groups of neurons, containing approximately 10 000 neurons each in the anteroventral hypothalamus situated just above the optic chiasm.2,3 Destruction of the SCN, either experimentally in laboratory animals or as a result of disease in humans, disrupts the ability to express any circadian rhythm.4 On the other hand, individual neurons from the SCN, when dissociated and held in vitro, retain a robust circadian rhythm in electrical firing that can be recorded for several weeks.5

The rhythms generated by the SCN are synchronized to a daily pattern by regularly recurring environmental stimuli or “zeitgebers.” In usual environmental conditions, circadian biological clocks are reset daily to 24-hour astronomical time by the day/night cycle, ie, through the influence of light, the main zeitgeber. Other environmental factors that can serve as zeitgebers are the availability of food, social schedules, and social exchanges.1,6

Light stimuli arriving at the nonvisual photoreceptive retinal ganglion cells are transmitted directly to the SCN by way of the retinohypothalamic tract (Figure 1), in which the putative neurotransmitter is glutamate. Another pathway that indirectly conveys light stimuli to the SCN is the geniculohypothalamic tract. This pathway, in which the principal neurotransmitters are γ-aminobutyric acid and neuropeptide Y, runs from the intergeniculate leaflet of the lateral geniculate complex. Moreover, the serotonergic pathway from the raphe nuclei acts as a synchronizer of the SCN (Figure 1).7,8 Indeed, the SCN is one of the important target areas of serotonergic projections.9 Serotonin (5-HT) is the principal neurotransmitter in the retino-raphe input pathway to the SCN. The serotonergic systemin the SCN is involved in themechanismof entrainment and rhythm modulation through its receptors, which respond to photic and nonphotic stimuli, and it thus plays a key role in circadian clock resetting.9 Binding studies have demonstrated the presence of different 5-HT receptors (5-HT1A, 5-HT1B, 5-HT2A, 5-HT2C, and 5-HT7) in the SCN with variable levels of expression.10 In particular, a high concentration of 5-HT2C receptors has been reported.11 In situ hybridization investigations in rats have reported that transcription of 5-HT2C messenger RNA is highest early in the morning, suggesting that 5-HT2C receptors also have a circadian rhythm of expression.12,13 In a recent study, it was shown that 5-HT1A receptors, possibly with co-activation of 5-HT7 receptors, are implicated in the nonphotic effects on the main clock. By contrast, 5-HT3 and 5-HT2C receptors are involved in photic-like effects and, for the 5-HT2C subtype only, in potentiation of photic resetting.8

Figure 1
Figure 1. Connections of the suprachiasmatic nuclei (SCN) within
the brain. PVT, paraventricular thalamic nucleus.

The main known afferent and efferent pathways from the SCN to various brain
regions are shown schematically.

The timing of external zeitgeber stimuli can phase-shift the SCN, and this can have an important impact on circadian rhythms. For instance, light during the early part of the night causes a phase delay in the SCN, while light in the early morning causes a phase advance. Other zeitgebers, such as social activity and work schedules, can also either directly or indirectly affect the SCN, as they influence the timing of food intake, physical exercise, light exposure, and sleep. In the absence of external zeitgebers, individuals express their endogenous period of circadian rhythms. This period is generally different from the 24-hour period, and is called the free-running period. Conditions without zeitgebers are, for example, constant darkness or constant light, in comparison with the usual light and darkness alternation, and a common example of the occurrence of this is in blind subjects. The free-running inherent rhythm of the SCN is slightly longer than 24 hours.1,14

Intercellular circadian rhythms

All of the aforementioned afferent pathways link the SCN to the daily changes in the external environment. In turn, the SCN act as the central pacemaker of the circadian system whereby daily rhythms are regulated according to external environmental changes. Indeed, it was gradually discovered that the SCN communicates with other brain regions to impart or entrain circadian rhythmicity in physiological and behavioral processes. For example, sleep/wake cycles are modulated by an efferent pathway via the paraventricular nucleus of the hypothalamus, and via a multisynaptic pathway to the pineal gland where melatonin is synthesized at night and suppressed by light during the day (Figure 1). Melatonin, secreted by the pineal gland, transmits information about the occurrence and duration of darkness; during short winter days, the duration of nocturnal melatonin secretion increases, whereas it decreases during long summer days.15,16 Moreover, melatonin itself has a zeitgeber function; in fact, melatonin, secreted under the hierarchical dependence of the SCN, influences the SCN in return by acting through specific receptors in this area (Figure 1).17 Indeed, preclinical studies have demonstrated that with respect to other areas in the brain, the SCN have a particularly high concentration of melatonergic MT1 and MT2 receptors, which are temporally expressed in a circadianmanner. It has been shown that expression of the MT1 receptor is regulated by both light and the central pacemaker, with a peak level of gene transcription occurring during the middle of the night.17

