Neurophysiology of the Circadian System

Research lines of the Meijer group

The suprachiasmatic nuclei (SCN) of the anterior hypothalamus drive circadian rhythms of about 24 h (circa: about; dies: day) in physiology and behavior. In addition, the SCN play a crucial role in seasonal rhythms as they measure the changes in day-length that occur throughout the day. A specialized projection from the retina to the SCN is responsible for the influence of light on the circadian pacemaker, and via this projection the pacemaker is synchronized to the day nigt cycle, as well as to the changing seasons (see Fig 1). Humans have circadian rhyhms in for instance: cognitive performance, blood pressure, temperature, hormone levels and sensitivity to medication. Also 10% of our genes is rhythmically expressed. Seasonal rhythms exist in our immune system, in the occurrence of several diseases, in fertility, birth rate and sleep need.  

Figure 1. The Suprachiasmatic nuclei (SCN) contain a circadian clock that drives daily rhythms. Kluver barrera staining of a coronal hypothalamic brain slice with the SCN visible as two nuclei directly above the optic chiasm. 

Circadian rhythms are generated in individual neurons by ‘clock genes’ via intertwined negative and positive transcription-translation feedback loops. Electrical impuls activity is a major output signal of the SCN. In our laboratory, we perform recordings of electrical activity by extracellular recording methods, both in vitro and in vivo. In vitro recordings are performed in acutely prepared slices of mice and rats that can kept alive and spontaneously active for about 2 cycles (48 h). This allows us to investigate after effects ex vivo in the slice, such as responses to behavioural manipulations, to medical treatment or to day length. In vivo recordings are performed in freely moving rodents with the aid of counter balanced rotating contacts. Such recordings can be performed for many days up to several weeks. The acute responsiveness to retinal stimulation, to neurotransmitter or drug injection and to behavioural manipulations can be investigated, which offers the important advantage to study the circadian pacemaker, as an integrated part of the central nervous system.

The Cryptochrome 1 and 2 genes are indispensable for the presence of behavioural rhythmicity in mice and in the absence of mCry1/mCry2 genes, mice reveal a-rhythmic behavioural activity patterns. We performed recording of electrical activity in slices and showed that Cryptochrome 1 and 2 genes are required for the presence of electrical activity rhythms in the SCN (see Fig 2). These results are an example on our in vitro recording studies, and show that the clock genes Cry1and Cry2 not only function to generate circadian rhythms, but are also required for the SCN to act as a circadian pacemaker (i.e. form a rhythmic output signal to drive rhythms in the central nervous system)

Figure 2. Firing-Rate Patterns in SCN Slices from Wild-Type and mCry-Deficient Mice Kept in Constant Darkness (DD)

The running-wheel activity patterns of the mice in the last 7 days prior to slice preparation are indicated above the records. The black and gray bars above each plot indicate the subjective night and day, respectively. CT, circadian time.

(A) Circadian rhythm in multiunit activity (MUA) in the SCN of a wild-type mouse.

(B) Representative example of the gradually decreasing firing frequency in the SCN of a mCry1-/- mCry2-/- mouse.

(Albus H, Bonnefont X, Chaves I, Yasui A, Doczy J, van der Horst GT, Meijer JH. 2002. Current Biology 12 p.1130-1133.)

Recently we performed in vivo recordings to investigate the ability of the SCN to encode photoperiodic information. We kept mice under long and short day length and investigated the waveform changes that are present in the SCN. In long days, the SCN electrical activity is high for long periods, reflecting the long days, while in short days, SCN electrical activity is high for short periods of time. After release in constant darkness these patterns remained consistent for at least 4 days, which indicate structural changes (or plasticity) in the SCN pacemaker (Fig 3).

Figure 3. SCN neuronal activity measured in vivo following entrainment to short or long day photoperiods.

(A,B) Two typical examples of recordings of multiunit electrical activity of freely-moving mice showing the last two days in an LD 8:16 h (A) or LD 16:8 h (B) photoperiod followed by the first 4 days in DD (dark indicated by grey background). Individual data points represent 10 s epochs. Smoothed data are indicated by a white line. Drinking activity is shown at the bottom of each plot. (C) Mean width of the peak, measured at half maximum electrical activity (± SEM) during LD and the first four days in DD. Grey bars represent data from animals kept in short day photoperiod and black bars represent data from long day animals (short day-length, n=4,4,4,4,2 on the consecutive days, and long day-length, n=4,5,5,5,4 on the consecutive days). Asterisks indicate a significant peak width difference between the recordings (t-test, *P<0.05, **P<0.001). (D) Smoothed waveforms of the recorded MUA on the first day in constant darkness. X-axis represents extrapolated external time (ExT 0 = mid-night, ExT 12 = mid-day). Top bar indicates the prior light-dark cycle, grey is lights-on. Data are normalized by setting the first trough value to zero and the first peak value to one. Grey lines represent individual recordings, black lines are the averaged waveform (short day-length n=4, long day-length n=5).

(VanderLeest HT, Houben T, Michel S, Deboer T, Albus H, Vansteensel MJ, Block GD, Meijer JH. 2007. Current Biology 17 p.468-473.)

