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The aim of the study was to develop a method for long-term non-invasive recording of the bioelectrical activity induced in isolated neuronal axons irradiated with short infrared (IR) pulses and to study the effect of radiation on the occurrence of action potentials in axons of a neuron culture in vitro ., Materials and Methods: Hippocampal cells of mouse embryos (E18) were cultured in microfluidic chips made of polydimethylsiloxane and containing microchannels for axonal growth at a distance of up to 800 μm. We studied the electrophysiological activity of a neuronal culture induced by pulses of focused laser radiation in the IR range (1907 and 2095 nm). The electrophysiological activity of the neuronal culture was recorded using a multichannel recording system (Multi Channel Systems, Germany)., Results: The developed microfluidic chip and the optical stimulation system combined with the multichannel registration system made it possible to non-invasively record the action potentials caused by pulsed IR radiation in isolated neuronal axons in vitro . The propagation of action potentials in axons was detected using extracellular microelectrodes when the cells were irradiated with a laser at a wavelength of 1907 nm with a radiation power of 0.2-0.5 W for pulses with a duration of 6 ms and 0.5 W for pulses with a duration of 10 ms. It was shown that the radiation power positively correlated with the occurrence rate of axonal response. Moreover, the probability of a response evoked by optical stimulation increased at short optical pulses. In addition, we found that more responses could be evoked by irradiating the neuronal cell culture itself rather than the axon-containing microchannels., Conclusion: The developed method makes it possible to isolate the axons growing from cultured neurons into a microfluidic chip, stimulate the neurons with infrared radiation, and non-invasively record the axonal spiking. The proposed approach allowed us to study the characteristics of neuronal responses in cell cultures over a long (weeks) period of time. The method can be used both in fundamental research into the brain signaling system and in the development of a non-invasive neuro-interface., Competing Interests: Сonflict of interest. There is no conflict of interest to be declared.