The Na+/H+-Exchanger in the Central Clock of the Rat Suprachiasmatic Nucleus: Immunofluorescent Characterization and Regulation of Intracellular Ph, [Ca2+], Membrane Excitability, and Neurotransmitter Responses

The central clock of the hypothalamic suprachiasmatic nucleus (SCN) coordinates the peripheral oscillators to control circadian rhythms in mammals. On the one hand, the SCN neurons exhibit circadian rhythms in spontaneous firing rate, [Ca2+]i, and Na+/Ca2+ exchanger NCX1 activity, and also metabolic rhythms in glucose uptake, cytochrome oxidase activity, and Na/K pump activity. On the other hand, the SCN neurons are metabolically active and sensitive to metabolic perturbation. While evidence suggests that metabolic regulation of the circadian clock is mediated by NPYergic inputs onto the VIP-positive SCN neurons, we recently demonstrate that the AVP-positive, but not VIP-positive, SCN neurons express KATP channels to act as a glucosensor to respond to glucose shortage. Most recently, we further show that mitochondria preferentially uptake Ca2+ entering via nimodipine-insensitive Ca2+ channels, which may provide a linkage between firing activity and energy metabolism. ATP hydrolysis during energy metabolism produces H+, which may cause intracellular and extracellular acidifications to impact H+ targets to regulate cellular activity. As the SCN is densely packed with neurons and has higher level of metabolic activity than extra-SCN areas, we hypothesized there be a standing extracellular acidification in the SCN in hypothalamic slices. Indeed, our unpublished result shows a standing extracellular acidification ~0.3 pH unit in the center of SCN, but not in extra-SCN areas. Furthermore, the standing extracellular acidification is partly mediated by H+ extrusion via the Na+/H+ exchanger (NHE). Although the SCN expresses the plasmalemmal-type NHE isoforms NHE1, NHE4, and NHE5, pharmacological studies suggested that NHE1 is the major NHE isoform in mediating extracellular acidification. In other words, our results suggest that NHE1 is constitutively active to extrude H+ to partly mediate the extracellular acidification in the SCN.The observation of constitutive activation of NHE1 to extrude H+ also suggests that NHE1 may help maintain a more alkaline intracellular pH in the SCN neurons. As intracellular pH is vital to proper protein functioning, including proteins involved in handling intracellular Ca2+ and the regulation of membrane conductance, the constitutive activation of NHE1 may play a role in the regulation of [Ca2+]i, membrane excitability, and neurotransmitter responses. This three-year project aims to investigate the role of NHE, particularly NHE1, in the central clock of the SCN. In the First and Second year, we will determine the role of NHE1 in the regulation of intracellular pH, [Ca2+]i, membrane excitability, and neurotransmitter responses in the SCN neurons. In the Second and Third year, we will use immunostaining to determine the distribution and localization of NHE1 in the SCN. We will particularly focus on the colocalization of NHE1 with the targets involved in the regulation of Ca2+ homeostasis and GABAergic signaling.

Project ID:PC10708-1045
External Project ID:MOST107-2320-B182-040