Cellular Mechanisms for Circadian Timing

Many physiological functions and behavior in almost all organisms are regulated in a daily fashion to preserve energy or avoid predators, to name just a few obvious reasons. These daily rhythms of sleep and wakefulness, body temperature, blood pressure etc, are controlled by an endogenous clock. This “circadian” clock works independently of environmental cues like light/dark cycle, generating a rhythm of about 24 hours (thus “circa-dian”).  In mammals, the “master” pacemaker resides in a brain region which is part of the hypothalamus and called suprachiasmatic nucleus (SCN, located just above the crossing of the two optic nerves). Neurons in this nucleus receive light information from specialized cells the retina via the retino-hypothamalic-tract.

This part of our group is investigating cellular mechanisms of circadian pacemaker neurons in mammals. We use electrophysiological and imaging technologies (see Methods) to measure membrane properties and intracellular messengers involved in pathways for entrainment and resetting.

  scn signaling

Figure 1: Simple sketch of some signaling pathways we studied. PACAP in co-transmitter and modulator of glutamatergic transmission form the retina to the SCN. VIP is expressed and released by ventral SCN cells and is a crucial factor for coupling and synchronization within the SCN. GABA is the main neurotransmitter found in the SCN and seems to play a role in ventral-dorsal synchronization.


During the past years, we investigated the role of peptides in the resetting of phase of the circadian system and the ionic conductances regulating circadian rhythmicity in the SCN.

The highlights of this research are:

  1. Mechanism how pituitary adenylyl cyclase activating peptide (PACAP) enhances light-induced phase shift in the SCN. PACAP is modulating glutamatergic synaptic transmission in the SCN. We developed and use PACAP-deficient mice to functionally test the role of PACAP in behavior. (Michel et al 2006a, Colwell et al., 2004)
  2. The role of vasointestinal peptide (VIP) on circadian rhythms in behavior and electrical activity in the SCN. VIP modifies synaptic transmission within the SCN and seems to affect coupling between pacemaker neurons. Development of VIP-deficient mice revealed a phenotype with many circadian deficits, up to arhythmicity (Itri et al. 2004, Colwell et al. 2003)
  3. Neurotrophins like brain derived neurotrophic factor (BDNF) modulate photic input by enhancing glutamatergic transmission. (Michel et al. 2006b)
  4. As least two distinct groups of ionic currents seem to be responsible for regulating circadian modulation of membrane potential on the one hand and frequency of action potentials on the other hand. We identified a conductance, the fast delayed rectifier K-current, which is regulated by the circadian clock, but also crucial for the maintenance of the circadian rhythmicity in action potential frequency (ref. Itri et al 2005, Michel et al. 1993, 1999)

Currently, we focus on three main questions:

  1. Which ionic conductances are responsible for generation and maintenance of circadian rhythmicity. (Itri et al., 2005)
  2. The role and mechanism of coupling between SCN neurons (Albus et al., 2005; Michel and Colwell, 2001)
  3. Interaction of membrane excitability and regulation of gene expression.

Collaborations

  • Prof. Michel D. Ferrari, Dept. of Neurology, LUMC
  • Arn M.J.M van den Maagdenberg, Dept Human Genetics, LUMC
  • Dr. Roeland Dirks, Molecular Cell Biology, LUMC
  • Prof. Rudi G. J. Westendorp, Dept. of Gerontology and Geriatrics, LUMC
  • Peter van Vliet, Dept. of Gerontology and Geriatrics, LUMC
  • Prof. Chris Colwell, University of California, Los Angeles, USA
  • Prof. Gene Block, University of California, Los Angeles, USA
     

