Neurophysiology of the Circadian System

Research lines of the Meijer group

   Joke Meijer

 

  1. Neuronal network organization of the circadian clock; basic research
  2. Decline of clock function in aging, depression, migraine and neurodegenerative diseases
  3. Circadian rhythms and the metabolic syndrome
  4. Retinal signaling to the SCN clock
  5. Chronobiological optimization of drug application in humans
  6. ADHD and the circadian system

I.      Neuronal network organization of the circadian clock; basic research

 

Most bodily functions show pronounced 24h rhythms. These rhythms have developed as an adaptation to the recurring changes in the environment, brought about by the rotation of the earth around its axis. In order to anticipate to these changes, innate clocks have evolved that allow organisms to prepare for the predictable onset of night and day. In humans, the central clock is located in the suprachiasmatic nucleus (SCN). The SCN is a bilateral structure, located at the base of the brain, with the ventral aspect immediately above the optic chiasm. The SCN contains about 10.000 neurons on each side and maintains direct and indirect connections with many parts of the central nervous system. Twenty-four hour rhythms exist in the activity of the central nervous system, hormonal levels, activity of organs such as lung, liver and kidney, cardiovascular function, as well as in the expression of at least 10% of our genes, throughout the body.

Light is the major external stimulus that synchronizes the endogenous clock to the external 24h cycle. It reaches the neurons of the SCN via a monosynaptic pathway, formed by melanopsin containing retinal ganglion cells, that project with glutamate and PACAP containing fibers to the SCN. Generation of circadian rhythmicity occurs at the single cell level and is based on an intertwined negative feedback loop between clock genes and their protein products. The genetic basis for rhythm generation can explain that isolated cells of the SCN are capable of generating circadian rhythms and do not require rhythmic input. The implication is that the SCN functions as a multi-oscillator structure in which the different neurons coordinate their activity patterns. In our laboratory we have investigated how the individual neurons determine the overall output of the clock. We have discovered that the individual neurons oscillate with small phase differences between them (Schaap et al, 2003). The phase distribution among the neurons determines the strength of the overall rhythm signal, rather than the rhythm strength of individual cells (Rohling et al, J.Biol. Rhythms 2006; VanderLeest, Curr Biol, 2007; vanderLeest PlosOne, 2009). The identification of coupling mechanisms in the circadian pacemaker is part of the project Complexity, NWO (co-PI: JHT Rohling) and includes neuronal network recordings, in vitro electrophysiology, per2 bioluminescence and patch clamp recordings, as well as computational biological approaches (co-PI: S. Gielen, Radboud University, Nijmegen).

The insight that the strength of the rhythm is determined by synchronization and communication among rhythmic neurons, rather than by the rhythm amplitude of the individual cell leads to the important implication that rhythm disorders, as observed in aging and disease (see projects II-IV) may be caused by a lack of communication within the neuronal network of the SCN.

II.      Decline of clock function in aging, depression, migraine and neurodegenerative diseases 

 

Disruptions in the circadian system, are commonly associated with aging (see Farajnia et al, 2012), depression (see Hampp et al, Curr Biol. 2008), Fraxile X syndrome (see Zhang et al, Am J Human Genet. 2008) and with neurodegenerative disorders including Alzheimer’s disease, Parkinson’s disease and Huntington’s disease. These patients as well as aged people have difficulty sleeping at night and staying awake during the day. While the underlying pathology leading to sleeping and rhythm disorders in these patients has not yet been identified, several studies have been carried out using mouse models of these neurodegenerative diseases. Most of these mouse models exhibit circadian disruptions and there is at least some evidence that treatments designed to stabilize these rhythms can improve other, non-motor symptoms of these mice (for review, see Meijer et al, Progress in Brain Research, in press). In humans, a stabilization of circadian rhythms and sleep-wake cycles, leads to a decrease in depression. We have shown that mice with clock gene mutations show differences in mood regulatory brain area, suggesting a causal relation between rhythm disturbances and depression (Hampp et al, Curr. Biol. 2008, discussed in Science, 2008). In patients suffering from migraine, a circadian rhythm exists in the migraine onset, and jet lag is a known trigger for migraine attacks. In collaboration with M Ferrari (dept Neurology) and A. van den Maggdenberg (dept. Human Genetics) we have shown that R192Q knock-in mice show reduced robustness of the circadian system and respond with extraordinary large phase shifts in response to environmental light (van Oosterhout et al, Ann Neurol 2008). We propose that migraineurs lack the physiological retardation of phase resetting, which could lead to acute imbalance of brain systems, leading to an attack (see further under T. de Boer, for related sleep studies).

