Submerged chamber

Brain slices were continuosly perfused in a submerged chamber (Warner Instruments, Hamden, CT, USA) with a recording solution containing (in mM): 120 NaCl, 3.2 KCl, 1 KH₂PO₄, 26 NaHCO₃, 1 MgCl₂, 2 CaCl₂, 10 glucose at pH 7.4 (with 5% CO₂/95% O₂) and Ca²⁺ signal images (512 × 512 pixels) were acquired by a TCS-SP5-RS confocal microscope (Leica Microsystem, Germany) equipped with a 20× water/objective (NA, 1.0) and a CCD camera for differential interference contrast. Time frame acquisitions from 314 to 491 ms (with 6–7 line averaging) were used. The Ca²⁺ responsiveness in neurons and astrocytes was determined on the basis of a threshold criterion. The onset was identified by the change in Δ F/F0 that should be more than two standard deviations over the average baseline and remained above this value in the successive frames for at least 2 s (two to six frames, depending on the frame acquisition rate). No background subtraction or other manipulations were applied to digitized Ca²⁺ signal images, with the exception of difference images in Figure 2 that were obtained by subtracting the pre-stimulation Ca²⁺ image from the post-stimulation Ca²⁺ image.