We performed additional in vitro recordings in mice from long and short days and questioned whether the behavior of the SCN in long and short days reflects a similar behavior of individual SCN neurons. By an analysis of single cell electrical activity patterns, with the aid of a principal component and cluster analysis of spike waveform, we followed several single units throughout the circadian cycle. Remarkably, the patterns of individual SCN neurons are very similar in long and short days (Fig 4). The essential difference between long and short days appeared to be the phase distribution among the neurons. In long days, the neurons show large differences in phase, which means that the show peak activity at various phases of the circadian cycle. This results in long periods of activity at the population level, and this in the output of the SCN. In short days, the units show a narrow distribution in phase, resulting in shorter periods of electrical activity at the population level. We conclude therefore that coding for photoperiod is a neuronal network property that depends on the consorted action of different neurons in the network. This is in contrast with rhythm generation, which is a single cell attribute.

Figure 4. Single units in short and long day-length.

Four representative examples of single unit electrical activity for animals housed under a short (A) and long (B) day light regime. On the left, circadian activity patterns with the grey background indicating the projected dark period. In the middle, the corresponding spikes with mean spike waveform (white line). On the right, the inter-spike interval histogram. The data indicate that single units show short activity patterns in long and short days.

(VanderLeest HT, Houben T, Michel S, Deboer T, Albus H, Vansteensel MJ, Block GD, Meijer JH. 2007. Current Biology 17 p.468-473.)

In general terms, our group is interested in the level of organization that codes for biological attributes, and we expect that some properties arise at the level of single cells, such as rhythm generation, while many other attributes require neuronal networks, such as photoperiodicity. In addition to the methodologies described above, strong interactions exist with Dr. S Michel, who adds patch clamp and imaging studies to our neurophysiology group, Dr. T. deBoer, who adds EEG recordings to our lines of expertise, and Dr. RJ van den Berg, who performs studies on isolated cultured neurons. We expect that together, we cover a broad range of technologies, which should allow for a broad and almost unique investigation of the properties of the circadian system.

In the neurophysiology group of Meijer, we have the following major projects and questions, and in most of these projects, the methodologies described above, are applied:

1. Investigation of the mechanism for photoperiod: what is the mechanism that introduces plasticity in the neuronal network and what (neuronal) coupling mechanism is modulated by day-length?
2. In the 24-hour society, an increasing part of the population is involved in shift work. However, shift work is associated with fatigue-related problems, and health risks in the long run. We investigate phase resetting to a shifted light-dark cycle, and mimic protocols applied in shift work. Which protocols and light-wavelength are optimal for rapid readjustment? What are the effects of different protocols for the immune system?
3. Medication at some moments is more optimal than at other moments, which is often referred to as ‘chronopharmacology’. There is surprisingly little systematic research on the mechanism underlying enhanced sensitivity to medication at some phases of the circadian day, and reduced sensitivity at other phases. This phenomenon is further investigated in our group.
4. It has recently become evident that clock-mutant mice are sensitive to the development of the metabolic syndrome. We have planned critical experiment to identify the causal relation between metabolic symdrome, sleep disturbances and the circadian clock.
5. Aging is associated with a decrease in circadian amplitude which explains that aged people fall asleep during the day, and have problems to sleep during the night. We want to investigate aging of the biological clock, and want to find ways to increase the circadian amplitude.

Highlights

1. VanderLeest, H.T., Houben, T., Michel, S., Deboer,T., Albus, H., Vansteensel, M.J., Block, G.D. and Meijer, J.H., Seasonal encoding by the circadian pacemaker of the SCN, Current Biol. 17 (2007) 468-473. 
2. Deboer, T., Détari, L., Meijer, J.H., Long term effects of sleep deprivation on the mammalian circadian pacemaker. Sleep, 30 (3) (2007) 257-262
3. Rohling, J., Wolters, L., Meijer, J.H., Simulation of day-length encoding in the SCN: from single-cell to  tissue-level organization. J. Biol. Rhythms, 4 (2006) 301-13.
4.  Itri, J.N., Michel, S., Vansteensel, M.J., Meijer, J.H., and Colwell, C.S., Fast delayed rectifier potassium current is required for circadian neural activity, Nature Neuroscience, 8(5) (2005) 650-656.
5. Albus, H., Vansteensel, M.J., Michel, S., Block, G.D. and Meijer, J.H., A GABAergic mechanism is necessary for coupling dissociable ventral and dorsal regional oscillators within the circadian clock., Current Biol., 15 (2005) 886-893.
6. Schaap, J., Albus, H., van der Leest, H.T., Eilers, P.H.C., Détari, L., Meijer, J.H., Heterogeneity of rhythmic suprachiasmatic nucleus neurons: implications for circadian waveform and photoperiodic encoding, PNAS, 26 (2003) 15994-15999.
7.  Deboer, T., Vansteensel, M.J., Détari, L., Meijer, J.H., Sleep states alter activity of suprachiasmatic nucleus neurons, Nature Neuroscience, 10 (2003) 1086-1090. see also: Colwell, C.S., Michel S (2003) Sleep and circadian rhythms: do sleep centers talk back to the clock? Nat Neurosci 6: 1005-1006
8. Vansteensel, M.J., Yamazaki, S., Albus, H., Deboer, T., Block G.D., Meijer J.H., Dissociation between circadian Per1 and neuronal and behavioral rhythms following a shifted environmental cycle, Current Biology, 13 (2003) 1538-1542. 
9. Albus, H., Bonnefont, X., Chaves, I. Yasui, A., Doczy, J., van der Horst, G.T.J., Meijer, J.H., Cryptochrome-deficient mice lack circadian electrical activity in the suprachiasmatic nuclei, Current Biology 12 (2002) 1130-1133.