Demonstration of ATP-responsive astroglial Ca2+ signalling in the NAc slice. A: Time-series of Fluo-4 pseudo-coloured fluorescence images (see also Additional file 1) show propagation of Ca2+ transients induced by pressure-ejection of 100 μM ATP onto the surface of the NAc slice through a 5-10 μm patch-pipette for 60 s (long puff). Time refers to the period passed from the beginning of the long ATP puff. Arrows indicate the origin and orientation of the ATP puff (red with X) and the direction of ACSF perfusion (white). Scale bar: 50 μm. B: The area initially covered by the short (left and middle) and long (right) ATP puffs was investigated to observe which cells get a direct ATP stimulus. Representative images of the glial marker dye SR101 (100 μM dissolved in ACSF; [19, 20]) applied together with 100 μM ATP to visualize propagation of ATP puff above 630 nm using a 543 nm laser for excitation. This was done in parallel with monitoring the ATP-responsive Fluo-4 Ca2+ transients in the NAc slice. The small azure circle indicates the border of the ATP+SR101 puff for 2 s (short puff) and the big azure circle shows the margin of the ATP+SR101 diffusion. Yellow circle beyond the small azure circle highlight the cell responded to the short puff application of ATP. Scale bar: 100 μm. C: Representative (dF/F0)max Fluo-4 fluorescence plot of the cell highlighted by the yellow circled cell in panel B recorded during ATP application for 2 s (short puff) and for 60 s (long puff, cf. B). D: Number of networking cells in long ATP puff-stimulated Ca2+ transients was significantly higher in the NAc when compared to the adjacent ventral pallidal area.