Offline sorter

Unit extracellular recordings were performed from RGCs with carbon microelectrodes (1MΩ; Kation Scientific LCC Minneapolis, MN, USA) attached to an AC differential amplifier (DAM80i, World Precision Instruments) and digitized with an analog-to-digital board (Digidata 1440a; Axon Instruments, Sunnyvale, CA, USA). RGC action potentials were recorded with Axoscope (Axon Instruments, Foster City, CA) with a sampling rate of 20 kHz. In some experiments, multielectrode extracellular electrophysiology was also performed by utilizing either a 60 or a 120 channel MEA system (Multichannel Systems Gmbh, Germany) that allowed for recording simultaneously from many RGCs. Analyses were performed offline using Spike 2 (Cambridge Electronics Design Ltd., Cambridge, UK), Off-line Sorter (Plexon, Dallas, TX) and NeuroExplorer 5 (Nex Technologies, Littleton, MA) softwares. Histograms and graphs were generated in Origin2018 (OriginLab, Northampton, MA, USA). Collected spikes were timestamped (bin size 10 ms) to generate peristimulus-time histograms (PSTH; NeuroExplorer 5, Nex Technologies, Littleton, MA). PSTHs then served to obtain response peak positions and delays. Response fadeout time (PSTHτ) and trial-to-trial variability were calculated in Microsoft© Office Excel, further data- and statistical analyses were performed in Origin 2018 (OriginLab, Northampton, MA, USA).

We simultaneously recorded the spiking activity from many single neurons isolated from the 96-channel multielectrode array using a Cerebus Neural Signal Processor (Blackrock Microsystems). A block of 32 channels could be recorded simultaneously over the course of a session. The raw signal was bandpass filtered (0.3 Hz to 7.5 kHz) and digitized (16 bit) at 30,000 samples/s. For each channel, spikes were detected every time the digitally high-pass filtered (250 Hz/4-pole) voltage trace crossed a threshold equivalent to approximately four times the root mean square of the noise amplitude. The extracted spikes and associated waveforms were sorted offline using both manual and semi-automatic techniques (Offline sorter, Plexon Inc.). Monkey “F” completed 27 recording sessions with a mean (STD) of 47.89 (6.67) simultaneously recorded neurons. Monkey “JL” completed 28 recording sessions with a mean of 51.89 (3.67) simultaneously recorded neurons.

Raw data was digitally filtered with a 125-Hz high pass filter (four-pole Butterworth) sorting spike events. A threshold of 6SD was set for each channel and 1 ms of data before and 4 ms after a threshold-crossing event were stored for each negative-slope event. These candidate spike waveforms were then sorted with Offline Sorter (Plexon, Denton, TX) using the first three principal components of the spike waveforms. Coincident events within 0.5 ms of each other that occurred on all electrodes were attributed to perfusion noise and removed. Clusters were first identified using an EM cluster algorithm by Shoham et al. [37 (link)] then manually edited for clustering errors. Typically, the activity of one to three cells was recorded by each electrode.