One mechanism by which aging and disease can alter the function of the circadian system is by reducing the strength of the inter-cellular coupling within the SCN circuit. We explored this possibility by investigating the effect of aging on the SCN neuronal network organization. Longitudinal studies in aging mice (0-2.5 years) showed phenotypic changes in behavioral rhythms and EEG characteristics. The phenotype for aging appeared fully developed at the age of 2 years. Electrophysiological recordings in the neuronal network of the SCN of 2 year old mice revealed characteristic differences in phase distribution, with a subpopulation oscillating in antiphase (Farajnia et al, 2012). This explains the reduction in the overall rhythm produced by the SCN. Patch clamp recordings from individual cells of the SCN revealed a near absence of rhythmicity and strong distortion of specific ionic membrane channels (FDR and IA, but not SDR). The aged phenotype in young cells could be mimicked by blockage of the specific ionic channel, indicating a causal relation, and offering tool for further investigation (Farajnia et al, 2012). An important implication is that the network appeared capable of compensating the deficiencies at the single cell level.

We have shown that the excitatory transmitter glutamate is present in the retinofugal patway to the SCN and that blockage of this transmitter blocks light effects on the SCN (see de Vries et al, 1993;1994). We have also shown that the inhibitory transmitter GABA is important for coupling within the SCN (see Albus et al, 2005) and that the peptide VIP is indispensable for phase synchronization and adaptation among SCN neurons (see Lucassen et al, 2012). So, one likely consequence of a number of diseases of the central nervous system is a change in the balance between excitation and inhibition synaptic transmission. A number of studies have found evidence that the levels of VIP in the SCN are reduced with aging and with neurodegenerative diseases in humans and rodents. This data adds key support to our suggestion that an important consequence of disease pathology will be to alter intra-SCN coupling within the circadian circuit and decrease the synchrony of the SCN population. Current research focusses on restoration of rhythm amplitude (see van Oosterhout et al, PloSONE, in press). See further under T. de Boer (sleep disturbances in aged mice), under S. Michel (patch clamp recordings in aged SCN neurons) and under J. Rohling (computational modeling studies of the aged SCN clock).

III.      Circadian rhythms and the metabolic syndrome

 

Obesity and type 2 diabetes mellitus (T2DM) are an increasing risk factor in modern society. In the past decade, a strong and potentially causal relation between metabolic disorders and disturbances of the circadian system has been elucidated. The output of the SCN clock is crucial for synchronization of many metabolic and endocrine parameters such as glucose, insulin, cortisol, leptin, ghrelin, neuropeptide Y, as well as for the synchronization of peripheral oscillations of clock genes. Animal models mutant for clock genes have indicated a link between clock genes and metabolic disturbances. For instance, CLOCK-/- mutant mice have a greatly attenuated diurnal feeding rhythm, are hyperphagic and obese and develop a metabolic syndrome of hyperleptinemia, hyperlipidemia, hepatic steatosis and hyperglycemia. As clock gene mutants are not specific to the SCN, the detrimental effects of disturbed rhythms may have their origin in targets in the body, other than the SCN. It is not clear, therefore, to what extent the SCN itself is involved in metabolic disorders. Given the accumulating evidence for disorders of SCN cellular organization in aging, neurodegenerative disorders and dementia, this question is also clinically relevant, as it would explain comorbidity between different disorders.

In this project we investigated the effects of ablation of the SCN, and show that such selective ablation leads to severe development of hepatic insulin resistance (see Coomans et al, submitted to Diabetes). Furthermore we investigate the influence of attenuated rhythm amplitude on metabolic parameters (see Coomans et al, submitted to FASEB). We use results from project I and reduce SCN rhythm amplitude by manipulation of the neuronal phase synchrony. In vivo recordings in the SCN show that the manipulation decreases the amplitude of the SCN rhythm by 50%. This decrease is similar to the decrease in rhythm amplitude observed in aging.  We use this manipulation to investigate the effects of physiological (i.e. naturally occurring) reduction in rhythm amplitude on metabolic parameters. The experiments are part of a TOPGO grant on aging (PI: Meijer), and are performed in collaboration with the department of Human Genetics (Willems van Dijk), the Dept of Endocrinology (N.R. Biemasz, P.C.N. Rensen), TNO (L.M.Havekes) and the NIN, Amsterdam (A. Kalsbeek). See under T. de Boer for related sleep studies.

