Glutamate-Evoked Ca2+ Responses in the Rat Suprachiasmatic Nucleus: Involvement of Na+/K+-ATPase and Na+/Ca2+-Exchanger.

Glutamate mediates photic entrainment of the central clock in the suprachiasmatic nucleus (SCN) by evoking intracellular Ca2+ signaling mechanisms. However, the detailed mechanisms of glutamate-evoked Ca2+ signals are not entirely clear. Here, we used a ratiometric Ca2+ and Na+ imaging technique to investigate glutamate-evoked Ca2+ responses. The comparison of Ca2+ responses to glutamate (100 µM) and high (20 mM) K+ solution indicated slower Ca2+ clearance, along with rebound Ca2+ suppression for glutamate-evoked Ca2+ transients. Increasing the length of exposure time in glutamate, but not in 20 mM K+, slowed Ca2+ clearance and increased rebound Ca2+ suppression, a result correlated with glutamate-induced Na+ loads. The rebound Ca2+ suppression was abolished by ouabain, monensin, Na+-free solution, or nimodipine, suggesting an origin of activated Na+/K+-ATPase (NKA) by glutamate-induced Na+ loads. Ouabain or Na+-free solution also slowed Ca2+ clearance, apparently by retarding Na+/Ca2+-exchanger (NCX)-mediated Ca2+ extrusion. Together, our results indicated the involvement of glutamate-induced Na+ loads, NKA, and NCX in shaping the Ca2+ response to glutamate. Nevertheless, in the absence of external Na+ (NMDG substituted), Ca2+ clearance was still slower for the Ca2+ response to glutamate than for 20 mM K+, suggesting participation of additional Ca2+ handlers to the slower Ca2+ clearance under this condition.

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.

Effects of losartan and exercise on muscle mass and exercise endurance of old mice.

This study evaluated the effects of angiotensin II type I receptor blocker (ARB) on muscle mass and exercise capacity in healthy older animals. The effects of combined ARB and exercise training were also determined. Eighty 18-month-old mice were randomized into the control group (C), exercise group (E), losartan group (L) and losartan plus exercise group (LE). Mice in the L and LE groups received losartan from drinking water every day. Mice in the E and LE groups trained on a treadmill 30 min per day, 3 days per week for 4 months. Exercise endurance and spontaneous physical activity of mice were measured at baseline and monthly for 4 months. After 4 months of intervention, serum interleukin-6 (IL-6) levels, muscle mass, and muscle fiber cross sectional area (CSA) were measured. Total antioxidant capacity (TAC), lipid peroxidation and IL-6 levels were determined in quadriceps. We found that exercise endurance only increased in the E and LE groups. Muscle TAC levels of E, L, and LE groups were greater than that in the C group. Serum IL-6 and lipid peroxidation levels were not different among groups. LE group, but not E and L groups, had greater muscle mass, larger muscle fiber CSA, and greater muscle IL-6 levels than that in the C group after 4 months of intervention. These results suggest that losartan promotes the adaptions of muscle mass with exercise training in healthy older animals.

Glycolytic metabolism and activation of Na+ pumping contribute to extracellular acidification in the central clock of the suprachiasmatic nucleus: Differential glucose sensitivity and utilization between oxidative and non-oxidative glycolytic pathways.

