5 μM tetrodotoxin. All drugs were obtained from Sigma or Tocris (UK). Chemicals were applied extracellularly by bath superfusion. Living neurons expressing eGFP were visualized in brain slices using an Olympus BX50WI upright microscope equipped with oblique illumination optics, a mercury lamp, and eGFP excitation and emission filters. Somatic recordings were carried out at 37°C using an EPC 10 patch-clamp

amplifier (HEKA Elektronik, Germany). Patch pipettes were made from borosilicate glass, and their tip-resistances ranged from 3 to 8 MΩ (3–5 MΩ with high-Cl and 5–8 MΩ with low-Cl pipette solution). Slices were placed in a submerged-type chamber (volume ∼2 ml, solution flow rate 2.5 ml/min) and anchored with a nylon string grid. Only cells with access resistances between 10 and 25 MΩ were accepted selleck products for analysis. In experiments where change in membrane potential was quantified, all cells were initially held at the same potential to facilitate see more comparison (−50 mV when depolarization was quantified, and

−40 mV when hyperpolarization was quantified), by applying a fixed holding current throughout experiment. Data were sampled and filtered using Pulse and Patchmaster software (HEKA Elektronik, Germany). Current-voltage (I-V) relationships were obtained by performing voltage-clamp ramps from −20 to −130 mV at a rate of 0.1 mV/ms ramp, which is sufficiently slow to allow leak-like K+ currents to reach steady state at each potential (Meuth et al., 2003). In cell-attached the mode (Figure 1H), the patch pipette was filled with ACSF and action potential frequency was measured in voltage-clamp at a command potential under which the holding current is 0 pA (Perkins, 2006). Breaks in some current-clamp traces correspond to moments when the recording

was paused (e.g., to take voltage-clamp measurements or inject cell with current for measurement of input resistance). In some current-clamp experiments (e.g., Figures 1D and 4A) the cells were periodically injected with hyperpolarizing current pulses to monitor membrane resistance. Statistical analyses were performed using Origin (Microcal, Northampton, MA) and Microsoft Excel (Microsoft, Redmond, WA) software. Averaged data are presented as mean ± SEM. Statistical significance was tested using the Student’s t test unless indicated otherwise. The following modified Hill equation was fitted to the data in Figures 1G and 3C: V=Rmax[AA]hEC50h+[AA]hwhere Rmax is the maximal change in membrane potential, EC50 is the concentration that gives half-maximal response, and h is the Hill coefficient. The fit shown in Figure 1G was obtained with Rmax = 20.4 mV, h = 1.79 and EC50 = 438.2 μM. The fit shown in Figure 3C was obtained with Rmax = 950.6 pA, h = 2.39, and EC50 = 3.19 mM. Subjects were 14-week-old C57BL/6 male mice (Charles River). The mice were maintained on a standard 12 hr light-dark cycle (lights on at 0700 hr).