Empirical validation of ephaptic coupling in printed human neural circuits

Ephaptic coupling is a phenomenon describing the influence of endogenous electric fields on neuronal activity^1,2. Although ephaptic coupling is deemed to contribute to computations in the brain^1,3,4, the olfactory system⁵, the retina^6,7, and to cardiac conduction^8,9, and being associated with diseases like epilepsy^10,11and arrhythmia^12,13, it is still poorly understood since it is notoriously difficult to investigatein vivoandin vitro.In vitroelectrophysiology allows accessible and flexible experimentation, but circuits form randomly, leading to a lack of precision and reproducibility. Here, we present a method for reproducibly constructing human neuronal networksin vitrowith single-cell precision. We constructed neuronal circuits from the bottom up by bringing their axons into close contact. This enabled us to measure the effects of ephaptic coupling and validate theoretical predictions, such as reduced action potential velocity, increased activity synchronization^14–16and reduced stimulation threshold¹⁷. Our precise measurements of electrophysiological activity support the importance of ephaptic coupling in neuronal circuit function. Printed neuronal circuits allow detailedin vitrostudies of neuronal interactions and may serve as a platform for disease modeling related to synaptic, ephaptic, or myelination processes.