Single-cell, single-shot stimulation reveals heterogeneous network recruitment not explained by standard functional properties.

Understanding how minimal perturbations influence cortical network dynamics remains a central challenge in neural engineering. While single-cell stimulation has been shown to affect population activity, the resulting variability is often treated as noise rather than an informative feature of network behavior. Here, we investigated how single-cell stimulation reflects heterogeneous modulation of neuronal recruitment and the extent to which these effects can be explained by the functional state of the stimulated cell. For this, we combined single-cell optogenetic stimulation with wide-field calcium imaging in cortical cultures. In each network, a single stimulation event was induced, and subsequent alterations in stimulus-coupled recruitment, synchrony, and pairwise correlations were quantified. Additionally, we evaluated whether the baseline functional state of the stimulated neurons, including their event activity levels and Pearson correlation structures, were linked to the observed network responses. Single cell stimulation induced effects were transient, and the network dynamics recovered over a few seconds within the responder population. Importantly, Our findings demonstrate that the observed direction and magnitude of recruitment changes were not significantly explained by the functional state of the stimulated neurons, indicating that these parameters do not capture the determinants of perturbation-induced network responses. This highlights a possibilities in the future approaches for characterizing network responsiveness and suggests that additional unobserved features govern the response of microcircuits to localized inputs.