Sodium and calcium currents in acutely dissociated neurons from rat suprachiasmatic nucleus

1. Neurons were acutely dissociated from the suprachiasmatic nucleus (SCN) of adult rats and studied with whole-cell and perforated-patch recordings at room temperature. 2. Acutely dissociated SCN neurons had spherical cell bodies of 12 μm in average diameter. The recorded cells were randomly selected and had either no process (38%), one (41%), two (19%), or three processes (2%). They had a resting potential of about 60 mV, an input resistance of ~5 GΩ, and a cell capacitance of ~7 pF. 3. The dissociated neurons had variable spontaneous firing rates, typically (76%) <1 Hz. 4. Under current clamp, continuous current injection elicited repetitive action potentials. 1 μM tetrodotoxin (TTX) reduced the amplitudes of the action potentials as well as the firing rate, whereas 200 μM Cd²⁺ stopped repetitive firing altogether. Action potentials were completely eliminated with Cd²⁺ and TTX present. These results suggest that both Na⁺ and Ca²⁺ contribute to the action potential in these cells. 5. With 200 μM Cd²⁺ present to block calcium currents, a train of brief depolarizing pulses could still elicit repetitive sodium action potentials, but these became attenuated at stimulating frequencies as low as 1 Hz. 6. Under voltage clamp, the sodium current was activated at about 40 mV and peaked at about -10 mV. It inactivated with a time constant of ~0.5 ms at 0 mV, and in steady state the current was half-inactivated at about 60 mV. Recovery of the current from inactivation showed two very different phases with time constants of ~30 and 600 ms at -60 mV. The slow phase was probably responsible for the very low firing rate of the sodium action potential. 7. In the absence of external sodium, depolarization-activated calcium action potentials were preferentially blocked by 20 μM Cd²⁺, whereas a posthyperpolarizing depolarization (or anode break) was preferentially reduced by 100 μM Ni²⁺. These differential effects hinted at the presence of both low-threshold and high-threshold calcium currents in these cells. 8. Voltage-clamp experiments confirmed the presence of a low-threshold, transient calcium current that was activated by depolarizations above -70 mV. It inactivated with a time constant of ~25 ms between -50 and -30 mV. Steady-state inactivation was half-complete at about -90 mV and complete at about -70 mV. This current was only weakly inhibited by 20 μM Cd²⁺ but almost completely blocked by 100 μM Ni²⁺. It was insensitive to nifedipine and ω-conotoxin GVIA. 9. High- threshold, slowly inactivating calcium currents were also observed, being activated by depolarizations positive to 30 mV. The currents were almost completely blocked by 20 μM Cd²⁺, whereas 100 μM Ni²⁺ was only partially effective. 10 μM nifedipine blocked 18% of the currents, 0.3 μM ω-conotoxin blocked 39% of the currents, and a combination of these two drugs blocked 50% of the currents. 10. In summary, I have examined the excitability of isolated SCN neurons and the underlying voltage-gated sodium and calcium currents. The sodium current has a slow phase of recovery from inactivation, which may impose a limit on the firing rate of these neurons. High-threshold calcium currents also appear to be important from maintaining repetitive firing of these cells owing to their weak inactivation. The findings reported here provide an initial basis for studying the circadian rhythm in excitability of these neurons.