We combine ultrasensitive electron paramagnetic resonance (EPR) spectroscopy, 4,096-channel high-density multielectrode array (HD-MEA) recordings, and advanced graph-theoretical analysis to study how NO contributes to retinal network formation during a critical period of synaptogenesis. Retinal waves recording P9-11 Long-Evans rats were submitted to rapid eye enucleation after decapitation. Retinas were isolated in aCSF solution containing (in mM) 126 NaCl, 26 NaHCO3, 10 D-glucose, 1.45 NaH2PO4, 2.5 KCl, 1 MgCl2, 2 CaCl2, continually oxygenated (5% CO2 – 95% O2), and placed on a nitrocellulose membrane (#AABP04700, Millipore, USA) with the ONL facing the membrane. Retinas were kept oxygenated at room temperature (~25°C) for at least one hour before electrophysiological recordings. For MEA recordings, retinas were placed over the MEA with the RGC layer facing the electrodes and maintained in position by a homemade harp. Retinas were maintained for ~1 hour with continuous perfusion of oxygenated aCSF (~2 mL/min at 33 °C) until the spontaneous activity reached stability. Using a high-resolution MEA system consisting of 4,096 active electrodes in a 5.12 x 5.12 mm area with an inter-electrode space of 81 µm (BioChip 4096 3Brain GmbH, Switzerland), 10 min recordings were obtained of stable retinal activity (baseline) followed by 30 min recordings with an L-NAME or bicuculline diluted in the aCSF (100 µM or 10-50 µM, respectively). All records were acquired at 7062.1 Hz and saved on hard drives for further analysis. Retinal waves analysis All recordings were submitted for offline analysis using Herding Spikes 2, a highly efficient spike detection method that uses C++ and Python routines (Muthmann, Amin et al. 2015). Only channels with more than 5 and fewer than 300 spikes were considered to characterize retinal waves. For each channel, only spikes in a burst were used, which are characterized by sequences of at least 4 events with inter-spike intervals (ISI) less than 1.0 second, whereas an ISI of >1 second denoted the end of the burst. After that, for each one-second window, the number of spikes in all channels was counted. The retinal wave starts when at least 4 spikes are counted in a row for 5 seconds and ends when fewer than 4 spikes are counted in a one-second window. Waves with fewer than 50 spikes were discarded. The burst analysis algorithm provided the wave duration, the number of spikes per second, the mean interspike interval (ISI), and the standard deviation of ISI within each wave. The python codes and bin data are included in NO_and_developing_retina.zip.