5–4 mm lateral) with

dental acrylic and dental cement. The chamber was filled with sterile cortex buffer (125 mM NaCl, 5 mM KCl, 10 mM glucose, 10 mM HEPES, 2 mM CaCl2, and 2 mM MgSO4 [pH 7.4]) and sealed with a glass coverslip. MK-2206 solubility dmso Intrinsic optical signals (Figures 1A and S1A–S1C) were imaged through the intact skull using an Imager 3001F (Optical Imaging, Mountainside, NJ, USA). For details see Supplemental Experimental Procedures. After imaging, a small, ∼1 × 1 mm piece of bone was removed using a dental drill (centered above the C2 whisker maximum intrinsic optical signal response). The dura was removed, and the craniotomy was covered with agarose (2% in cortex buffer). A glass coverslip was positioned over the agarose (covering more than half of the craniotomy) to reduce heartbeat and breathing-induced motion of the cortex. Whole-cell “blind” patch-clamp recordings were obtained as previously described by Brecht et al. (2003). High-positive pressure (200–300 selleck screening library mbar) was applied to the pipette (5–8 MΩ) to prevent tip occlusion while penetrating the agarose and the pia.

After passing the pia the positive pressure was immediately reduced to prevent cortical damage. The pipette was then advanced in 2 μm steps, and pipette resistance was monitored in the conventional voltage-clamp configuration. When the pipette resistance suddenly increased, positive pressure was relieved to obtain a 3–5 gigaohm seal. After break-in, Vm was measured, and dialysis was allowed to occur for at least 5 min before deflecting the whisker. Data were acquired using a Multiclamp 700B Amplifier (Molecular CYTH4 Devices), and digitized at 10 kHz (National Instruments), using MATLAB (MathWorks)-based Ephus software (http://research.janelia.org/labs/display/ephus;

The Janelia Farm Research Center). Offline analysis was performed using custom routines written in IGOR Pro (WaveMetrics). All neurons were located between 100 and 275 μm below the pia (Supplemental Experimental Procedures). Current-clamp recordings were made using a potassium-based internal solution (135 mM potassium gluconate, 4 mM KCl, 10 mM HEPES, 10 mM Na2-phosphocreatine, 4 mM Mg-ATP, 0.3 mM Na-GTP, 3 mM biocytin, 0.1 mM spermine, pH adjusted to 7.25 with KOH, 285 mOsm). Rs and input resistance (Rin, not including Rs) were monitored with a 100 ms long-lasting hyperpolarizing square pulse 400 ms prior to each whisker deflection, and extracted offline by using a double-exponential fit. Initial Rs and Rin were not different between CTRL and DWE cells (Supplemental Experimental Procedures). Recordings were discarded if one of the following conditions occurred: (1) Vm and Rs exceeded −50mV and 50 MΩ, respectively; (2) spontaneously occurring spikes were not overshooting; and (3) Rs or Rin changed more than 30% over the duration of the experiment. The bridge was usually not balanced, and liquid junction potential was not corrected.