The lamina terminalis (LT) contains three main regions, namely the subfornical organ (SFO), the median preoptic nucleus (MnPO) and the vascular organ of the LT (OVLT). Although LT is recognized of paramount importance in the regulation of hydromineral homeostasis, identity of the neurocircuits interconnecting the SFO and OVLT to the MnPO is not known. Furthermore, the phenotype of neuronal populations activated during acute hydromineral challenge is not yet determined. By using the high cellular resolution of the in situ hybridization histochemistry (ISHH), we investigated whether a furosemide-induced fluid and electrolyte depletion might modify both putative GABAergic and glutamatergic systems within the LT. We show that acute furosemide treatment (4 h) significantly reduced the expression of GAD67 mRNA, the active holoenzyme predictive of GABA synthesis, within the SFO. A strong tendency toward a reduction of GAD67 signal was also observed in the OVLT and MnPO. The hydromineral challenge did not alter the expression of GAD65 and type 2 vesicular glutamate transporter (vGlut2) mRNA in all the structures of the LT. Furosemide treatment was associated with a reduction in the population of GAD67-containing neurons in the periphery of the SFO and dorsal part of the MnPO. Contrastingly, GAD65-containing cells were shown to be increased in the OVLT and no change was observed for the vGlut2-containing neurons in the whole LT. By combining ISHH with immunohistochemistry (Fos immunoreactivity), we report that furosemide-induced water and sodium depletion did essentially recruit a glutamatergic network throughout the LT, although GABAergic neurons were specifically activated in the ring of the SFO and in the OVLT. The MnPO, the region of the LT that is considered as being an integrative area for sensory inputs arising from the SFO and OVLT, showed exclusive activation of excitatory neuronal populations. Taken together these results suggest that acute water and Na(+) depletion diminish the efficacy of the GABAergic system and mainly activates excitatory neuronal pathways in the regions of the LT.