Nitric oxide refines retinal circuit architecture independently of retinal wave dynamics.

How diffusible neuromodulators control neural circuit assembly remains a key open question in neuroscience. While nitric oxide (NO) is known to regulate mature synaptic plasticity, its role during development, specifically whether it shapes circuits through activity-dependent or activity-independent pathways, has not been fully understood. Here, we combine ultrasensitive electron paramagnetic resonance (EPR) spectroscopy, 4096-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. In the rat retina, nNOS expression begins at postnatal day 10 in two distinct amacrine cell subtypes, coinciding with the first detectable NO production. Acute or selective nNOS inhibition preserved the spatiotemporal features of Stage III retinal waves but significantly changed network topology, increasing network degree and density. Molecular profiling showed that nNOS blockade lowered the expression of chemical (SYN, SYP) and electrical (Cx36, Cx45) synaptic genes, disrupted their laminar distribution in vivo, and increased neurite length in primary retinal cultures without altering branching complexity. By combining ultrasensitive NO detection, large-scale electrophysiology, and mathematical network analysis, our results identify NO as a key regulator of circuit refinement that operates largely independently of the spatiotemporal dynamics of retinal waves during development. These findings reveal a molecular mechanism by which diffusible modulators shape neural networks independently of patterned activity and offer a framework for understanding how altered NO signaling might contribute to neurodevelopmental disorders characterized by impaired synaptic organization.