NO potentiates GABA-mediated IPSPs in the NTS (Wang et al. 2004). We hypothesize that NO may potentiate GABA release via elevation of [Ca²⁺]_i in NTS GABAergic interneurons. P8 rats were humanely killed and used to prepare brainstem organotypic slice cultures which were transfected with adenoviral vector containing 3.7 kb of the GAD67 promoter driving expression of enhanced green fluorescent protein (Teschemacher et al. 2005). Fluorescent GABAergic neurons were visualized by combined confocal and DIC optics and recorded in whole-cell patch clamp mode. The intracellular solution contained a red-shifted calcium indicator Rhod-2. The NO donor diethylamine NONOate (DEA/NO) concentration-dependently (1 and 10 μM) increased [Ca²⁺]_i as assessed by fluorescence intensity (FI) in different compartments of GABAergic neurons and caused a slight depolarization in the majority of neurons tested (+3.4 ± 0.9 mV; ±SEM with 10 μM, 6/9 cells). The most dramatic FI increase (+40±11%, P<0.05) was observed in 5 of 6 putative axons. sGC blocker-ODQ completely blocked DEA/NO actions. The IP₃ receptor inhibitor – 2-APB was unable to block DEA/NO-induced [Ca²⁺]_i rise indicating that IP₃-sensitive Ca²⁺ stores are not essential for NO action on [Ca²⁺]_i. An antagonist of cyclic ADP-ribose (cADPR) receptors, 8-Bromo-cyclic adenosine diphosphate ribose, introduced via patch pipette, completely abolished DEA/NO-induced [Ca²⁺]_i increase. Therefore, the sGC/cGMP signalling cascade may lead to activation of ADP-ribosyl cyclase via cGMP-dependent protein phosphorylation and subsequent production of cADPR. This leads to ryanodine receptor-mediated Ca²⁺ release from stores thus promoting GABA exocytosis.