BACKGROUND: The central clock of the suprachiasmatic nucleus (SCN) controls the metabolism of glucose and is sensitive to glucose shortage. However, it is only beginning to be understood how metabolic signals such as glucose availability regulate the SCN physiology. We previously showed that the ATP-sensitive K+ channel plays a glucose-sensing role in regulating SCN neuronal firing at times of glucose shortage. Nevertheless, it is unknown whether the energy-demanding Na+/K+-ATPase (NKA) is also sensitive to glucose availability. Furthermore, we recently showed that the metabolically active SCN constantly extrudes H+ to acidify extracellular pH (pHe). This study investigated whether the standing acidification is associated with Na+ pumping activity, energy metabolism, and glucose utilization, and whether glycolysis- and mitochondria-fueled NKAs may be differentially sensitive to glucose shortage. METHODS: Double-barreled pH-selective microelectrodes were used to determine the pHe in the SCN in hypothalamic slices. RESULTS: NKA inhibition with K+-free (0-K+) solution rapidly and reversibly alkalinized the pHe, an effect abolished by ouabain. Mitochondrial inhibition with cyanide acidified the pHe but did not inhibit 0-K+-induced alkalinization. Glycolytic inhibition with iodoacetate alkalinized the pHe, completely blocked cyanide-induced acidification, and nearly completely blocked 0-K+-induced alkalinization. The results indicate that glycolytic metabolism and activation of Na+ pumping contribute to the standing acidification. Glucoprivation also alkalinized the pHe, nearly completely eliminated cyanide-induced acidification, but only partially reduced 0-K+-induced alkalinization. In contrast, hypoglycemia preferentially and partially blocked cyanide-induced acidification. The result indicates sensitivity to glucose shortage for the mitochondria-associated oxidative glycolytic pathway. CONCLUSION: Glycolytic metabolism and activation of glycolysis-fueled NKA Na+ pumping activity contribute to the standing acidification in the SCN. Furthermore, the oxidative and non-oxidative glycolytic pathways differ in their glucose sensitivity and utilization, with the oxidative glycolytic pathway susceptible to glucose shortage, and the non-oxidative glycolytic pathway able to maintain Na+ pumping even in glucoprivation.

Role of Intracellular Na+ in the Regulation of [Ca2+]i in the Rat Suprachiasmatic Nucleus Neurons.

Transmembrane Ca2+ influx is essential to the proper functioning of the central clock in the suprachiasmatic nucleus (SCN). In the rat SCN neurons, the clearance of somatic Ca2+ following depolarization-induced Ca2+ transients involves Ca2+ extrusion via Na+/Ca2+ exchanger (NCX) and mitochondrial Ca2+ buffering. Here we show an important role of intracellular Na+ in the regulation of [Ca2+]i in these neurons. The effect of Na+ loading on [Ca2+]i was determined with the Na+ ionophore monensin and the cardiac glycoside ouabain to block Na+/K+-ATPase (NKA). Ratiometric Na+ and Ca2+ imaging was used to measure the change in [Na+]i and [Ca2+]i, and cell-attached recordings to investigate the effects of monensin and ouabain on spontaneous firing. Our results show that in spite of opposite effects on spontaneous firing and basal [Ca2+], both monensin and ouabain induced Na+ loading, and increased the peak amplitude, slowed the fast decay rate, and enhanced the slow decay phase of 20 mM K+-evoked Ca2+ transients. Furthermore, both ouabain and monensin preferentially enhanced nimodipine-insensitive Ca2+ transients. Together, our results indicate that in the SCN neurons the NKA plays an important role in regulating [Ca2+]i, in particular, associated with nimodipine-insensitive Ca2+ channels.

The Na+/H+-Exchanger NHE1 Regulates Extra- and Intracellular pH and Nimodipine-sensitive [Ca2+]i in the Suprachiasmatic Nucleus.

The central clock in the suprachiasmatic nucleus (SCN) has higher metabolic activity than extra-SCN areas in the anterior hypothalamus. Here we investigated whether the Na+/H+ exchanger (NHE) may regulate extracellular pH (pHe), intracellular pH (pHi) and [Ca2+]i in the SCN. In hypothalamic slices bathed in HEPES-buffered solution a standing acidification of ~0.3 pH units was recorded with pH-sensitive microelectrodes in the SCN but not extra-SCN areas. The NHE blocker amiloride alkalinised the pHe. RT-PCR revealed mRNA for plasmalemmal-type NHE1, NHE4, and NHE5 isoforms, whereas the NHE1-specific antagonist cariporide alkalinised the pHe. Real-time PCR and western blotting failed to detect day-night variation in NHE1 mRNA and protein levels. Cariporide induced intracellular acidosis, increased basal [Ca2+]i, and decreased depolarisation-induced Ca2+ rise, with the latter two effects being abolished with nimodipine blocking the L-type Ca2+ channels. Immunofluorescent staining revealed high levels of punctate colocalisation of NHE1 with serotonin transporter (SERT) or CaV1.2, as well as triple staining of NHE1, CaV1.2, and SERT or the presynaptic marker Bassoon. Our results indicate that NHE1 actively extrudes H+ to regulate pHi and nimodipine-sensitive [Ca2+]i in the soma, and along with CaV1.2 may also regulate presynaptic Ca2+ levels and, perhaps at least serotonergic, neurotransmission in the SCN.

