Mea 1060bc

After 1 h preincubation in oxygenated aCSF at room temperature, each slice was transferred to a 60 channels MEA probe (Multi Channel Systems, Reutlingen, Germany). To cover the ACC, an 8 × 8 MEA with 100 μm electrode spacing was used in the experiment. The slice was positioned above the recording area on the MEA probe, and the upper region was aligned with one site of the ACC. A silver weight was placed on a net above the slice to provide mechanical stabilization. The chamber was kept at 30°C under continuous perfusion (2 ml/min) of oxygenated aCSF. Local field potentials were simultaneously recorded from 60 electrodes with high spatial and temporal resolution (inter-recording leads, 200 μm; total covered area, ∼1400 μm × 1400 μm). Local field potentials at each electrode were recorded against the bath electrode. 4AP (250 μM final concentration) and Bic (5 and 50 μM final concentrations) in aCSF were applied in the perfusion system to induce seizure activity. A 60-channel amplifier was used with a band-pass filter set between 1 Hz and 3 kHz (MEA-1060-BC, Multi Channel Systems, Reutlingen, Germany). The data were acquired using MC Rack software (Multi Channel Systems, Reutlingen, Germany) with continuous recording at a sampling rate of 10 kHz.

We used the same procedures previously described in Gullo et al. (2009 (link), 2010 (link)). Briefly, analog signals sampled at 40 kHz were recorded at 36°C in CO₂-controlled incubators using MEA-1060BC or 1060INV pre-amplifiers (bandwidth 1–8,000 Hz, Multichannel Systems, Germany) connected to a MEA Workstation (bandwidth 100–8,000 Hz, Plexon Inc., USA). Data were sorted into timestamp files by the MEAWorkstation Sorter software (MEAWS, see details below) and cleaned of artifacts using the OFFLine Sorter program (Plexon Inc., USA). Next, during the PCA-based waveform sorting and for multi-unit electrodes, we applied one of the following procedures: (i) spike removal with a Mahalanobis threshold in the range 1.8 to 1.4; we concurrently checked that the P-value of multivariate ANOVA sorting quality statistics was <0.01 among the identified units; (ii) when the previous procedure led to excessive spike invalidation, we manually removed the spikes invading the adjacent unit ellipsoids (the latter method was very effective in decreasing the P-values, with only a limited number of erased spikes).

Recordings of neural activity were conducted from 21 to 26 DIV using a commercial multichannel signal amplifier (MEA 1060BC, Multi Channel Systems, Inc gain 1200×, sampling rate 25 KHz, Bandwidth 1 Hz–10 KHz). Raw electrophysiological signals were filtered (300 Hz–3 KHz) and putative action potentials (spikes) were detected online using MEABench (Wagenaar et al., 2005 ) as positive or negative excursions beyond 5.0× estimated root-mean-square (RMS) noise gathered for each electrode during the first 15 s of each recording. Electrodes that produced spikes with rates less than 0.01 spikes per second are typically due to noise alone and were dropped from further analysis.
Spikes were then sorted using the surrounding ±1 ms of each spike’s waveform using the first three components from a principal components analysis (PCA) followed by unsupervised k-means based on the KlustaKwik method (Harris et al., 2000 (link)). There were no significant differences between groups in the average number of neurons per electrode (m = 1.37 ± 0.04, p > 0.06) but Layer I was slightly elevated relative to Layer II (1.46 ± 0.13 vs. 1.28 ± 0.11, Layer I and II respectively, p < 0.05).