The manuscript explores the development and functional evaluation of asymmetric microchannel motifs to guide and direct axonal growth between neuronal populations. Using microfluidic devices combined with microelectrode arrays (MEA), the authors studied the structure-function relationship in engineered neuronal circuits. The study introduces a novel "Rams" microchannel design that effectively biases unidirectional axonal outgrowth and ensures functional connectivity between "Source" and "Target" neuron populations. The findings highlight the role of engineered microenvironments in studying neuronal connectivity and suggest potential applications in modeling neurological diseases and preclinical drug screening.

Following suggestions fo the authors:
1) Provide more details on why previous designs (e.g., "Arrowhead") underperformed compared to expectations. This could guide the scientific community in replicating and improving upon earlier findings.
2) Hihgligths importance of the implementation of brain-on-chip alike models to replace animal model and consideration about "engineering a sustainable future"
3) comment on possible drawbakcs may arise if this system woulf become a standard and PDMS replaces with other materials (if need be)
4) Incorporate case studies or hypothetical examples of how these circuits could be applied in disease models or pharmacological testing.
5) cite and comment the event abstract "Optimization of asymmetric microchannels for unidirectional synaptic coupling of neuronal cultures with high efficiency"

The authors successfully implemented the suggestion if the reviewers and now the article in my opinion is more critical and clear and ready to be published