A major development in research in recent years has been the discovery that beside the SCN, various other circadian clocks are present in organisms.18,19 We now know that various nonneuronal tissues and non-SCN brain regions (eg, hypothalamic nuclei, forebrain, olfactory bulb, pineal gland, and the cortex) contain autonomous oscillators and are capable of generating circadian rhythms when isolated from the organism and cultured in vitro (Figure 1).1,4,19-21 These peripheral oscillators (as opposed to the central SCN clock) rely on feedback loops composed of clock genes and proteins, just as in the SCN clock. In all tissues studied to date, 5%-10% of the transcriptome displays circadian rhythms (ie, up to 10% of the genes are clock-controlled genes), but the subset of rhythmic transcripts is almost entirely distinct among tissues. This implies that the role of the clocks found in the SCN and those in the different peripheral tissues is distinct, and must reflect the particular functions of each tissue.4 The diversity of secondary clocks in the brain, their specific sensitivities to timegiving cues, as well as their differential coupling to the master SCN clock, may allow more plasticity in the ability of the circadian timing system to integrate a wide range of temporal information. Furthermore, this raises the possibility that pathophysiological alterations of internal timing that are deleterious for health may result from internal desynchronization within the network of cerebral clocks.19

Interestingly, a novel SCN output pathway to the ventral tegmental area via the median preoptic nucleus has been recently described (Figure 1).22 This projection may function as the circadian regulator of behavioral processes such as arousal and motivation, further linking well-known behavioral observations to reward-related actions and circadian rhythmicity. Another example of a circadian regulator is the hippocampus, pivotal in neuronal plasticity, learning, and memory processes, which shows rhythmic gene expression relatively independent of the SCN. In this context, it has been recently demonstrated that clock-related genes are highly expressed in hippocampal pyramidal cell layers, and that the expression of both protein and mRNA varies with a circadian rhythm, independent from that of the SCN, since it is detectable in isolated hippocampal slices maintained in culture. This can allow for the initiation of intrinsic rhythms necessary for timeof- day–dependent memory formation, which can be—and probably need to be—desynchronized fromthe SCN rhythm.23

Intracellular clock mechanisms

At the molecular level, circadian clocks use clock genes to generate self-sustained rhythmicity. Clock genes are expressed not only in the SCN, but also in extra-SCN brain regions as in most peripheral tissues. At their core, the clocks contain a cell autonomous oscillator that is generated by a transcription-translation negative feedback loop with a crucial delay between stimulus and response. In mammals, the circadian clock mechanism comprises a core set of genes that is highly conserved among species24-26: Circadian Locomoter Output Cycles Kaput (Clock; and its paralogue neuronal PAS domain protein 2, NPAS2), Bmal1 (also known as aryl hydrocarbon receptor nuclear translocator-like; Arntl), period homologue 1 (Per1), Per2, Cryptochrome 1 (Cry1) and Cry2 (Figure 2). During the day, the basic helix-loop-helix PAS-domain containing transcription factor Clock (or NPAS2) interacts with Bmal1 to activate transcription of a large number of output genes. Clock and Bmal1 also activate the transcription of the Per and Cry genes via E-Box sequences in their promoter, resulting in high levels of these transcripts (Figure 2).