Publications cited

  • Albus H, Vansteensel MJ, Michel S, Block GD and JH Meijer (2005) A GABAergic mechanism is necessary for coupling dissociable ventral and dorsal regional oscillators within the circadian clock. Curr Biol 15: 886-893
  • Colwell CS, Michel S, Itri J, Rodriguez W, Tam J, Lelievre V, Hu Z, Liu X and JA Waschek (2003) Disrupted circadian rhythms in VIP and PHI deficient mice. Am J Physiol Regul Integr Comp Physiol.  285: R939-49 
  • Colwell CS, Michel S, Itri J, Rodriguez W, Tam J, Lelievre V, Hu Z, Waschek JA. (2004) Selective Deficits in the Circadian Light Response in Mice Lacking PACAP. Am J Physiol Regul Integr Comp Physiol. 287: R1197-1201
  • Itri J, , Michel S, Waschek, JA and CS Colwell (2004) Circadian rhythm in inhibitory synaptic transmission in the mouse suprachiasmatic nucleus. J Neurophysiol. 92: 311-1 
  • Itri JN, Michel S, Vansteensel MJ, Meijer JH and CS Colwell (2005) Fast delayed rectifier potassium current is required for circadian neural activity. Nat Neurosci 8:650-656.
  • Michel S, Geusz ME, Zaritzky JJ and GD Block (1993) Circadian rhythm in membrane conductance expressed in isolated neurons. Science 259: 239-41
  • Michel S, Manivannan K, Zaritzky JJ and GD Block (1999) A delayed rectifier current is modulated by the circadian pacemaker in Bulla. J Biol Rhythms 14: 141-50
  • Michel S and CS Colwell (2001) Cellular communication and coupling within the suprachiasmatic nucleus. Chronobiol Int 18: 579-600
  • Michel S and NL Wayne (2002) Neurohormone secretion persists following post-afterdischarge membrane depolarization and cytosolic calcium elevation in peptidergic neurons in intact nervous tissue. J Neurosci  22: 9063-69
  • Michel S, Itri J, Han JH, Gniotczynski K and CS Colwell (2006a) PACAP in the Mammalian Circadian System: Physiological Mechanisms of Action. BMC Neuroscience 7: 15
  • Michel S, Clark JP, Ding JM, and CS Colwell (2006b) BDNF and Neurotrophin Receptors Modulate Glutamate-Induced Phase Shifts of the Suprachiasmatic Nucleus. Eur J Neurosci. 24 (4): 1109-1116.

Methods

1. In vitro long-term recording of electrical activity of hypothalamic slices containing the SCN

Customized recording chambers with controlled temperature, flow-through and gas supply maintain the slice viable for several days. Extracellular recordings with stationary metal electrodes allow for simultaneous long-term recording of multiunit and subpopulation activity at two locations within the SCN or adjacent areas.

In vitro long-term recording of electrical activity of hypothalamic slices containing the SCN 

In vitro long-term recording of electrical activity of hypothalamic slices containing the SCN

In vitro long-term recording of electrical activity of hypothalamic slices containing the SCN

2. Patch clamp recording of SCN neurons using a IR-videomicroscope

IR-DIC (Infrared-differential interference contrast) microscopy makes neurons within thick (350 um) brain slices visible. This allows for identification of the neurons under investigation, easier access of patch electrodes, dual recordings and simultaneous Calcium-imaging (see below). Patch electrode are positioned using highly stable manipulators. Microscope is mounted on a vibration-damped table. Recordings are performed using a HEKA EPC-10 double patch amplifier. Solution can be controlled in bath and locally.  

  Patch clamp recording of SCN neurons using a IR-videomicroscope

Patch clamp recording of SCN neurons using a IR-videomicroscope

Patch clamp recording of SCN neurons using a IR-videomicroscope


3. Calcium imaging in SCN neurons.

Using the calcium indicator dye Fura-2 AM, Ca-concentrations of SCN neurons in hypothalamic slcies can be recorded. We use a monochromator as the  excitation light-source and a Peltier-cooled CCD camera for imaging (TILL-Photonics imaging system). This setup can be synchronized with the patch recording and simultaneous recordings of  electrophysilogical data and intracellular messenger responses can be recorded (c.f. Michel and Wayne, 2002)

  Calcium imaging in SCN neurons

Calcium imaging in SCN neurons