Afterhyperpolarization potential modulated by local [K+]o in K+ diffusion-restricted extracellular space in the central clock of suprachiasmatic nucleus.

BACKGROUND: Intercellular coupling is essential for the suprachiasmatic nucleus (SCN) to serve as a coherent central clock. Synaptic release of neurotransmitters and neuropeptides is critical for synchronizing SCN neurons. However, intercellular coupling via non-synaptic mechanisms has also been demonstrated. In particular, the abundant perikaryal appositions with morphological specializations in the narrow extracellular space (ECS) may hinder molecular diffusion to allow for ion accumulation or depletion. METHODS: The SCN neurons were recorded in the whole-cell current-clamp mode, with pipette filled with high (26 mM)-Na+ or low (6 mM)-Na+ solution. RESULTS: Cells recorded with high-Na+ pipette solution could fire spontaneous action potentials (AP) with peak AHP more negative than the calculated value of K+ equilibrium potential (EK) and with peak AP more positive than calculated ENa. Cells recorded with low-Na+ pipette solution could also have peak AHP more negative than calculated EK. In contrast, the resting membrane potential (RMP) was always less negative to calculated EK. The distribution and the averaged amplitude of peak AHP, peak AP, or RMP was similar between cells recorded with high-Na+ and low-Na+ solution pipette. In a number of cells, the peak AHP could increase from more positive to become more negative than calculated EK spontaneously or after treatments to hyperpolarize the RMP. TTX blocked the Na+ -dependent APs and tetraethylammonium (TEA), but not Ba2+ or Cd2+, markedly reduced the peak AHP. Perforated-patch cells could also but rarely fire APs with peak AHP more negative than calculated EK. CONCLUSION: The result of peak AHP negative to calculated EK indicates that local [K+]o sensed by the TEA-sensitive AHP K+ channels must be lower than bulk [K+]o, most likely due to K+ clearance from K+ diffusion-restricted ECS by the Na+/K+-ATPase. The K+ diffusion-restricted ECS may allow for K+-mediated ionic interactions among neurons to regulate SCN excitability.

Differential regulation of nimodipine-sensitive and -insensitive Ca2+ influx by the Na+/Ca2+ exchanger and mitochondria in the rat suprachiasmatic nucleus neurons.

BACKGROUND: Transmembrane Ca2+ influx is critical for molecular rhythmicity, metabolic activity, and neuropeptide release in the central clock of the suprachiasmatic nucleus (SCN). We previously reported that both the Na+/Ca2+ exchanger (NCX) and mitochondria play a role in regulating intracellular Ca2+ homeostasis in the rat SCN neurons. Here we present evidence to show differential regulation by NCX and mitochondria of nimodipine-sensitive and -insensitive Ca2+ influx. METHODS: Ratiometric Ca2+ imaging was used to measure change in [Ca2+]i and patch clamp recordings to study spontaneous firing, membrane potential, and voltage-dependent Ca2+ channels in neurons from reduced SCN slice preparations. Immunofluorescent staining was used to determine the distribution pattern of CaV1.2 and CaV1.3 and their colocalization with NCX1. RESULTS: Ratiometric Ca2+ imaging indicates that nimodipine (2 µM) blocked most of 20 (mM) K+-induced, but less so of 50 K+-induced, Ca2+ rise. The nimodipine-sensitive 50 K+-induced Ca2+ transient rose more rapidly but decayed similarly with the nimodipine-insensitive component, suggesting both components were extruded by NCX. Immunofluorescent stains showed the expression of both CaV1.2 and CaV1.3 and their colocalization with NCX1, whereas functional studies suggest that CaV1.2 mediated most of the nimodipine-sensitive Ca2+ rise but had insignificant effect on spontaneous firing. After normalization relative to the Ca2+-free solution, nimodipine reduced ~ 65% of basal Ca2+ influx, and TTX lowered it by ~ 35%, leaving ~ 25% basal Ca2+ influx in the combined presence of TTX and nimodipine. With the mitochondrial uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) to inhibit mitochondrial Ca2+ uptake, 20 K+-induced Ca2+ transients became larger and slower, both in the absence and presence of nimodipine. FCCP markedly enhanced nimodipine-insensitive, but not nimodipine-sensitive, Ca2+ transients, suggesting that mitochondria preferentially buffer nimodipine-insensitive Ca2+ influx. Results from using CaV2 channel blockers further indicate that FCCP enhanced Ca2+ transients mediated by N-, P/Q-, and the blocker cocktail-insensitive Ca2+ channels. CONCLUSIONS: The differential regulation of transmembrane Ca2+ influx by NCX and mitochondria suggests that Ca2+ entry via different sources may be regulated differently to play different roles in SCN physiology.

