Heterogeneous chloride homeostasis and GABA responses in the median preoptic nucleus of the rat

The median preoptic nucleus (MnPO) is an integrative structure of the hypothalamus receiving periphery-derived information pertinent to hydromineral and cardiovascular homeostasis. In this context, excitability of MnPO neurones is controlled by fast GABAergic, glutamatergic and angiotensinergic projection from the subfornical organ (SFO). Taking advantage of a brain slice preparation preserving synaptic connection between the SFO and the MnPO, and appropriate bicarbonate-free artificial cerebrospinal fluid (CSF), we investigated a possible implication of an active outward Cl − transport in regulating efficacy of the GABA A receptor-mediated inhibitory response at the SFO–MnPO synapse. When somata of the MnPO neurones was loaded with 18 m m chloride, stimulation of the SFO evoked outward inhibitory postsynaptic currents (IPSCs) in 81% of the MnPO neurones held at −60 mV. Accordingly, E IPSC was found 25 mV hyperpolarized from the theoretical value calculated from the Nernst equation, indicating that IPSC polarity and amplitude were driven by an active Cl − extrusion system in these neurones. E IPSC estimated with gramicidin-based perforated-patch recordings amounted −89.2 ± 4.3 mV. Furosemide (100 μ m ), a pharmacological compound known to block the activity of the neurone-specific K + –Cl − cotransporter, KCC2, reversed IPSC polarity and shifted E IPSC towards its theoretical value. Presence of the KCC2 protein in the MnPO was further detected with immunohistochemistry, revealing a dense network of KCC2-positive intermingled fibres. In the presence of a GABA B receptor antagonist, high-frequency stimulation (5 Hz) of the SFO evoked a train of IPSCs or inhibitory postsynaptic potentials (IPSPs), whose amplitude was maintained throughout the sustained stimulation. Contrastingly, similar 5 Hz stimulation carried out in the presence of furosemide (50 μ m ) evoked IPSCs/IPSPs, whose amplitude collapsed during the high-frequency stimulation. Similar reduction in inhibitory neurotransmission was also observed in MnPO neurones lacking the functional Cl − extrusion mechanism. We conclude that a majority of MnPO neurones were characterized by a functional Cl − transporter that ensured an efficient activity-dependent Cl − transport rate, allowing sustained synaptic inhibition of these neurones. Pharmacological and anatomical data strongly suggested the involvement of KCC2, as an essential postsynaptic determinant of the inhibitory neurotransmis