This study explores the structure-function relationship in neuronal circuits, focusing on how unidirectional connectivity, as seen in the hippocampus, supports efficient information transfer. The authors use asymmetric microchannels in microfluidics to direct axonal growth, emulating natural brain organization. They tested several designs and identified a new “Rams” variant, which achieved a 76% probability of unidirectional connections after 14 days in vitro. Using microelectrode arrays, they confirmed that up to 94% of spiking activity along “Rams” microchannels directed signals toward the “Target” population, with network-level synchronization. Overall, this work demonstrates that asymmetric microchannels can successfully establish both structural and functional unidirectional connectivity. The overall design of this study is sufficient to induce axonal directionality and unidirectional connectivity. Here are just a few minor suggestions

1. Please identify the primary embryonic rat hippocampal neurons.
2. As shown in Fig. 1(B), could the authors also provide a Widefield microscopy mosaic image with the “Tesla v2” motif for comparison?
3. In addition to measuring action potentials, incorporating assessments of calcium ion transmission or synaptic vesicle release and recycling following stimulation would provide further insights into synaptic functionality. This would enhance the functional analysis of the synaptic connections.

Although some experiments will be conducted in future work, the existing data should be sufficient to validate the design. Publication is recommended.