Abstract
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.
Highlights
Sensitive microelectrodes in the suprachiasmatic nucleus (SCN) but not extra-SCN areas
Such knowledge is important, as we previously demonstrate that the SCN neurones are sensitive to mild extracellular acidification and express acid-sensing ion channels (ASIC), which contain high pH sensitivity of ASIC3 and ASIC1a subunits[18]
Our results show that NHE1 actively extrudes H+ to cause extracellular acidification in hypothalamic SCN slices and maintain a more alkaline pHi to regulate [Ca2+]i in the soma
Summary
Sensitive microelectrodes in the SCN but not extra-SCN areas. The NHE blocker amiloride alkalinised the pHe. While there is only limited knowledge available about how metabolic stress, such as glucose shortage, regulates the SCN, recent evidence indicates an important role of energy metabolism in the regulation of membrane excitability in the SCN neurones via the Na/K pump[12] and the ATP-sensitive K+ channel[13]. Tissue and the superfusate has been demonstrated in various neural tissues (see refs16,17), it is not known if this exists in the SCN Such knowledge is important, as we previously demonstrate that the SCN neurones are sensitive to mild extracellular acidification and express acid-sensing ion channels (ASIC), which contain high pH sensitivity of ASIC3 and ASIC1a subunits[18]. Membrane conductances involved in neurotransmission such as NMDA receptors, GABAA receptors, and voltage-gated calcium channels are sensitive to intra- and extracellular protons[4,16] and play a role in the regulation of circadian clock (see ref.[19])
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