The resulting Per and Cry proteins heterodimerize, translocate to the nucleus, and interact with the Clock–Bmal1 complex to inhibit their own transcription.27 The Cry proteins impair phosphorylation of Clock/Bmal1, thus reducing transcriptional activity of the dimer. During the night, the Per–Cry repressor complex is degraded; this leads to a reduction in the inhibitory complex through turnover, and the cycle starts again with a new round of Clock/Bmal1–activated transcription (Figure 2). Selectively in forebrain regions, NPAS2, a protein very similar to Clock, can bind Bmal1 and induce Per and Cry gene expression.20 NPAS2 may also function in the place of Clock in the SCN if the Clock protein is genetically disrupted.28

In addition to the primary loop, there is a second negative feedback loop involving nuclear orphan receptor genes, such as Rev-erbα, Rev-erbβ, Rorα, and Rorβ, whose transcription is activated by Clock–Bmal1 dimers (Figure 2). The result is the production of Rev-erbs and Rors with negative and positive regulatory effects on Bmal1 transcription, respectively.25 This secondary loop does not seem to be essential, but it is thought to add strength to the molecular clock. Finally, a number of other candidate clock components such as Timeless, Dec1, Dec2 and E4bp4 are involved, but their roles have not yet been clearly elucidated.24

Figure 2
Figure 2. Molecular mechanism of the core mammalian circadian clock.

The figure depicts a simplified scheme of the mammalian circadian rhythms core clock that is a transcription–translation negative-feedback loop with a delay between transcription and the negative feedback. In the nucleus the Clock/Bmal1 dimer binds to a specific chromosomal site (E-box) thus activating the expression of several genes, among them their regulators, Per1, Per2, Cry1, and Cry2. The Per/Cry protein dimers are phosphorylated in the cytoplasm by kinases such as casein kinase Iε /δ (CKIε /δ ) or glycogen synthase kinase 3β (GSK3β). Then the Per/Cry dimers translocate to the nucleus in a phosphorylation-regulated manner where they interact with the Clock/Bmal1 complex to repress their own
activators. At the end of the circadian cycle, the Per and Cry proteins are degraded in a CKI-dependent
manner, which releases the repression of the transcription and allows the next cycle to start. An additional
stabilizing feedback loop involves the activator Rorα and the inhibitor Rev-Erbα, which control Bmal1
expression and reinforce the oscillations. The black continuous lines indicate activation, the dotted black
lines indicate translocation between nucleus and cytoplasm, and the red continuous lines indicate inhibition.
After reference 25: Gallego M, Virshup DM. Nat Rev Mol Cel Biol. 2007;8:139-148. Copyright © 2006,
Nature Publishing Group.

The entire cycle takes about 24 hours to complete; however, not much is known about the stoichiometry and kinetics of this feedback loop. Light acts through the retina and direct neural pathways to the SCN to stimulate Per1 and Per2 gene expression (Figure 3). Whereas the genes coding for Clock and Bmal1 are turned on permanently, expression of Per and Cry genes is rhythmic, being highest in the first part of the day before being suppressed later.29,30 Light transmission to the SCN via the retinohypothalamic tract, mainly through glutamate and pituitary adenylate cyclase–activating polypeptide (PACAP), leads to activation of the N-methyl-D-aspartic acid (NMDA) and metabotropic glutamate (mGLU) receptors and PAC1 and VPAC2 receptors, resulting in membrane depolarization and an influx of Ca2+ into targeted SCN neurons (Figure 3). The first responders in the postsynaptic SCN neurons during phase resetting are a group of immediate early genes that includes Per1, Per2, c-fos, and arc. Induction of these genes involves an array of signaling pathways that seem, at least in part, to converge at the Ca2+/cAMP response element binding (CREB) protein pathway (Figure 3). Various kinases have been implicated in CREB signaling, including PKA, PKG, Ca2+/Calmodulin dependent kinases (CaMK) and the mitogen activated protein kinases (MAPK) Erk1/2. It has been suggested that the different kinases function at distinct times for the temporal progression of the clock: for instance, the cGMP/PKG pathway seems to be important for nightto- day progression.25,27 Recently, molecular insights into the mechanisms of circadian rhythms have provided evidence that different posttranslational modifications work in association with transcriptional regulation to finely tune our rhythms.

Indeed, posttranslational modifications have a key role in the maintenance of the delay in the negative feedback loop that is required to give the clock a circadian period. Among the different posttranslational modifications, a major role is played by phosphorylation events.25 Many of the kinases that contribute to the regulation of clock proteins are key cellular signaling components such as casein kinase I, glycogen synthase kinase 3 (GSK3), CaMKII, and MAPK, which also control vital cellular activities, eg, metabolism, development, differentiation, proliferation, and memory formation. Since at least some of these kinases and signaling pathways are clock-controlled themselves, the study of their circadian function may be of help in understanding the crosstalk between the circadian clock and cellular signaling, which likely contributes to the synchronization of physiology and the robustness of circadian oscillations.25,27

Figure 3
Figure 3. Molecular regulation of
the light signal to the suprachiasmatic nuclei (SCN).