IV.      Retinal signaling to the SCN clock

 

Light is the primary cue (Zeitgeber) in the synchronization of endogenous circadian rhythms with the environment. Light information reaches the brain exclusively via ocular photoreceptors and is transmitted to the SCN via the retinohypothalamic tract. We have characterized the response properties of the SCN by in vivo recordings in anaesthetized animals (Meijer et al, 1989), and are now able to perform such recordings in freely-moving mice (see Meijer et al, J. Neurosci 1998; deBoer et al, Nature Neurosci, 2003). We have established that SCN neurons respond in a sustained way to light information, with a discharge level that is dependent on light intensity and the time of the cycle. In 2002, a new photopigment, melanopsin, was identified, that is present in about 1% of the retinal ganglion cells. Melanopsin-containing ganglion cells form the major input to the SCN clock, as well as to the pretectum for the regulation of pupil diameter. The identification of melanopsin has triggered new lines of investigation on the contribution of different retinal photopigments in regulating the SCN, the use of different wavelengths of light for the treatment of depressive disorders, and for optimization shift work adjustment.

As the spectral sensitivity of melanopsin shows maximal sensitivity to blue light, the prevailing view is that blue light is of major importance to regulate circadian rhythmicity. None of the studies, however, explored the specific contribution of UV light to the responsiveness of the SCN neurons to light. We obtained new data indicating that UV light can fully replicate melanopsin mediated light responses at the level of circadian behaviour and SCN electrophysiology. Our results also showed that UV light was as effective as white light in inducing sleep in mice and that in the absence of the melanopsin there was no attenuation of UV induced sleep induction compared to Opn4+/+ mice. This study is the first to demonstrate that UV light exposure induces sleep in mammals during their habitual wake period, and suggests that UV irradiance detection may be an important feature of additional non-image forming responses to light (see Current Biol 2012, in press).

We are part of a larger consortium of the Welcome Trust (title: Melanopsin signaling: phototransduction, behavioural regulation and clinical relevance) and help identify the contribution of photopigments to SCN neuronal responses by using retinal transgenic animals constructed at Oxford University, and by performing in vivo electrophysiological SCN recordings.

V.      Chronobiological optimization of drug application in humans

 

Chronopharmacological studies have revealed circadian variability in kinetics and dynamics of drugs, including analgetica, anti-inflammatory drugs, anticancer drugs, antibiotics, benzodiazepines, and cardiovascular drugs. Drug administration at the right time of the day, adjusted to the individual circadian phase, is likely to improve the outcome of pharmacotherapy, leading to improved therapeutic efficiency, decreased toxicity (less adverse effects), and/or lower drug dosing. A chronotherapeutic approach would be beneficial particularly to diseases that manifest according to a circadian pattern (such as asthma, migraine, heart stroke) and/or diseases (possibly) associated with circadian disfunctioning like cancer, metabolic syndrome, depression, hypertension, gastrointestinal diseases. Despite growing interest in this topic, the moment of drug administration is often neglected in clinical settings, and little is known on the underlying mechanisms.

Time-dependent changes in pharmacokinetics may proceed from 24h changes in absorption, distribution, metabolism and/or excretion. Ideally, research on circadian rhythmicity should address all these determinants, which could be done by a novel approach, including ‘semi-simultaneous’ oral and intravenous drug administration at different time points throughout a 24h cycle. We plan to use probe drugs, investigating the relative contribution of all relevant parameters to overall pharmacokinetics of the compound.

For the central nervous system (CNS), the chronopharmacological profile of a drug with CNS response (desired / toxic) is the result of pharmacokinetic and pharmacodynamic processes that all may be subjected to diurnal variation. The pharmacokinetic processes that determine the CNS target site pharmacokinetics include plasma pharmacokinetics, blood-brain barrier transport and intra-brain distribution. The pharmacodynamic processes encompass interaction of the drug with its target, target activation and subsequent signal transduction that ultimately leads to the CNS response. Diurnal variation in CNS drug effects has been reported for a number of CNS drugs and for treatment of a number of CNS diseases, however, without explicit distinction between target site pharmacokinetics and the pharmacodynamics.

