Nitric oxide (NO) is a freely diffusible signalling which modulates both, excitatory and inhibitory neurotransmission. Spatial and temporal characteristics of NO signalling in the brain remain unclear. At the same time, it is very important to have a realistic idea of NO spread in the brain in order to explain how it may convey any meaningful biological message. We visualized NO production in real time using ratiometric confocal imaging of a NO-sensitive fluorescent indicator DAA (1,2-diaminoanthraquinone sulfate) and Alexa 633, used as a reference. In order to ensure that NO is produced by identifiable cellular sources, we introduced nNOS selectively into the neurones of the hypoglossal motor nucleus (HN) of the rat using retrograde transfection with adenoviral vectors. Under deep halothane anaesthesia P3-5 rat pups were injected into the tongue with either a mixture of Ad hCMV-nNOS and/or Ad hCMV-eGFP viral vectors (~5×10¹¹ PFU/ml, 6 μl/pup). After 1 – 5 days acute brainstem slices containing the HN were prepared. The slices were transferred into a recording chamber and continually perfused with oxygenated artificial cerebrospinal fluid. eGFP-fluorescent neurones were identified and the mixture of DAA and Alexa was introduced into the adjacent parenchyma using a patch pipette. Dyes were imaged using Leica SP system with two separate channels (500-545 and 620-660 nm) for DAA and Alexa 633, correspondingly. We first established that under these imaging conditions the emission spectra of the dyes do not overlap and that the DAA/Alexa ratio increases reliably when the dyes are exposed to exogenously produced NO. A robust increase of the DAA/Alexa ratio (~+30%) occurred at areas immediately adjacent (<5 μm) to the membranes of nNOS/EGFP-transfected motoneurones (n=5) after stimulation with L-glutamate (500 μM applied in the bath solution). This was not observed in the eGFP-only transfected neurones. While the increase in the ratio was the highest directly at the membrane, it dropped as a function of the distance and became insignificant at ~15-20 μm. A NOS inhibitor L-NAME(0.2-2 mM) blocked glutamate-induced NO release (n=7). We conclude that DAA/Alexa combination can be used for ratiometric measurements of NO production in living brain slices. High levels of nNOS expression in neurones may result in activity-dependent generation of NO signals which are detectable within the surrounding brain parenchyma at distances of at least 10-15 μm.