Light is transduced into a neuronal signal conveyed to the SCN through
the retino-hypothalamic tract (RHT), resulting in the release of glutamate and
pituitary adenylate cyclase–activating peptide (PACAP) onto retino-recipient cells in
the SCN core. The activation of glutamatergic or PAC1 and VPAC2 receptors,
results in membrane depolarization and an influx of Ca2+. The resulting activation
of kinases such as mitogen-activated protein kinase (MAPK-Erk1/2), results in
the phosphorylation of cAMP response element binding (CREB) protein.
Activated CREB binds to the Ca2+/cAMP response element (CRE) in the
promoter region of both Per genes, activating their transcription.

As an example, several studies have pointed out a role for Erk-MAPK in the regulation of the circadian system in the SCN. Early studies showed that the MAPK cascade functions as one of the first transduction steps leading from light stimulation to rapid transcriptional activation, an essential event in the entrainment process.31 More recently, it was shown that MAPK is autonomously activated in the SCN, and that inhibition of MAPK activity results in dampened rhythms and reduced basal levels in circadian clock gene expression at the SCN single neuron level. Furthermore, MAPK inhibition attenuates autonomous circadian neuronal firing rhythms in the SCN, thus suggesting that light-independent MAPK activity contributes to the robustness of the SCN autonomous circadian system.32

Temporal abundance and activity of Per are regulated by casein kinases Iä and Iε (CKIä/å), which through phosphorylation, lead tomodulation of degradation and cellular localization of the Per protein. Recent work demonstrated that circadian rhythms were completely disrupted by two different approaches targeting the kinase activity and specific interaction between the kinases and the substrate, thus indicating that CKIä/å are essential for rhythm generation.33

Biochemical studies revealed that GSK-3β phosphorylates Per2 for nuclear localization, Cry2 for proteasomal degradation, and Rev-Erbα for stabilization.33,34

Recently, it was also shown that chromatin modifications through acetylation, deacetylation, andmethylation of histones bound to promoter regions of core clock genes participate in the regulation of oscillating transcription. Moreover, it was reported that the Clock gene possesses intrinsic histone acetyltransferase activity, and that the activation of core clock genes by Bmal1/Clock heterodimers is indeed preferentially coupled to histone acetylation.35

A role for brain-derived neurotrophic factor and related signaling in the regulation of circadian rhythms

Recent observations have suggested that circadian cycling of the activity of ERK-MAPK in the hippocampus profoundly regulates the capacity of novel experiences to trigger lasting memory formation.36 Interestingly, these data suggest that ongoing circadian cycling of ERK activation in the hippocampus, through the transcription-regulating cAMP/ERK/CREB pathway, is necessary for long-term memory stability (that is, repetitive reactivation of signaling cascades that were used in the initial formation of a memory is required for the persistence of that memory). This work suggests that ongoing cyclical reactivation of memory-associated signaling cascades is a necessary part of the memory stabilization and storage mechanism. Although it is widely accepted that circadian rhythms in general, and sleep cycles in particular, regulate the robustness of memory capacity, this study suggests that the circadian cycling of specific molecular signaling pathways may underlie these general cognitive phenomena.36,37 Brain-derived neurotrophic factor (BDNF) and its cognate receptor, the TrkB tyrosine kinase, are well-known mediators of synaptic plasticity in both developing and mature neurons. The neurotrophin BDNF has been implicated in the regulation of neuroplasticity, gene expression, and synaptic function in the adult brain, as well as in the pathophysiology of neuropsychiatric disorders and the mechanism of action of antidepressants.38,39 A growing body of evidence also supports a role for BDNF and TrkB in the modulation and mediation of circadian rhythms. As a starting point, high levels of expression of BDNF and TrkB were demonstrated in the rat SCN.40 It was reported that BDNF protein and mRNA levels in the rat SCN showed evident signs of variation over the course of a circadian cycle. The SCN content of BDNF protein remained low throughout the subjective day, began to rise early in the subjective night, and reached peak levels near the middle of the subjective night. BDNF mRNA levels in the SCN reached maximal values during the early subjective day, approximately 16 hours before the peak in protein content. After declining during the middle of the subjective day, the content of BDNF mRNA in the SCN remained at basal levels until the late subjective night.40 Diurnal variation in BDNF protein expression levels was demonstrated in the cerebellum, hippocampus, and cerebral cortex.41 In the same study, it was shown that CREB, a transcription factor regulating BDNF expression, was greatly activated by the phase advance in the entorhinal and visual cortex, suggesting the existence of CREB-mediated pathways of BDNF synthesis that are responsive to external light input.41

