Here we propose a methodology to analyze volumetric electrical activity of neuronal masses in the somatosensory barrel field of Wistar rats. robustness of our methodology to unavoidable physiological noise and electrode configuration. We compared the accuracy to reconstruct neocortical current sources with that obtained with a previous method. This constitutes a type of electrophysiological microscopy with high spatial and temporal resolution, which could switch the way we analyze the activity of cortical neurons in the future. extracellular electric recording from these barrels provides information about the activity of large populations of neurons with an excellent temporal resolution. Although the extracellular electric recording technique 83602-39-5 IC50 was launched in the middle of the 19th century, it is now recapitulating its role with the quick development of silicon-based microelectrode arrays (MEA). 83602-39-5 IC50 With the technological advances in the micro-electromechanic systems (e.g., deposition, lithography, etching, die-preparation, Wise, 2005), MEAs with high spatial resolution are gradually being built with a variety of not only microelectrode local configurations (e.g., tetrodes, octodes, polytrodes) but also shank spatial plans (e.g., linear or laminar, planar and three-dimensional) (Ulbert et al., 2001; Csicsvari et al., 2003; Buzski, 2004; Blanche et al., 2005; Kipke et al., 2008; Du et al., 2009; Ogawa et al., 2011; Riera et al., 2012). MEAs with three-dimensional types are ideal to obtain volumetric recordings from multiple barrels, a crucial step to understand trans-laminar and tangential interactions in the cortical microcircuits with an acceptable spatial and temporal resolution (Riera et al., 2012). Regrettably, the extracellular electric potentials do not represent directly the ionic flows generated by excitable membranes in active 83602-39-5 IC50 neuronal Mrc2 ensembles, i.e., the volumetric density of current sources = ?50 ? 100 ms) evoked by whisker deflections were calculated by averaging LFPs over 100 trials. Another band-pass filter with cut-off frequency of 500 Hz and 8 kHz was applied to the raw data. From the resulting high frequency components, we extracted MUA by negative edge detection threshold of 4 times the standard deviation and 1.5 ms dead time. Twenty samples (i.e., eight and twelve samples prior and posterior to the spike troughs, respectively) of the detected spikes were used for classification. Spikes at each microelectrode were divided into putative excitatory pyramidal cells (PCs) and interneurons (INs) by two-step clustering strategy (Ogawa et al., 2011). First, we represented the spikes using four-level Haar wavelets. From the resulting 20 wavelet coefficients, 10 representative coefficients were selected as the input for cluster analysis using the KolmogorovCSmirnov test. The cluster analysis was performed using the superparamagnetic clustering method (Blatt et al., 1996) followed by a manual clustering strategy to avoid obvious outliers and misclassifications. The aforementioned data processing was carried out using the free-downloaded MATLAB toolbox, Wave Clus (Quiroga et al., 2004). Second, we extracted three features from the mean waveform of each classified spike cluster, i.e., the peak amplitude asymmetry, half width and trough peak. We applied k-means clustering method to these features and we finally obtained two spike clusters (Figure ?(Figure2).2). Based on the three features, we assumed that spikes whose waveforms show wide and narrow shapes were generated by putative PCs and INs, respectively (Sakata and Harris, 2009). The separability of these clusters was tested by the Hotelling’s T-squared test (= 0.022). It is well known that spiny stellate (SS) cells in Layer 4 are one of the INs in the neocortex. The spike’s duration for SS cells is around 0.6 ms, which is within the range of that for the INs (i.e., 0.27C0.65 ms) but different from that for the PCs, i.e., from 0.70 to 1 1.50 ms (Tierney et al., 2004). Therefore, based only on its duration it is difficult to distinguish a spike fired by a SS cell from one fired by a GABAergic INs. Meanwhile, a study using intracellular recording showed that SS cells in the stimulated barrel respond around 6C8 ms after the deflection (Armstrong-James et al., 1992). Based on this criterion, we selected the microelectrodes located around layer 4 of the barrel corresponding to the stimulated whisker. We picked up IN-like spikes observed at these microelectrodes in the post-stimulus period from 6 to 8 8 ms, and defined them as putative SS cells. The spiking times of PCs, INs and SS cells at each microelectrode were.