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Glass coverslips bearing microglia were placed in a recording chamber (approximately 3 ml volume), where the external solutions were perfused at a rate of 3.5�C5 ml min?1 throughout the experiments. Proton currents were evoked by depolarization pulses (2�C4 s) applied at a holding potential of ?80 mV every 20�C30 s. Local anaesthetics were added to the perfusing solutions. In some experiments, http://www.selleck.cn/products/AZD6244.html a gravity-fed U-tube system was utilized for short application of local anaesthetics on the steady-state proton currents. The U-tube was placed within 30�C50 ��m of the target cell. The timing and duration of each application were controlled by computer-driven solaenoid valves (General Valve, Fairfield, NJ, USA). This permitted us to exchange the external solution around the cell within 50 ms (Mori et al. 2001; Nakanishi et al. 2007). Leak currents were estimated from the linear portion of the current�Cvoltage (I�CV) relationship at voltages lower than the threshold potential for proton channels and were subtracted from the current records. All experiments were performed at room temperature (22�C24��C; Kuno et al. 2009). The proton currents activated by depolarization pulses were fitted with a single-exponential function after a delay, giving estimates of the steady-state currents and the activation time constant (��act). The reversal potentials http://www.selleckchem.com/products/lee011.html (Vrev) were estimated using the repolarization-pulse method (Gordienko et al. 1996); the I�CV relationships were obtained by applying 20 ms repolarization voltage ramps at the end of a 3 s depolarization pulse and at the end of a mock 10 ms depolarization pulse. The subtracted currents yielded the net I�CV curves for the proton currents. Curve fittings were performed using SigmaPlot (Systat Software Inc., San Jose, CA, USA). Data are means �� SD unless described otherwise. The statistical significances (P http://www.selleckchem.com/products/bmn-673.html (and -6) carboxyfluorescein (BCECF). Cells were plated on glass coverslips for 10Cells were plated on glass coverslips for 10�C24 h and loaded with the acetoxymethylester form of BCECF (BCECF AM; 1 ��m) for 30 min at 37��C. After washout of the dye, the ratios of fluorescence images (the emission wavelength ��520 nm) excited at two wavelengths (488 and 460 nm) were measured every 10 s with 30�C100 ms exposures. Data (80�C120 pixels for each cell) for each illumination were averaged and plotted against time. The external solution contained the following (mm): 140 NaCl, 4 KCl, 10 Hepes, 1 CaCl2 and 1 MgCl2 (pH 7.3). To measure the lidocaine or bupivacaine-induced change in pHi at low pHi, cells were loaded with NH4Cl and then washed by the Na+-free solution.