Structure-Function Dynamics of Engineered, Modular Neuronal Networks with Controllable Afferent-Efferent Connectivity

Microfluidic devices interfaced with microelectrode arrays have in recent years emerged as powerful platforms for studying and manipulating in vitro neuronal networks at the micro- and mesoscale. By segregating neuronal populations using microchannels only permissible to axons, neuronal networks can be designed to mimic the highly organized and modular topology of neuronal assemblies in the brain. In vivo, the development of such neuronal assemblies is tightly orchestrated by reciprocal, dynamic structure-function relationships shaped by an interplay between intrinsic neuronal self-organizing properties and spatiotemporally regulated chemical and physical guidance cues. Engineered neuronal networks represent reductionist paradigms that can help recapitulate such dynamics in vitro. However, little is known about how the underlying topological features of such engineered neuronal networks contribute to their functional profile. To start addressing this question, a key parameter is control of afferent or efferent connectivity within the engineered network. In this study, we show that a microfluidic device featuring axon guiding channels with geometrical constraints inspired by a Tesla valve effectively promotes unidirectional axonal outgrowth between neuronal nodes, thereby enabling us to control afferent connectivity. We verified this by applying designer viruses to fluorescently label the neurons to visualise the structure of the networks, combined with extracellular electrophysiological recordings using embedded nanoporous microelectrodes to study the functional dynamics of these networks during maturation. We furthermore show that electrical stimulations of the networks induce signals selectively transmitted in a feedforward fashion between the neuronal populations. This model system has the potential to provide novel insights into the development, topological organization, and neuroplasticity mechanisms of neuronal assemblies at the micro- and mesoscale in healthy and perturbed conditions.