cAMP, Ca2+, pHi, and NO Regulate H-like Cation Channels That Underlie Feeding and Locomotion in the Predatory Sea Slug Pleurobranchaea californica.

A systems approach to regulation of neuronal excitation in the mollusc Pleurobranchaea has described novel interactions of cyclic AMP-gated cation current (INa,cAMP), Ca2+, pHi, and NO. INa,cAMP appears in many neurons of feeding and locomotor neuronal networks. It is likely one of the family of hyperpolarization-activated, cyclic-nucleotide-gated currents (h-current) of vertebrate and invertebrate pacemaker networks. There are two isoforms. Ca2+ regulates both voltage dependence and depolarization-sensitive inactivation in both isoforms. The Type 1 INa,cAMP of the feeding network is enhanced by intracellular acidification. A direct dependence of INa,cAMP on cAMP allows the current to be used as a reporter on cAMP concentrations in the cell, and from there to the intrinsic activities of the synthetic adenyl cyclase and the degradative phosphodiesterase. Type 2 INa,cAMP of the locomotor system is activated by serotonergic inputs, while Type 1 of the feeding network is thought to be regulated peptidergically. NO synthase activity is high in the CNS, where it differs from standard neuronal NO synthase in not being Ca2+ sensitive. NO acidifies pHi, potentiating Type 1, and may act to open proton channels. A cGMP pathway does not mediate NO effects as in other systems. Rather, nitrosylation likely mediates its actions. An integrated model of the action of cAMP, Ca2+, pHi, and NO in the feeding network postulates that NO regulates proton conductance to cause neuronal excitation in the cell body on the one hand, and relief of activity-induced hyperacidification in fine dendritic processes on the other.

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.

The discoveries of molecular mechanisms for the circadian rhythm: The 2017 Nobel Prize in Physiology or Medicine.

Circadian clocks evolved to allow plants and animals to adapt their behaviors to the 24-hr change in the external environment due to the Earth's rotation. While the first scientific observation of circadian rhythm in the plant leaf movement may be dated back to the early 18th century, it took 200 years to realize that the leaf movement is controlled by an endogenous circadian clock. The cloning and characterization of the first Drosophila clock gene period in the early 1980s, independently by Jeffery C. Hall and Michael Rosbash at Brandeis University and Michael Young at Rockefeller University, paved the way for their further discoveries of additional genes and proteins, culminating in establishing the so-called transcriptional translational feedback loop (TTFL) model for the generation of autonomous oscillator with a period of ∼24 h. The 2017 Nobel Prize in Physiology or Medicine was awarded to honor their discoveries of molecular mechanisms controlling the circadian rhythm.

Na⁺/Ca²âº Exchanger 2 (NCX2) in the Circadian Clock of the Rat Suprachiasmatic Nucleus: Colocalization with Neuropeptides and Daily Profiles of Gene Expression and Protein Levels.

