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By interfacing microElectrode arrays with microFluidics (\u03bcEF devices), it is possible to compartmentalize neuronal cultures with a specified alignment of axons and microelectrodes. This setup allows the extracellular recording of spike propagation with a high signal-to-noise ratio over the course of several weeks. Addressing these \u03bcEF devices, we developed an advanced yet easy-to-use publically available computational tool, \u03bcSpikeHunter, which provides a detailed quantification of several communication-related properties such as propagation velocity, conduction failure, spike timings, and coding mechanisms. The combination of \u03bcEF devices and \u03bcSpikeHunter can be used in the context of standard neuronal cultures or with co-culture configurations where, for example, communication between sensory neurons and other cell types is monitored and assessed. The ability to analyze axonal signals (in a user-friendly, time-efficient, high-throughput manner) opens the door to new approaches in studies of peripheral innervation, neural coding, and neuroregeneration, among many others. We demonstrate the use of \u03bcSpikeHunter in dorsal root ganglion neurons where we analyze the presence of both anterograde and retrograde signals in \u03bcEF devices. A fully functional version of \u00b5SpikeHunter is publically available for download from <jats:ext-link xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" ext-link-type=\"uri\" xlink:href=\"https:\/\/github.com\/uSpikeHunter\">https:\/\/github.com\/uSpikeHunter<\/jats:ext-link>.<\/jats:p>","DOI":"10.1038\/s41598-019-42148-3","type":"journal-article","created":{"date-parts":[[2019,4,8]],"date-time":"2019-04-08T10:04:10Z","timestamp":1554717850000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":10,"title":["\u00b5SpikeHunter: An advanced computational tool for the analysis of neuronal communication and action potential propagation in microfluidic platforms"],"prefix":"10.1038","volume":"9","author":[{"given":"Kristine","family":"Heiney","sequence":"first","affiliation":[]},{"given":"Jos\u00e9 C.","family":"Mateus","sequence":"additional","affiliation":[]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7467-6498","authenticated-orcid":false,"given":"C\u00e1tia D. 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