Ticking Clocks and Changing Times

Published in Lab Times 03-2009.

The circadian pacemaker encodes the body’s response to seasonal change. Johanna Meijer and her group have a twisting tale of how their work on pacemaker cells revealed exceptions to the limit cycle oscillator theory. As we arrive at a consensus, do the results have more profound implications?

lt_2009_03_32_33What do the thirsty cries of a migratory swallow, the summer mating call of an oyster toad-fish, the squeaks of a nocturnal Syrian Hamster and the groan of a tired globe-trotter have in common? Their internal, rhythmic molecular clocks. Dictated by the rhythm pacemaker, biological clocks present a way of fine-tuning the physiological functions of an organism to suit the environment or precisely, the amount of daylight in its area. The molecular clocks endow migratory birds and polar animals with the ability to respond to changing seasons, nocturnal fauna to sense day-light and of course, humans to organize their work shifts and recover from a jet lag.

With the strong foothold provided by studies on the molecular mechanisms underlying circadian rhythms, recent research in this field has applied techniques in neurophysiology to address the function of the “pacemaker” for newer insights from a different level of organization. Johanna Meijer’s group has been caught up for over a decade with the question of how the suprachiasmatic nuclei (SCN), the mammalian version of the circadian pacemaker, respond to changes in day-length. Their “counterintuitive” findings coupled with the studies from other contemporary groups have given a new direction to the understanding of biological rhythms.

The Molecular Clock ticks in all organisms alike

From single-cell organisms to mammals and man, the circadian clock serves the same function – to equip the organism with an anticipatory behavior towards on-coming changes in seasons and hence offer a cue for various physiological functions viz. metabolism, mating and reproduction and predator-defense. The “pacemaker” that receives input mainly in the form of light, has evolved to be the sole structure responsible for driving circadian rhythms. The electrical activity of the pineal gland in lizards, the optic lobes in Drosophila, the retina in molluscs and the SCN in mammals is thus, modulated by light.

The first episodes

Having completed her doctoral thesis with Dr. G. A. Groos, Leiden University, The Netherlands and Prof. B. Rusak, Dalhousie University, Canada in 1989, Johanna Meijer undertook post-doctoral training in Leiden and Biological Center, Haren, The Netherlands. Currently a professor in Neurophysiology at the Department of Molecular Cell Biology, Leiden University Medical Center (LUMC), The Netherlands, with her own research group, Johanna has focused on elucidating the contribution of individual neurons in the SCN to the electrical activity of the functioning pacemaker. Their first observations came from extracellular recordings of electrical activity in SCN slices. “Extracellular or ex vivo recordings are simple and non-invasive but require extremely low noise levels”, admits Johanna. The technique enabled the simultaneous quantification of the circadian discharge pattern of many neuronal subpopulations within the SCN. The results showed discrepancies in the phase of the activity peaks of small neuronal subpopulations. While large phase differences were conspicuous among very small groups of neurons, within the subpopulations the neurons were mutually synchronized.

It came as a surprise when Johanna and colleagues determined, using cluster analysis, the activity profile of single cells from the multiunit signal of the SCN ensemble. The single cells of the 24-hour molecular clock showed only short durations (5-7h) of increased neuronal activity! “Our results did not comply with the ‘half-conscious’ expectation that SCN neurons are active throughout the day and our manuscript was at first rejected by many journals”, recalls Johanna. “Only with the timely help of Prof. Joseph Takahashi, the paper finally made its appearance in PNAS” (PNAS vol.100(26):15994-99), she sighs, recollecting the relieving moment.

Despite the controversies, the determined group proceeded to confirm their hypothesis, “changes in phase distribution among oscillating neurons is the most effective mechanism to code for the response of the SCN to day-length”.  They began their studies with simulations (Journal of Biological Rhythms vol.21(4):301-313) and thereafter took to in vitro and in vivo approaches to make multiunit subpopulation and single-unit recordings and confirmed the plausibility of their prediction (Current Biology vol.17:468-73). Individual cells of the SCN peak at different times of the day with small periods of neuronal activity. The activity peak of the ensemble of single neurons, as both their simulations and single-cell recordings revealed, is condensed in shorter day-lengths implying a higher degree of synchronization among the contributing neurons. Conversely, oscillating neurons in long days are desynchronized producing a broader peak of activity of the ensemble. The response to day-length is thus, in every means, a network property.

Setting a trend after a second stumbling block

Even as the group was drawing to a conclusion about the “intrinsic” “network” property of the SCN and its response to day-length, they were beset with making another tough hypothesis on phase shifts and photoperiodicity (PloS ONE vol.4(3):e4976). It has for long been believed that high-amplitude rhythms show reduced phase shifting responses than do low-amplitude rhythms. But again, with the Meijer group, it had worked the opposite! They observed that high-amplitude rhythms characteristic of short days (when neurons are highly synchronized resulting in a high frequency in multiunit activity) exhibit a large overall shift in response to a light input signal. They excluded a lack of response to NMDA as a factor for decreased phase-shifting capacity among neurons from long days as the group observed no difference in the acute response to increased concentrations of the neurotransmitter. Though their observations vividly explained the photoperiodic modulation of the phase shifting capacity of the circadian rhythm, Johanna and colleagues had to fight it hard to proclaim their discovery owing to the general disconcert of their predictions to the classical “limit cycle oscillator theory”. “The theory holds valid in explaining rhythms in single-cell organisms, but complex neuronal networks are governed by different rules”, affirms Johanna. She adds on that phase-shifting responses are not explained by merely accounting for the responsiveness to light but it also takes neuronal network analysis to decipher the mechanism.

Johanna is now pleased that there are many other groups that have confirmed her observations using molecular and neurophysiological approaches and have now drawn to a consensus on the short-term activity of single cells and their effect on the concomitant output of the SCN. Despite the challenges that she was confronted with, Johanna explains that the heart of her interest in studying how behavioural traits are encoded by the brain kept her spirit alive and lent her the hue of persistence. “What makes biological rhythm research attractive is”, she peps up with excitement, “it is here that one can quantify rhythms in terms of period or revolution time, shifts in phase along the X-axis and amplitudes and this enables a comparative physiological research across different species. Moreover, even within the same organism it allows comparison of different levels of organization – from molecular and cellular to networks and all the way up to the behavioural level”. Johanna ascertains that despite the reductionist approach in Science, it is many times worthwhile to look at higher levels of organization since many attributes, including those of the circadian system, originate here and not all properties are encoded for at the genetic level.

Untrodden paths

The Meijer group has a sound combination of technical expertise with specialists in extracellular electrophysiology in live animals and slices, patch clamping, sleep recordings and mathematical modelling. Johanna and her colleagues also collaborate with international scientists, Profs. Gene Block and Chriss Colwell, UCLA and Prof. Bill Schwartz, UMASS Medical School, United States.

The inquisitive team is now all set to apply their studies in addressing the question – is ageing associated with those changes in the SCN that are observable during long days? The similarities, they justify, between ageing and long day-length include a greater degree of de-synchronization between neurons and hence, a decreased phase-shifting capability; and a decrease in the amplitude of the rhythms. One cannot but agree to their rationale. Ageing is indeed manifested as increased difficulties in sleeping at night and staying awake during the day; and problems with adjusting to new time zones and working in shift hours. Does the circadian rhythm, as we age, act out of order? Do the contributing neurons stop acting in harmony? Does the SCN become refractory to seasonal changes? The Meijer group is game to crack them all.

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