The plasmalemmal Na⁺/Ca²âº changer (NCX) regulates intracellular Ca²âº by exchanging 3 Na⁺ for 1 Ca²âº in either the Ca²âº exit or Ca²âº entry mode. All three NCX isoforms NCX1, NCX2, and NCX3 are expressed in the rat brain, with isoform-specific differential distribution. In the central clock of suprachiasmatic nucleus (SCN), intracellular Ca²âº controls the circadian release of major neuropeptides, which are the arginine vasopressin (AVP), vasoactive intestinal peptide (VIP) and gastrin releasing peptide (GRP), and the NCX, most likely NCX1, rapidly clears depolarization-induced somatic Ca²âº influx. However, the role of NCX2 in the SCN remains unknown. This study aimed to investigate the colocalization of NCX2 with neuropeptides and daily expression profiles of NCX2 in mRNA and protein levels. Consistent with the restricted distribution of NCX2 in the retinorecipient ventral SCN, the immunostaining results showed colocalization of NCX2 with VIP, GRP and VIP/GRP in the ventral SCN, but not with AVP in the dorsal SCN, or markers for astrocyte and major input pathways. Importantly, the presynaptic marker Bassoon was found to colocalize with NCX2/GRP and NCX2/ VIP, indicating localization of both VIP/NCX2 and GRP/NCX2 at the presynaptic sites. Furthermore, real-time PCR and western blotting revealed no day-night difference in NCX2 mRNA and protein levels, in contrast to a robust circadian rhythm in the expression of clock genes Per1 and Per2. Together the results suggest a role of NCX2 in the regulation of the release of VIP and GRP.

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.

Role of Na⁺/Ca²âº exchanger in Ca²âº homeostasis in rat suprachiasmatic nucleus neurons.

Intracellular Ca(2+) is critical to the central clock of the suprachiasmatic nucleus (SCN). However, the role of Na(+)/Ca(2+) exchanger (NCX) in intracellular Ca(2+) concentration ([Ca(2+)]i) homeostasis in the SCN is unknown. Here we show that NCX is an important mechanism for somatic Ca(2+) clearance in SCN neurons. In control conditions Na(+)-free solution lowered [Ca(2+)]i by inhibiting TTX-sensitive as well as nimodipine-sensitive Ca(2+) influx. With use of the Na(+) ionophore monensin to raise intracellular Na(+) concentration ([Na(+)]i), Na(+)-free solution provoked rapid Ca(2+) uptake via reverse NCX. The peak amplitude of 0 Na(+)-induced [Ca(2+)]i increase was larger during the day than at night, with no difference between dorsal and ventral SCN neurons. Ca(2+) extrusion via forward NCX was studied by determining the effect of Na(+) removal on Ca(2+) clearance after high-K(+)-induced Ca(2+) loads. The clearance of Ca(2+) proceeded with two exponential decay phases, with the fast decay having total signal amplitude of ∼85% and a time constant of ∼7 s. Na(+)-free solution slowed the fast decay rate threefold, whereas mitochondrial protonophore prolonged mostly the slow decay. In contrast, blockade of plasmalemmal and sarco(endo)plasmic reticulum Ca(2+) pumps had little effect on the kinetics of Ca(2+) clearance. RT-PCR indicated the expression of NCX1 and NCX2 mRNAs. Immunohistochemical staining showed the presence of NCX1 immunoreactivity in the whole SCN but restricted distribution of NCX2 immunoreactivity in the ventrolateral SCN. Together our results demonstrate an important role of NCX, most likely NCX1, as well as mitochondrial Ca(2+) uptake in clearing somatic Ca(2+) after depolarization-induced Ca(2+) influx in SCN neurons.

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.

Acid-sensing ion channels in neurones of the rat suprachiasmatic nucleus.

