Wang YC, Chen YS, Cheng RC, Huang RC. Role of Na ϩ /Ca 2ϩ exchanger in Ca 2ϩ homeostasis in rat suprachiasmatic nucleus neurons. J Neurophysiol 113: 2114 -2126, 2015. First published January 7, 2015 doi:10.1152/jn.00404.2014 is critical to the central clock of the suprachiasmatic nucleus (SCN). However, the role of Na ϩ /Ca 2ϩ exchanger (NCX) in intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) homeostasis in the SCN is unknown. Here we show that NCX is an important mechanism for somatic Ca 2ϩ clearance in SCN neurons. In control conditions Na ϩ -free solution lowered [Ca 2ϩ ] i by inhibiting TTX-sensitive as well as nimodipine-sensitive Ca 2ϩ influx. With use of the Na ϩ ionophore monensin to raise intracellular Na ϩ concentration ([Na ϩ ] i ), Na ϩ -free solution provoked rapid Ca 2ϩ uptake via reverse NCX. The peak amplitude of 0 Na ϩ -induced [Ca 2ϩ ] i increase was larger during the day than at night, with no difference between dorsal and ventral SCN neurons. Ca 2ϩ extrusion via forward NCX was studied by determining the effect of Na ϩ removal on Ca 2ϩ clearance after high-K ϩ -induced Ca 2ϩ loads. The clearance of Ca 2ϩ proceeded with two exponential decay phases, with the fast decay having total signal amplitude of ϳ85% and a time constant of ϳ7 s. Na ϩ -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 2ϩ pumps had little effect on the kinetics of Ca 2ϩ 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 2ϩ uptake in clearing somatic Ca 2ϩ after depolarization-induced Ca 2ϩ influx in SCN neurons. Ca 2ϩ homeostasis; Ca 2ϩ imaging; circadian rhythm; Na ϩ /Ca 2ϩ exchanger; suprachiasmatic nucleus

BackgroundTransmembrane 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.MethodsRatiometric 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.ResultsRatiometric 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.ConclusionsThe 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 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 [Ca 2+ ] 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 [Ca 2+ ] i , and decreased depolarisation-induced Ca 2+ rise, with the latter two effects being abolished with nimodipine blocking the L-type Ca 2+ 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 [Ca 2+ ] i in the soma, and along with CaV1.2 may also regulate presynaptic Ca 2+ levels and, perhaps at least serotonergic, neurotransmission in the SCN.

The plasmalemmal Na⁺/Ca²⁺ changer (NCX) regulates intracellular Ca²⁺ by exchanging 3 Na⁺ for 1 Ca²⁺ in either the Ca²⁺ exit or Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ 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.