Mc rack

Isolated retinas were placed photoreceptor side up on a perforated 60 electrode array (60pMEA200/30iR-Ti using a MEA2100 system: Multichannel systems, Reutlingen, Germany) in a recording chamber mounted on an Nikon Optiphot-2 upright microscope and viewed under IR with an Olympus 2x objective and video camera (Abus TVCC 20530). Extracellular multiunit GC activity was recorded at 25 kHz in MC rack (Multichannel systems, Reutlingen, Germany), zero-phase bandpass filtered (250–6,250 Hz) with a fourth-order Butterworth filter in Matlab (MathWorks, Natick, MA, USA), and sorted into single-unit activity with “offline spike sorter” (Plexon, Dallas, TX, USA). Spikes were detected using an amplitude threshold > 4σ_n where σ_n is an estimation of the background noise
${\sigma }_n=median\left\{\frac{\left|x\right|}{0.6745}\right\}$
with x being the bandpass-filtered signal [43 (link)]. The detected spikes were manually sorted into single units based on principal component or amplitude variables versus time. The clustering versus time approach allowed us to track changes in extracted features of single units occurring over extended recording periods and with differing firing rates.

MEA with 60 electrodes of 30 μm in diameter, spaced 200 μm from each other were used to record spontaneous network activity starting at 21 days-in vitro. N-methyl- D- aspartic acid (NMDA, Sigma-Aldrich) was used to increase corticostriatal bursting (Vergara et al., 2003 (link)). Typically cells were recorded in their own incubation medium or in artificial cerebrospinal fluid (ACSF; in mM: 136 sodium chloride; 5 potassium chloride; 1 magnesium chloride; 2.5 calcium chloride; 10 Hepes-sodium; 10 glucose) and no differences in recorded activity were seen between the two media. MEA temperature was kept at 37°C by a temperature controller (Multichannel Systems TC01/02 Rev D). Cell activity from each electrode was fed into the commercial 60- channel amplifier (Multi Channel Systems, MC-rack, Reutlingen, Germany) at 25 kHz sampling rate. To keep medium pH stable cells continually received carbogen (5–95% CO₂/O₂) bubbled in sterile water during recordings.
We examined three experimental conditions. First neurons were continuously recorded in their incubation medium for 3–6 min. Second, NMDA (250–300 nM) was then added to the medium and recordings were consequently performed for 3 min every 5 min. Finally a cut between cortical and striatal neurons was performed 15 min after NMDA was first introduced. At least 10 min elapsed before effects of the cut were recorded.

The recording data in vitro and in vivo were directly digitized at 20 kHz without filtering. The data were acquired and transformed using MC Rack and MC Data Tool software (Multi Channel Systems, Reutlingen, Germany). The data were analyzed using a custom MATLAB program (MathWorks, Natick, MA, USA). Briefly, event activity was first evaluated and filtered with a 200 Hz high-cut. To detect oscillatory events, we set two to four standard deviations (SDs) of the noise level as the threshold which is according to each background noise. The amplitudes of the peaks during an oscillation event and the seizure activities that surpassed this threshold were automatically detected by MC Rack software.

After 48 h of culturing, transduced retinal pieces with post-mortem times <20 h were placed photoreceptor side up on a perforated 60 electrode array (60pMEA200/30iR-Ti using a MEA2100 system: Multichannel systems, Reutlingen, Germany) in a recording chamber continuously superfused with oxygenated Ames’ medium (37 °C) mounted on a Nikon Optiphot-2 upright microscope and viewed under IR with an Olympus 2× objective and video camera (Abus TVCC 20530). Extracellular multiunit GC activity was recorded at 25 kHz in MC rack (Multichannel systems, Reutlingen, Germany), zero-phase bandpass filtered (250–6250 Hz) with a fourth-order Butterworth filter in Matlab (MathWorks, Natick, MA, USA), and sorted into single-unit activity with “offline spike sorter” (Plexon, Dallas, TX, USA). Spikes were detected using an amplitude threshold > 5 σ_n where σ_n is an estimation of the background noise
${\sigma }_n=median\left\{\frac{\left|x\right|}{0.6745}\right\}$
with x being the bandpass-filtered signal [71 (link)]. The detected spikes were manually sorted into single units based on principal components.

MEAs and associated equipment were acquired from Multi Channel Systems. We used a 60-channel MEA-1060-Inv-BC amplifier with MEAs that had 59 TiN electrodes with 30 μm radius and 200 μm spacing, and referenced recorded potentials against a larger internal reference electrode. Recordings were made at 37°C in room atmosphere. Before recording, cultures were allowed to equilibrate at least 5 min in room atmosphere after being taken out of the incubator.
Spontaneous activity on MEAs (see Fig. 2) was recorded for 10 min at a 32 kHz sampling frequency and 1200× gain. We recorded from all cultures three times per week for the first 48 d, where we changed to recording only twice every week. Data acquisition was performed using MC Rack (Multi Channel Systems), and files were then converted to the HDF5 file format using MC Data Manager (Multi Channel Systems). The exported files were opened in MATLAB (MathWorks) using the third-party publicly available MATLAB plugin MCSMATLABDataTools (2022; Armin Walter, McsMATLABDataTools) where all further analyses were performed using custom-written software.

Multi-electrode array (MEA) chips from Multi Channel Systems were coated with fibronectin (Sigma-Aldrich). Beating EBs were plated and incubated at 37°C, and MEA measurements were performed at 37°C, as previously described [7] (link). The signals were initially processed, and the obtained data were subsequently analyzed with MC Rack (Multi Channel Systems). Data for analysis were extracted from 2–5 min of the obtained data. The recorded extracellular electrograms were used to determine local field potential duration (FPD), defined as the time interval between the initial deflection of the field potential(FP) and the maximum local T wave. FPD measurements were normalized (corrected FPD: cFPD) to the activation rate using Bazett's correction formulae: cFPD = FPD/(RR interval)^1/2, where RR represents the time interval (in seconds) between two consecutive beats. Isoproterenol hydrochloride (Sigma-Aldrich), E4031 (Sigma-Aldrich), and verapamil hydrochloride (Sigma-Aldrich) were prepared as 1 or 10 mM stock solutions. The FPs were recorded for 5 min, and then drug was added to the medium. After 2–3 min of incubation, the FPs were again measured for 5 min.

Bursting raw data were analyzed with MC Rack (Multichannel Systems, Germany). Detection of bursts was performed using the “Spike Sorter” algorithm, which sets a threshold based on multiples of standard deviation of the noise (5-fold) calculated over the first 500 ms of recording free of electrical activity. A fivefold standard deviation threshold was used to automatically detect each event, which could be modified in real-time by the operator on visual check if needed. Bursts were arbitrarily defined as discharges shorter than 5 s in duration. Typically, bursts were characterized by fast voltage oscillations followed by slow oscillations or negative shifts. To analyze seizure activity, data were exported to Neuroexplorer v4 (Nex Technologies, USA). Paroxysmal events were identified as discharges lasting more than 5 s; successive paroxysmal discharges were considered separate events based on their waveform and on the presence of a minimum (>10 s) interval of silent or bursting activity between them^{84 (link)}.