AACIMP 2011 Summer School. Neuroscience Stream. Lecture by Nikolai Kononenko.

1. Searching of a membrane target for mammalian circadian clock responsible for circadian modulation of firing rate Nikolai I. Kononenko Department of General Physiology of Nervous System, Bogomoletz Institute of Physiology, Kiev, Ukraine

2. 1. The suprachiasmatic nucleus (SCN) of the hypothalamus is the primary biological clock regulating circadian rhythms in mammals

3. 2. Individual SCN neurons express self-sustained circadian oscillations. As a result of internal coupling, the SCN generates a coherent output signal

4. Circadian-clock core Messenger Membrane target Hz

6. Multielectrode array dish Hz 0 1 2 3 4 5 (Days) 1 min

7. Spike-associated currents are key targets for circadian modulation of firing rate First of all, we asked whether spike-associated currents (i.e., currents active only during action potentials) are indeed primary membrane target(s) responsible for circadian modulation of firing rate (CMFR).

8. B A Action potentials across the circadian cycle Thus, result allowed preliminarily to suggest that spike-associated currents are not a key targets for circadian modulation of firing rate.

9. Nature, 2002, 16 , 286-290

10. Effect of nifedipine on circadian firing rhythms in SCN neurons

11. Nature Neuroscience, 2005, 8 , 650-656

12. Effect of 4-aminopyridine on circadian rhythms of firing rate in SCN neurons

13. A B Cd 2+ does not suppress immediately spontaneous activity and its circadian modulation (n=13 neurons)

14. Spike-associated channels are not the principal determinants of circadian modulation of firing rate

15. Two acutely isolated SCN neurons on the bottom of a Petri dish 6 days after isolation 100  m

16. On-cell recording of single channels 2 pA 100 ms

17. Spontaneous firing of isolated SCN neurons and circadian modulation of their firing rate Noon Midnight Noon of the next day

18. 2 s (A) 5 s (B) 7 pA 20 mV A B 0 pA C V h = -90 mV V h = -65 mV 2 s 5 pA Fluctuation of membrane potential produces spontaneous activity of SCN neuron

19. The numerous single-channel inward currents we observed during on-cell recording of spontaneous electrical firing in isolated SCN neurons led us to study the properties of the corresponding channels in their relation to spontaneous electrical firing

20. 20 ms 2 pA 8 pA 20 mV A1 A2 A3 B C

21. We have revealed a novel set of subthreshold, voltage-dependent cation (SVC) channels that are active at resting potential and increase their open probability with membrane depolarization

22. A C B 20 pA 0.016 2 s 10 s 10 pA 0.032 -15 s 15 s 0 -100 s 0 100 s * * 2 pA 200 ms

23. Correlation between Po of single-channel activity and spontaneous firing in isolated SCN neuron 1 s 10 pA 2 pA 0.06 200 ms

24. Voltage dependence of persistent single channel

25. We hypothesize that a sufficient number of SVC channel openings would result in threshold depolarization of the SCN neuronal membrane and spontaneous electrical firing

26. A B db-cGMP application evokes spontaneous electrical activity in acutely isolated SCN neurons

28. g C =40 pS; E C =0 mV  o =0.2 ms;  fc =1 ms Single-channel openings (N=1)

29. 36 channels ~0.017 Hz 44 channels ~3.2 Hz 55 channels ~10 Hz B A 40 mV Single-channel openings produce spontaneous firing in a model SCN neuron 1 s

30. Minimal mammalian circadian clockwork model (circadian oscillator) From Scheper et al., J Neurosci 1999 Period Period Period*

31. A B Circadian modulation of the number of single channels produces modulation of the action potential firing rate Circadian peaks of 3 intact SCN neurons in MED

33. Acknowledgments: The author thanks Dr. F.E. Dudek (University of Utah School of Medicine, Salt Lake City, USA) and Dr. S. Honma (Hokkaido University Graduate School of Medicine, Sapporo, Japan), in whose laboratories experimental data were obtained, and Dr. N.M. Berezetskaya (Institute of Physics, Kiev, Ukraine), who converted differential equations into computer programs.