We used reduced slice reparations to study ASIC-like currents in the rat central clock suprachiasmatic nucleus (SCN). In reduced SCN preparations, a drop of extracellular pH evoked a desensitizing inward current to excite SCN neurones to fire at higher rates. Under voltage-clamped conditions, all SCN neurones responded to a 5 s pH step to 6.4 with an inward current that decayed with an average time constant of 1.2 s to 10% of the peak at the end of step. The current was blocked by amiloride with an IC(50) of 14 microm and was carried mainly by Na(+), suggesting an origin of ASIC-like channels. The SCN neurones were sensitive to neutral pH, with 94% of cells responding to pH 7.0 with an inward current. The study of sensitivity to pH between 7.0 and 4.4 revealed a two-component dose-dependent H(+) activation in most SCN neurones, with the first component (85% in amplitude) having a pH(50) of 6.6, and the second (15%) a pH(50) of 5. The ASIC-like currents were potentiated by lactate and low Ca(2+), but were inhibited by Zn(2+). RT-PCR analysis demonstrated the presence of mRNA for ASIC1a, 2a, 2b, and 3 in SCN. Compared to other central neurones, the unique presence of ASIC3 along with ASIC1a in SCN neurones may contribute to the high pH sensitivity and unusual inhibition by Zn(2+). The high pH sensitivity suggests that the SCN neurones are susceptive to extracellular acidification of physiological origins and that the ASIC current might play a role in regulating SCN excitability.

Effects of sodium pump activity on spontaneous firing in neurons of the rat suprachiasmatic nucleus.

Cell-attached and whole cell recording techniques were used to study the effects of electrogenic sodium pump on the excitability of rat suprachiasmatic nucleus (SCN) neurons. Blocking the sodium pump with the cardiac steroid strophanthidin or zero K+ increased the spontaneous firing of SCN neurons to different degrees with different recording modes, whereas turning the sodium pump into a nonselective cation channel with the marine toxin palytoxin invariably increased the spontaneous firing to the point of total blockade. Current-clamp recordings indicated that strophanthidin increased the rate of membrane depolarization and reduced the peak afterhyperpolarization potential (AHP), whereas zero K+ also increased the rate of depolarization, but enhanced the peak AHP. The dual effect of zero K+ was reflected by the biphasic time course of voltage responses to zero K+: an inhibitory phase with enhanced peak AHP and slower firing, followed by a delayed excitatory phase with faster rate of membrane depolarization and faster firing. In the presence of strophanthidin to block the sodium pump, zero K+ consistently decreased firing by enhancing the peak AHP. Repetitive applications of K+ -free solution gradually turned the biphasic inhibitory-followed-by-excitatory voltage response into a monophasic inhibitory response in cells recorded with the whole cell (but not the cell-attached) mode, suggesting rundown of sodium pump activity. Taken together, the results suggest that spontaneous firing of SCN neurons is regulated by sodium pump activity as well as the AHP, and that sodium pump activity is modulated by intracellular soluble substances subject to rundown under the whole cell conditions.

Diurnal modulation of the Na+/K+-ATPase and spontaneous firing in the rat retinorecipient clock neurons.

The ventral "core" suprachiasmatic nucleus (vSCN) neurons are the retinorecipient neurons in the mammalian circadian clock and maintain a diurnal firing rhythm in reduced preparations. We tested the possibility that daily changes in Na+/K+-ATPase accompany diurnal variation in spontaneous electrical activity. In control, bath application of 9 microM strophanthidin increased the spontaneous firing both at day and night but to different extents. In the presence of 1 mM Ni2+ to block spontaneous firing, addition of 9 microM strophanthidin, but not higher concentrations (6.5-20 mM) of external K+, induced the silenced cells to fire action potentials in a diurnal rhythm, suggesting a diurnal change in Na+/K+-ATPase activity. Consistently, voltage-clamp recordings demonstrated that the pump current blocked by 9 microM strophanthidin was approximately three times larger in daytime than nighttime and was little affected by the presence of 1 mM Ni2+. Experiments with various concentrations of strophanthidin further suggests day-night differences in maximum Na+/K+-ATPase activity, amounting to 6 pA of pump current at day and down to 3.5 pA at night, and in its half-block concentrations, changing from a daytime value of 4 microM to a nighttime value of 8 microM. Our results indicate that the vSCN neurons exhibit a diurnal rhythm in the Na+/K+-ATPase the activity of which is higher during the day when the firing rate is also higher. Mechanistically, the modulation could be accounted for in terms of changes in the maximum activity of Na+/K+-ATPase and its ability to block by strophanthidin.