KATP Channels Mediate Differential Metabolic Responses to Glucose Shortage of the Dorsomedial and Ventrolateral Oscillators in the Central Clock.

The suprachiasmatic nucleus (SCN) central clock comprises two coupled oscillators, with light entraining the retinorecipient vasoactive intestinal peptide (VIP)-positive ventrolateral oscillator, which then entrains the arginine vasopressin (AVP)-positive dorsomedial oscillator. While glucose availability is known to alter photic entrainment, it is unclear how the SCN neurones respond to metabolic regulation and whether the two oscillators respond differently. Here we show that the ATP-sensitive K+ (KATP) channel mediates differential responses to glucose shortage of the two oscillators. RT-PCR and electrophysiological results suggested the presence of Kir6.2/SUR1 KATP channels in the SCN neurones. Immunostaining revealed preferential distribution of Kir6.2 in the dorsomedial subregion and selective colocalization with AVP. Whole cell recordings with ATP-free pipette solution indicated larger tolbutamide-induced depolarisation and tolbutamide-sensitive conductance in dorsal SCN (dSCN) than ventral SCN (vSCN) neurones. Tolbutamide-sensitive conductance was low under perforated patch conditions but markedly enhanced by cyanide inhibition of mitochondrial respiration. Glucoprivation produced a larger steady-state inhibition in dSCN than vSCN neurones, and importantly hypoglycemia via opening KATP channels selectively inhibited the KATP-expressing neurones. Our results suggest that the AVP-SCN oscillator may act as a glucose sensor to respond to glucose shortage while sparing the VIP-SCN oscillator to remain in synch with external light-dark cycle.

Intracellular Na(+) and metabolic modulation of Na/K pump and excitability in the rat suprachiasmatic nucleus neurons.

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

Cholinergic modulation of neuronal excitability in the rat suprachiasmatic nucleus.

The central cholinergic system regulates both the circadian clock and sleep-wake cycle and may participate in the feedback control of vigilance states on neural excitability in the suprachiasmatic nucleus (SCN) that houses the circadian clock. Here we investigate the mechanisms for cholinergic modulation of SCN neuron excitability. Cell-attached recordings indicate that the nonspecific cholinergic agonist carbachol (CCh) inhibited 55% and excited 21% SCN neurons, leaving 24% nonresponsive. Similar response proportions were produced by two muscarinic receptor [muscarinic acetylcholine receptor (mAChR)] agonists, muscarine and McN-A-343 (M1/4 agonist), but not by two nicotinic receptor (nAChR) agonists, nicotine and choline (alpha7-nAChR agonist), which, however, produced similar response proportions. Whole cell and perforated-patch recordings indicate that CCh inhibition of firing was mediated by membrane hyperpolarization due to activation of background K(+) currents, which were sensitive to submillimolar concentrations of Ba(2+) and to millimolar concentrations of TEA. RT-PCR analysis demonstrated the presence of mRNA for M1 to M5 mAChRs in SCN. The CCh-induced hyperpolarization and activation of background K(+) currents were blocked by M4 antagonists and to a lesser degree by M1 antagonists but were insensitive to the antagonists for M2 or M3, suggesting the involvement of M4 and M1 mAChRs in mediating CCh inhibition of firing. CCh enhancement of firing was mediated by membrane depolarization, as a result of postsynaptic inhibition of background K(+) currents. The multiple actions of cholinergic modulation via multiple receptors and ion channels may allow acetylcholine to finely control SCN neuron excitability in different physiological settings.