Bio-imaging of nitric oxide-producing neurones in slices of rat brain using 4,5-diaminofluorescein

4,5-Diaminofluorescein (DAF-2) was used to identify individual nitric oxide (NO)-producing neurones in brain slices in vitro. Coronal slices of midbra in or hippocampus, 300 mu m thick from young adult rats, were incubated for 30 min in 1 mu M DAF-2 diacetate (DAF-2 DA) and maintained in ACSF at 33 d egrees C. Illumination at 450-490 nm revealed punctate fluorescence in neur ones in the lateral tegmental nucleus, dorsal raphe nucleus, dorsolateral p eriaqueductal grey matter, deep collicular layers and cortical areas. Neuro nes in the hippocampal pyramidal cell layer, molecular layer of the dentate gyrus and the hilus fluoresced also. The fluorescence was abolished by pre -incubation of slices with L-NAME (100 mu M-1 mM), the inhibitor of constit utive nitric oxide synthase (NOS), but not by D-NAME (100 mu M) or L-NIL (5 -50 mu M), an inhibitor of inducible NOS. In some superficially located art erioles, there were small regions of bright fluorescence close to the outer smooth muscle wall and diffuse fluorescence within the adjacent smooth mus cle cells. A diffuse fluorescence was also seen in some superficially locat ed capillaries. Basal production of NO was not seen within deeper blood ves sels. DAF-2, DA offers a sensitive indicator for visualising basal producti on of NO with high spatial resolution and could provide a means of identify ing NOS-containing neurones in brain slices in vitro prior to neurophysiolo gical study. (C) 1999 Elsevier Science B.V. All rights reserved.