Abstract Self-sustained recurrent activity in cortical networks is thought to be important for multiple crucial processes, including circuit development and homeostasis. Yet, the precise relationship between the synaptic input patterns and the spiking output of individual neurons remains largely unresolved. Here, we developed, validated and applied a novel in vitro experimental platform and analytical procedures that provide – for individual neurons – simultaneous excitatory and inhibitory synaptic activity estimates during recurrent network activity. Our approach combines whole-network high-density microelectrode array (HD-MEA) recordings from rat neuronal cultures with patch clamping and enables a comprehensive mapping and characterization of active incoming connections to single postsynaptic neurons. We found that, during network states with excitation(E)-inhibition(I) balance, postsynaptic spiking coincided precisely with the maxima of fast fluctuations in the input E/I ratio. These spike-associated E/I ratio escalations were largely due to a rapid bidirectional change in synaptic inhibition that was modulated by the network-activity level. Our approach also uncovered the underlying circuit architecture and we show that individual neurons received a few key inhibitory connections – often from special hub neurons – that were instrumental in controlling postsynaptic spiking. Balanced network theory predicts dynamical regimes governed by small and rapid input fluctuation and featuring a fast neuronal responsiveness. Our findings – obtained in self-organized neuronal cultures – suggest that the emergence of these favorable regimes and associated network architectures is an inherent property of cortical networks in general.

Parallel reconstruction of the excitatory and inhibitory inputs received by single neurons reveals the synaptic basis of recurrent spiking