Whole-cell recording from a PPN before and after a calcium wave evoked by electrical stimulation. (A) PPNs were recorded under different experimental conditions. nSICs recorded 100 s before and 100 s after an evoked calcium response were used for analysis. Preincubation with the aCSF (alone, modified, or modified + drugs) was always performed. The calcium response was evoked with current pulses (100 µA) at 10 Hz in 4 s. Square shows an nSIC after stimulation. Arrow shows magnification of the recorded nSIC. (B) Calcium response evoked by electrical stimulation. Left: false-color image of the MNTB region. Numbers 1–4 show some active astrocytes. The position of the stimulation electrode is illustrated. Right: calcium responses (F/F₀) of the four astrocytes indicated on the left. (C) Summary of nSICs before and after stimulation in aCSF (n = 16); aCSF + TTX (1 µM) (n = 16); Mg²⁺-free aCSF; Mg²⁺-free aCSF + TTX (1 µM) (n = 17); Mg²⁺-free aCSF + strychnine (1 µM) + gabazine (10 µM) + TTX (1 µM) (n = 20); Mg²⁺-free aCSF + strychnine (1 µM) + gabazine (10 µM) + CNQX (25 µM) + TTX (1 µM) (n = 24); Mg²⁺-free aCSF + strychnine (1 µM) + gabazine (10 µM) + CNQX (25 µM) + ifenprodil (100 µM) (n = 17) + TTX (1 µM); Mg²⁺-free aCSF + strychnine (1 µM) + gabazine (10 µM) + DAAO+ TTX (1 µM) (n = 15); and Mg²⁺-free aCSF + strychnine (1 µM) + gabazine + TTX (1 µM) in a slice where an astrocyte was dialyzed with BAPTA 20 min before (n = 8). See the text and Table S1 for values.