Ryanodine-sensitive intracellular Ca2+ channels are involved in the output from the SCN circadian clock.

The suprachiasmatic nuclei (SCN) contain the major circadian clock responsible for generation of circadian rhythms in mammals. The time measured by the molecular circadian clock must eventually be translated into a neuronal firing rate pattern to transmit a meaningful signal to other tissues and organs in the animal. Previous observations suggest that circadian modulation of ryanodine receptors (RyR) is a key element of the output pathway from the molecular circadian clock. To directly test this hypothesis, we studied the effects of RyR activation and inhibition on real time expression of PERIOD2::LUCIFERASE, intracellular calcium levels and spontaneous firing frequency in mouse SCN neurons. Furthermore, we determined whether the RyR-2 mRNA is expressed with a daily variation in SCN neurons. We provide evidence that pharmacological manipulation of RyR in mice SCN neurons alters the free [Ca2+ ]i in the cytoplasm and the spontaneous firing without affecting the molecular clock mechanism. Our data also show a daily variation in RyR-2 mRNA from single mouse SCN neurons with highest levels during the day. Together, these results confirm the hypothesis that RyR-2 is a key element of the circadian clock output from SCN neurons.

Circadian modulation of the Cl(-) equilibrium potential in the rat suprachiasmatic nuclei.

The suprachiasmatic nuclei (SCN) constitute a circadian clock in mammals, where γ-amino-butyric acid (GABA) neurotransmission prevails and participates in different aspects of circadian regulation. Evidence suggests that GABA has an excitatory function in the SCN in addition to its typical inhibitory role. To examine this possibility further, we determined the equilibrium potential of GABAergic postsynaptic currents (E(GABA)) at different times of the day and in different regions of the SCN, using either perforated or whole cell patch clamp. Our results indicate that during the day most neurons in the dorsal SCN have an E(GABA) close to -30 mV while in the ventral SCN they have an E(GABA) close to -60 mV; this difference reverses during the night, in the dorsal SCN neurons have an E(GABA) of -60 mV and in the ventral SCN they have an E(GABA) of -30 mV. The depolarized equilibrium potential can be attributed to the activity of the Na(+)-K(+)-2Cl(-) (NKCC) cotransporter since the equilibrium potential becomes more negative following addition of the NKCC blocker bumetanide. Our results suggest an excitatory role for GABA in the SCN and further indicate both time (day versus night) and regional (dorsal versus ventral) modulation of E(GABA) in the SCN.