In human subjects we investigate the effects of administration at different clock times of oral and intravenous administration of very low doses of midazolam. Midazolam is a model drug for cytochrome P4503A (CYP3A4)-mediated drug metabolism which occurs in the liver and intestine. Our results indicate that the PK of oral midazolam are subject to significant diurnal variation, whereas the PK of iv midazolam does not differ throughout the 24 hour period. These data suggest that the route of drug administration is important in chrono-pharmacological experiments. As CYP3A4 is involved in the metabolism of 50-60% of all clinically available drugs, these findings have important implications for our understanding of the circadian regulation of xenobiotics.

This research was subsidized initially by “vrije beleidsruimte” LUMC board, and is now subject of the STW grant, entitled “On TIME”, Chronobiological optimization of drug application in humans, and is a collaboration with Dr. K. Burggraaf (CDR, co-PI), and Dr. L. de Lange (LACDR). The project is associated with the Leiden Center for Translational Drug Discovery and Development (LCTD3) neuropharmacology group focusing on chronification of disease and treatment development.

VI.      ADHD and the circadian system

 

Attention-Deficit Hyperactivity disorder (ADHD) is characterized by inattention, hyperactivity and impulsiveness, symptoms that closely resemble those observed following sleep deprivation.  Children and adults with ADHD frequently exhibit sleep problems, including shorter sleep duration, difficulty waking up, daytime sleepiness, increased nocturnal movements, longer sleep latencies, and more nocturnal awakening.  These symptoms affect a large proportion of adults with ADHD, with 65% complaining of always having trouble sleeping `patients with sleep onset insomnia, additional symptoms are observed, most notably delayed onset and offset of sleep, delayed dim-light melatonin onset, and an attenuated amplitude of the rest-activity cycle , all symptoms consistent with a disruption of the circadian clock.  Supporting this is the recent observation that a polymorphism associated with the gene clock, one of the molecular “gears” of the intracellular circadian clock, may be a contributing factor for adult ADHD.

Methylphenidate is an amphetamine derivative and is a preferred treatment for ADHD in both adults and children.  It blocks both dopamine and norepinephrine transporters, increasing levels of these neurotransmitters in the synapse.  While methylphenidate is quite effective at managing the inattention, impulsiveness and hyperactivity associated with ADHD, it may not help the co-morbid sleep problems, and in fact methylphenidate may exacerbate these symptoms.  In children, placebo-controlled studies have demonstrated that methylphenidate leads to longer sleep latency, lower sleep efficiency, decreased total sleep time, increased motor activity at sleep onset, decreased circadian amplitude, and phase delayed daily rhythms.  Many of these symptoms are consistent with either a phase delayed circadian clock, or a circadian clock with a lengthened period.  It has long been appreciated that amphetamines, such as methamphetamine, can have a pronounced affect on the rat circadian clock, lengthening period, and disrupting entrainment to light-dark cycles.  A few studies have demonstrated that methylphenidate can alter overt circadian rhythm patterns in rats, but the circadian clock located in the suprachiasmatic nucleus was not examined.  We hypothesize that methylphenidate alone will lead to changes of the circadian clock that are consistent with a phase delayed circadian clock. Our findings indicate that methylphenidate alters the electrical firing rate rhythms in the suprachiasmatic nucleus, and induced (i) a delay in the trough of the rhythm, (ii) an increment in rhythm amplitude and (iii) a reduction in rhythm variability. EEG recordings showed a delay in the timing of their sleep-wake cycle (see further under T. de Boer). These observations suggest that methylphenidate alters the underlying circadian clock, and these changes are consistent with clock alterations that would promote sleep-onset insomnia. If this is true, then it is possible that methylphenidate may also contribute to ADHD symptoms by decreasing the quality of sleep.  A thorough screen of other ADHD medications may reveal better alternatives for managing ADHD symptoms that do not alter the underlying clock in such a way so as to increase the likelihood and severity of insomnia. Our present findings are currently under minor revision (Antle et al, Neuropsychopharmacology). Future studies are planned in collaboration with Antle (Univ Calgary) and Mendoza (Strasbourg, France)) to evaluate the effects in diurnal animal species. Our aim is to evaluate the side effects of methylphenidate as a function of time of day, leading to recommendations for patients.

The project is performed in collaboration with Strasbourg is part of a “European Lab” (established and honored by French government in 2012), with Dr. E. Challet (coordinator), A. Kalsbeek, NIN, Amsterdam, and Meijer, LUMC, to translate circadian findings from nocturnal to diurnal species.