1 2 3 Intracellular Na + and Metabolic Modulation of Na / K Pump and Excitability in the Rat 4 Suprachiasmatic Nucleus Neurons

40 41 Na/K pump activity and metabolic rate are both higher at day in the suprachiasmatic nucleus 42 (SCN) that houses the circadian clock. Here we investigated the role of intracellular Na and 43 energy metabolism in regulating Na/K pump activity and neuronal excitability. Removal of 44 extracellular K to block the Na/K pump excited SCN neurons to fire at higher rates and return 45 to normal K to reactivate the pump produced rebound hyperpolarization to inhibit firing. In the 46 presence of TTX to block the action potentials, both zero K-induced depolarization and 47 rebound hyperpolarization were blocked by the cardiac glycoside strophanthidin. Ratiometric 48 Na imaging with a Na-sensitive fluorescent dye indicated saturating accumulation of 49 intracellular Na in response to pump blockade with zero K. The Na ionophore monensin also 50 induced Na loading and hyperpolarized the membrane potential, with the hyperpolarizing effect 51 of monensin abolished in zero Na or by pump blockade. Conversely, Na depletion with Na52 free pipette solution depolarized membrane potential but retained residual Na/K pump activity. 53 Cyanide inhibition of oxidative phosphorylation blocked the Na/K pump to depolarize resting 54 potential and increase spontaneous firing in most cells, and raise intracellular Na levels in all 55 cells. Nonetheless, the Na/K pump was incompletely blocked by cyanide but completely 56 blocked by iodoacetate to inhibit glycolysis, indicating the involvement of both oxidative 57 phosphorylation and glycolysis in fuelling the Na/K pump. Together the results indicate the 58 importance of intracellular Na and energy metabolism in regulating Na/K pump activity as well 59 as neuronal excitability in the SCN neurons. 60 61