Real-time lactate measurements within slices were acquired with a calibrated enzyme-based lactate electrode using the FAST16 mKII amperometry system (Quanteon). Slices were transferred to a recording chamber located on an upright microscope (Axioskop, Zeiss) and perfused with aCSF (2 ml/min) aerated with 95% O2/5% CO2. fEPSPs were evoked by stimulation of the Schaffer collateral

pathway using a bipolar tungsten-stimulating electrode. fEPSPs were recorded with glass micropipettes filled with aCSF (4–6 MΩ), positioned in stratum radiatum of the CA1 region, and signals were acquired via an Axopatch 200B amplifier (Molecular Devices). Baseline synaptic responses were established by evoking fEPSPs every 30 s (0.03 Hz) for at least 20 min. Data were analyzed using Clampfit buy Vemurafenib 9.0 (Molecular Devices). Trains of electrical stimulation (20 Hz, 30 s at 5V) of the Schaffer

collaterals were performed with a concentric bipolar electrode (Frederick Haer) positioned ∼100 μm upstream of the NADH imaging area using a Grass S88× stimulator. Images were collected using 512 × 512 pixels and the scanning frame rate was 393.2 ms or 983.4 ms, depending on the area scanned, and 8 line averaging was utilized. For intracellular pH imaging, both BCECF and SR-101 fluorescence signals were defined as ΔF/F = [(F1 − B1) − (F0 − B0)]/(F0 − B0), where F1 and F0 are fluorescence inside of the cell plasma at any given time point and at the beginning of the experiment,

respectively, and B1 and learn more B0 are the background fluorescence at the same time point and at the beginning of the experiment, respectively. Background values were taken from an adjacent area of the imaged cell. Normalized BCECF values were calculated as [ΔF/FBCECF]/[ΔF/FSR101] at the same time point. Quantification Suplatast tosilate of BCECF and SR-101 fluorescence was performed with Zeiss LSM (version 2.8) software and ImageJ. Experimental values are presented as the mean ± SEM, expressed in percent from 100% baseline. The “n” value represents the number of experiments conducted for analysis. Statistical analyses were performed using a two-tailed Student’s t test or ANOVA. p < 0.05 was accepted as statistically significant (∗p < 0.05, ∗∗p < 0.01). Sodium-Oxamate (2.5 mM), Sodium-Iodoacetate (200 μM), and 4-CIN were applied continuously with different concentrations of [K+]ext. TTX (1 μM) (Alamone Laboratories) and IBMX (100 μM) (Sigma) were always present during the release assays. KH7, 2-OH (Steraloids), and DDA (Sigma) were simultaneously applied to brain slices with different concentrations of [K+]ext. This work was supported by an operating grant from the Canadian Institutes of Health Research and Transatlantic Networks of Excellence Program from the Foundation Leducq. B.A.M. is a Canada Research Chair. H.B.C. is supported by postdoctoral fellowships from the Arthur and June Wilms fellowship and the Heart and Stroke Foundation of Canada. G.R.J.G.