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.

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.

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.

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.

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.