Helix neuronal ensembles with controlled cell type composition and placement develop functional polysynaptic circuits on Micro-Electrode Arrays

Neuronal cell cultures on Micro-Electrode Arrays (MEAs) provide an essential experimental tool for studying the connectivity and long-term activity of complex neuronal networks. MEA studies are generally based on the analysis of mixed neuronal populations constituted by a large number of cultured cells with cell type composition and connectivity patterns which are quite unpredictable a priori. In this work, we propose a different approach which consists of assembling on MEAs neuronal circuits formed by individually identifiable C1, C3, and B2 Helix neurons. Cells were plated under conditions of controlled number and position to form neuronal networks of defined composition. We performed multi-site electrophysiological recordings, and we characterized the firing dynamics. By means of cross-correlation analysis, we studied the electrophysiological properties of MEA-coupled microcircuits and characterized their activity patterns. We showed how the synaptic connectivity, actually observed in polysynaptic circuits of C1, C3 and B2 neurons, correlates well with the expected connectivity of C1–B2, B2–B2 and B2–C3 cell pairs as previously reported in conventional electrophysiological studies in culture.