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Objective Cofilin is the major actin-depolymerizing factor in the CNS for regulation of actin dynamics.Neurodegenerative stimuli can induce the formation of cofilin rod, a pathological structure composed of cofilin and actin, which disrupts synapse function and causes neurite loss.Methods In this study, we used live cell imaging to monitor the whole process of cofilin rod formation in cultured hippocampal neurons overexpressing EGFP-tagged wild type cofilin, and the results were confirmed by immunostaining.Results We found a two-phase rod formation induced by glutamate stimulation.The first phase occurred shortly after stimulation (~1 h) but soon dissolved, and the second phase happened at a much longer time window (>9 h).Immunostaining of cofilin in neurons without cofilin overexpression also confirmed the two-phase formation pattern.Administration of either NMDA receptor antagonist or non-NMDA receptor antagonist before glutamate stimulation suppressed both phases of rod formation.The regulatory mechanism under this cofilin rod formation is under on-going investigation.Conclusion Cofilin rod formation induced by glutamate stimulation has a two-phase pattern and is both NMDA and non-NMDA receptor-dependent.