Converging evidence supports the hypothesis that BDNF serves to gate photic phase shifts: (i) blocking TrkB receptors inhibits light- and glutamate-induced phase-shifts; (ii) light-induced phase shifts are substantially attenuated in BDNF+⁄- mice; and (iii) exogenous BDNF administration during the subjective day allows light and glutamate to induce phase-shifts in the daytime in vivo and in vitro, respectively.42,43

More recently it was shown that as in the hippocampus, proteins from the plasminogen activation cascade responsible for BDNF activation are also present in the SCN, such as plasmin, plasminogen, tissue-type plasminogen activator, etc. The data support the hypothesis that these proteins regulate the conversion of proBDNF to mature BDNF (mBDNF) in the SCN, and that mBDNF availability acts as a gating mechanism for photic phase resetting. It is noteworthy that these proteins generally interact extracellularly, often bound to the extracellular matrix. As such, the consideration of processes thatmodulate SCN circadian clock phase-resetting should be expanded to include extracellular as well as intracellular mechanisms.44

Interestingly, the presence of a diurnal BDNF rhythm was also recently demonstrated in humans: plasma BDNF in human healthy males displays highest concentrations in the morn- ing, followed by a substantial decrease throughout the day, and the lowest values at midnight. Moreover, plasma BDNF levels were positively correlated with those of cortisol.45

Data from the same group recently showed the existence of a correlation between the daily levels of plasma BDNF and cortisol in women, corroborating the hypothesis of coregulation of cortisol, BDNF, and sex steroids in humans. This correlation suggests the possibility that glucocorticoid and neurotrophic tone may play a synergic role in the homeostasis of cerebral functions.46

It is known that one of the most common features of depressed patients is an altered hypothalamic-pituitary-adrenal axis, with high glucocorticoid secretion. It is possible to hypothesize that variations in BDNF levels, such as those observed in psychiatric patients, may also be related to disturbances in the function of those structures involved in determining circadian rhythms either directly (SCN function), and/or indirectly (altered release of glucocorticoids that are modulated by glutamatergic innervation of the SCN).

It should be reminded that the transcription-regulating cAMP/ ERK/CREB pathway, together with other signaling pathways, in particular the CaMKIV mediated signaling, is a major regulator of BDNF modulation in hippocampus, and has been suggested as having a role in both the pathophysiology of depression and the mechanism of action of antidepressants.39,47-49 During the last few years, it has been largely demonstrated that BDNF is involved in the mechanism of action of antidepressant drugs; in particular, an increase in BDNF expression— both mRNA and protein—follows antidepressant administration in both experimental animals and human patients.39,49

Recognition that circadian rhythm disruption also plays a key role in mood disorders has led to the development of the new antidepressant agomelatine, which is endowed with a novel mechanism of action distinct from that of currently available antidepressants. Agomelatine is an agonist of the melatonergic MT1 and MT2 receptors, and an antagonist of 5-HT2C receptors. The antidepressant activity of agomelatine is proposed to stem from the synergy between these sets of receptors, which are key components of the circadian timing system. Recent data from various groups, including ours, showed that agomelatine led to an increase in BDNF expression in treated animals, and that this effect follows a specific temporal profile and is mediated by a functional interaction between the melatonergic MT1/MT2 receptors and the serotonergic 5-HT2C receptors.50


In conclusion, alteration of circadian timing plays a crucial role in mood disorders. Since intercellular and intracellular processes in the brain implicated in the pathophysiology of psychiatric diseases follow a circadian rhythm regulation, this phenomenon may have important implications in the development of new agents in psychiatry. _


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Keywords: suprachiasmatic nucleus; circadian rhythm; clock gene; transcription factor; brain-derived neurotrophic factor; MAP kinase