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 system ⁵ , the retina ^6,7 , and to cardiac conduction ^8,9 , and being associated with diseases like epilepsy ^10,11 and arrhythmia ^12,13 , it is still poorly understood since it is notoriously difficult to investigate in vivo and in vitro . In vitro electrophysiology 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 networks in vitro with 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 ^14–16 and reduced stimulation threshold ¹⁷ . Our precise measurements of electrophysiological activity support the importance of ephaptic coupling in neuronal circuit function. Printed neuronal circuits allow detailed in vitro studies of neuronal interactions and may serve as a platform for disease modeling related to synaptic, ephaptic, or myelination processes.