Neuronal Sodium Leak Channel Is Responsible for the Detection of Sodium in the Rat Median Preoptic Nucleus

Abstract

. Sodium (Na ϩ ) ions are of primary importance for hydromineral and cardiovascular homeostasis, and the level of Na ϩ in the body fluid compartments [plasma and cerebrospinal fluid (CSF)] is precisely monitored in the hypothalamus. Glial cells seem to play a critical role in the mechanism of Na ϩ detection. However, the precise role of neurons in the detection of extracellular Na ϩ concentration ([Na ϩ ] out ) remains unclear. Here we demonstrate that neurons of the median preoptic nucleus (MnPO), a structure in close contact with the CSF, are specific Na ϩ sensors. Electrophysiological recordings were performed on dissociated rat MnPO neurons under isotonic [Na ϩ ] (100 mM NaCl) with local application of hypernatriuric (150, 180 mM NaCl) or hyponatriuric (50 mM NaCl) external solution. The hyper-and hyponatriuric conditions triggered an in-and an outward current, respectively. The reversal potential of the current matched the equilibrium potential of Na ϩ , indicating that a change in [Na ϩ ] out modified the influx of Na ϩ in the MnPO neurons. The conductance of the Na ϩ current was not affected by either the membrane potential or the [Na ϩ ] out . Moreover, the channel was highly selective for lithium over guanidinium. Together, these data identified the channel as a Na ϩ leak channel. A high correlation between the electrophysiological recordings and immunofluorescent labeling for the Na X channel in dissociated MnPO neurons strongly supports this channel as a candidate for the Na ϩ leak channel responsible for the Na ϩ -sensing ability of rat MnPO neurons. The absence of Na X labeling and of a specific current evoked by a change in [Na ϩ ] out in mouse MnPO neurons suggests species specificity in the hypothalamus structures participating in central Na ϩ detection.

Regulation of Central Na+Detection Requires the Cooperative Action of the NaXChannel and α1 Isoform of Na+/K+-ATPase in the Na+-Sensor Neuronal Population

“…A majority of neurons in the MnPO of adult rats have been demonstrated to specifically respond to changes in extracellular Na ϩ concentration (Grob et al, 2004;Tremblay et al, 2011). These neurons are Na ϩ sensors as they respond to changes in extracellular Na ϩ concentration but not to changes in osmolality.…”

“…These neurons are Na ϩ sensors as they respond to changes in extracellular Na ϩ concentration but not to changes in osmolality. The leak Na ϩ channel Na X has been demonstrated to be responsible for Na ϩ sensing in this neural population (Tremblay et al, 2011). To characterize the response of MnPO neurons to altered extracellular [Na ϩ ], we recorded from dissociated MnPO neurons in voltage-clamp mode a repeatedly increased [Na ϩ ] ext .…”

“…The condition allows separating the Na ϩ response of the membrane background noise. Please note also that MnPO neurons do not respond to osmolality changes (Grob et al, 2004;Tremblay et al, 2011). To test the Na ϩ /K ϩ -ATPase implication in Na ϩ regulation, pharmacological tests were performed, and stock solution of ouabain (10 mM) and strophanthidin (50 mM) were directly diluted in control aCSF.…”

“…The dissociated cells were fixed for 60 min in a paraformaldehyde solution ( pH 7.4) and immediately used for immunocytochemistry. The fixed cells were incubated overnight at 4°C in PBS containing 5% native goat serum, 1% BSA with rabbit anti-Na X antibody (1:250; for details, see Tremblay et al, 2011), and mouse anti-NeuN antibody (1:500, clone A60; Millipore) or mouse anti-Na ϩ /K ϩ -ATPase ␣3 subunit (1:10, clone XVIF9-G10; Sigma-Aldrich) or mouse anti-Na ϩ /K ϩ -ATPase ␣1 subunit (1:10, clone C464.6; Millipore). The slides were first washed in PBS and then incubated for 2 h at room temperature in PBS containing Alexa Fluor-488 goat anti-rabbit (1:500, green; Invitrogen) and Alexa Fluor-555 goat anti-mouse (1:500, red; Invitrogen) as secondary fluorescent antibodies to visualize the Na X and NeuN or Na ϩ /K ϩ -ATPase ␣3 or ␣1 proteins, respectively.…”

“…This ability to detect [Na ϩ ] ext variations is attributable to the presence of the Na X channel, an atypical Na ϩ channel described as a specific Na ϩ sensor (Hiyama et al, 2002(Hiyama et al, , 2004Noda, 2006). The Na X channel has recently been characterized as a Na ϩ leak channel passing Na ϩ ions through the membrane and detecting changes in [Na ϩ ] ext (Tremblay et al, 2011). However, the mechanism by which the Na X channel controls Na ϩ accumulation remains unclear.…”