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Due to their small dimensions, electrophysiology on thin and intricate axonal branches in support of understanding their role in normal and diseased brain function poses experimental challenges. To reduce experimental complexity, we coupled microelectrode arrays (MEAs) to bi-level microchannel devices for the long-term  in vitro  tracking of axonal morphology and activity with high spatiotemporal resolution. Our model allowed the long-term multisite recording from pure axonal branches in a microscopy-compatible environment. Compartmentalizing the network structure into interconnected subpopulations simplified access to the locations of interest. Electrophysiological data over 95 days  in vitro  (DIV) showed an age-dependent increase of axonal conduction velocity, which was positively correlated with, but independent of evolving burst activity over time. Conduction velocity remained constant at chemically increased network activity levels. In contrast, low frequency (1 Hz, 180 repetitions) electrical stimulation of axons or network subpopulations evoked amplitude-dependent direct (5–35 ms peri-stimulus) and polysynaptic (35–1,000 ms peri-stimulus) activity with temporarily (<35 ms) elevated propagation velocities along the perisomatic branches. Furthermore, effective stimulation amplitudes were found to be significantly lower (>250 mV) in microchannels when compared with those reported for unconfined cultures (>800 mV). The experimental paradigm may lead to new insights into stimulation-induced axonal plasticity.

A multielectrode array microchannel platform reveals both transient and slow changes in axonal conduction velocity