Presynaptic chloride-dependent regulation of spontaneous glutamate release in the rat medial preoptic nucleus

Neurosteroids and inhibitory neurotransmitters can modulate neurotransmitter release from presynaptic terminals, yet the mechanisms underlying such modulation remain unclear. In this study, we investigated how presynaptic glycine and GABA_AR-receptors (GlyRs and GABA_ARs) regulate glutamate release onto neurons in the medial preoptic nucleus (MPN), a hypothalamic region critically involved in reproductive and social behaviors. Using patch-clamp recordings from mechanically dissociated MPN neurons with functionally preserved presynaptic terminals, we selectively examined local presynaptic effects of receptor activation. Both the neurosteroid allopregnanolone and the selective GABA_AR agonist muscimol consistently increased the frequency of glutamate-mediated spontaneous excitatory postsynaptic currents (sEPSCs). This facilitation was sensitive to the GABA_AR-blocker picrotoxin, abolished by inhibition of sodium-potassium-chloride cotransporter 1 (NKCC1) or by the sodium-channel blocker tetrodotoxin, consistent with a mechanism involving depolarizing chloride efflux driven by a high intraterminal chloride ion concentration and subsequent sodium-dependent recruitment of presynaptic calcium channels. In contrast, activation of presynaptic GlyRs produced bidirectional effects on glutamate release: facilitation in some terminals and inhibition in others. We demonstrate that the inhibitory effect likely depends on low intraterminal chloride concentration maintained by the potassium-chloride cotransporter 2 (KCC2), which enables chloride influx and hyperpolarization upon GlyR activation. Consistent with this mechanism, pharmacological blockade of chloride extrusion abolished glycine-induced inhibition, and immunogold labeling revealed KCC2 presence in a subset of presynaptic terminals innervating MPN neurons. Together, these findings suggest functional presynaptic KCC2 in central neurons and identify presynaptic chloride homeostasis as a key determinant of synapse-specific modulation of glutamate release in the MPN.