Reorganization of Burst Initiation Zones as a Signature of Stimulation-Induced Plasticity in in vitro Cortical Networks

The spontaneous activity of neuronal cultures is dominated by periods of coherent activity known as network bursts . The dynamics of the initiation and propagation of these bursts are believed to depend on the structural connectivity of the neuronal network. Specifically, the spatial loci of burst initiation, referred to as initiation zones (IZs), are shaped by a complex interplay between dynamics and connectivity, with the particular structure of the latter playing a crucial role. Thus, alterations in network structure, such as those following from plasticity mechanisms activated by perturbations, are expected to lead to changes in network burst dynamics. To shed light on this point, we investigated the effect of localized electrical stimulation on the initiation of network bursts in in vitro cortical networks from embryonic rats of either sex cultured on high density micro-electrode arrays (HD-MEAs), and observed that electrical stimulation significantly altered the spatial patterning of IZs. To explore the mechanisms underlying these alterations, we assessed changes in effective connectivity (EC) using transfer entropy. Although both IZs and EC exhibited similar remodeling following stimulation, EC alone could not fully account for the observed IZ shifts. Moreover, IZ remodeling was largely unpredictable and independent of the stimulation site, suggesting that the observed changes were due to large-scale remodeling of connectivity structure, likely involving local potentiation/depotentiation and homeostatic mechanisms. Our findings provide evidence that electrical stimulation can remodel burst initiation and effective connectivity, although achieving precise local control appears unattainable. Significant Statement Spontaneous bursts in neuronal cultures emerge from a complex interplay between connectivity and intrinsic neuronal dynamics. Here, we show that brief electrical stimulation can modulate this interplay by shifting the spatial zones where bursts originate. Using high-density MEAs, we find that burst initiation zones are highly sensitive to stimulation, exhibiting pronounced and lasting spatial changes even when global firing rates remain stable. Effective connectivity maps reveal a coordinated but subtle network remodeling, suggesting that widespread synaptic adjustments and homeostatic mechanisms underlie these changes. Our results highlight both the responsiveness of spontaneous network activity to perturbations and the difficulty of achieving precise spatial control using simple stimulation protocols.