On average the Vm was most hyperpolarized during quiet waking (Q; mean ± standard deviation [SD] −60.5 ± 7.5 mV; median −60.7 mV; range −73.8 to −44.7 mV), depolarized during free whisking (W; mean ± SD

−58.4 ± Transmembrane Transporters activator 8.3 mV; median −57.7 mV; range −73.7 to −36.9 mV), and was significantly more depolarized during active touch (T; mean ± SD −55.4 ± 7.7 mV; median −57.2 mV; range −70.6 to −37.0 mV) (Figure 2A and Table S2). Compared to free whisking, during an active touch sequence the Vm of layer 2/3 neurons on average depolarized by 3.0 ± 2.9 mV (median 3.0 mV; range −2.2 to +6.9 mV). Vm variance was significantly lower during free whisking than during quiet wakefulness or active touch (Figure 2B). The low Vm variance during free whisking (when there is no incoming

touch information) may help provide a reduced noise background enhancing the detection of sensory-evoked signals during active touch. The mean action potential firing rates (Figure 2C and Table S2) indicate that spike rates increased during active touch (1.7 ± 5.0 Hz; median 0.2 Hz; range 0.0 to 20.8 Hz) as compared to quiet wakefulness (0.2 ± 0.2 Hz; median 0.1 Hz; range 0.0 to 0.5 Hz) and free whisking (0.3 ± 0.9 Hz; median 0.04 Hz; range 0.0 to 3.9 Hz). For most neurons the firing rate of layer 2/3 pyramidal cells remained low Bafilomycin A1 datasheet in all conditions, in good agreement with recent awake extracellular recordings of identified layer 2/3 pyramidal cells (de Kock and Sakmann, 2009 and Sakata and Harris, 2009)

and awake two-photon calcium imaging in layer 2/3 (Greenberg et al., 2008 and O’Connor et al., 2010). Low-frequency Vm dynamics dominated the Fast Fourier Transform (FFT) during all behavioral periods, with a near linear decrease at higher frequencies when plotted on log-log scale axes (Figure 2D) similar to observations from EEG recordings (Buzsáki and Draguhn, 2004). Slow Vm fluctuations (1–5 Hz) were significantly more prominent during quiet wakefulness than during free whisking (Crochet and Petersen, 2006, Poulet and Petersen, 2008 and Gentet et al., 2010) or active touch (Figures 2D and 2E). High-frequency Vm changes (30 to 100 Hz) were significantly increased during active touch compared to quiet wakefulness or free whisking (Figures 2D and 2E). These higher-frequency PDK4 Vm dynamics are likely to be driven by the rapid and large-amplitude depolarizations evoked by individual touch responses. Analysis on the millisecond timescale revealed further important correlations between the C2 whisker-related behavior and neuronal Vm. We averaged the Vm across many individual whisking cycles aligned to the peak of protraction during free whisking and found small-amplitude phase-locked Vm fluctuations, which weakly influenced action potential firing (Figure 3A and Figure S1) (Fee et al., 1997, Crochet and Petersen, 2006, Poulet and Petersen, 2008, Curtis and Kleinfeld, 2009 and de Kock and Sakmann, 2009).