Microfluidics-guided localized low-temperature modulation of axonal signal propagation

Low-temperature stimulation is recognized as a promising approach for neuromodulation, with the potential to suppress or slow neural activity. However, its impact on the spatial and electrophysiological properties of axonal conduction remains poorly understood. Conventional methods have lacked the spatial resolution necessary to isolate axon-specific responses to localized cooling. To overcome these limitations, we developed a microfluidic platform that integrates a microelectrode array (MEA) with a rapid and spatially confined cooling module. This platform enables real-time, phase-resolved monitoring of cooling-induced signal propagation between neuronal populations via unidirectionally guided axons, while maintaining structural integrity and enabling targeted thermal modulation. Using the microfluidic-MEA platform, we observed that one-minute cooling induced reversible suppression of both neuronal and axonal activity, followed by complete functional recovery. In contrast, five-minute cooling resulted in full recovery of neural network activity but persistent conduction delays in axons after rewarming, indicating selective vulnerability of axonal pathways and incomplete restoration of conduction dynamics. These outcomes were quantitatively validated through high-resolution electrophysiological recordings. Our findings demonstrate that localized cooling significantly modulates axonal conduction by altering ion channel kinetics and membrane excitability. The proposed platform offers a robust in vitro platform for dissecting cold-induced neuromodulation with axonal resolution, and lays the groundwork for precision-targeted neuromodulatory strategies in neuroengineering, brain-on-a-chip systems, and potential therapeutic applications for neurodegenerative disorders.