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Objective In the past 10 years, a technique allowing simultaneous, multisite and many neuron recordings in freely moving and behaving animals (e.g., monkeys, rats and mice) and even in human subjects has been well developed and successfully applied in many regions of the brain.The state-of-the-art technique of in vivo simultaneous multisite recordings of neural ensemble responses in the brain enables a good combination with various behavioral assays in freely moving and behaving animals.In this report, Ⅰ would like to introduce the use of this technique in study of pain in the cerebral cortex of behaving rats.Methods Microwire electrode arrays (2×4 or 2×8) were implanted into the target brain area of a rat in the aid of stereotaxic surgical procedures.After at least 7 days of recovery, simultaneous recordings of cerebral cortical neural ensemble responses in a behaving rat were carried out using the Plexon multichannel Acquisition Processor (MAP) system and an overhead video tracker system processed by Plexon CinePlex software that enables continuous real-time behavioral tracking and synchronization of video and neural signals through interaction with the MAP system.A Plexon MAP system receives and processes the pre-amplified and filtered neural ensemble signals by RASPUTIN Software for online displaying, sorting, recording and synchronizing of the neural ensemble data.Various nociceptive stimuli, including mechanical, thermal and chemical modalities, were applied to the rats hindpaws.Results Long-term unitary stability was confirmed by various statistical analyses.Nociceptive neuronal units were identified by online or offline sorting from the primary somatosensory (S1) cortex and the motor cortex (MCx).The neuronal units identified in the S 1 area were wide-dynamic-range (WDR) and nociceptive-specific (NS) in nature, while those in the MCx were also responsive to noxious heating.The neural ensemble responses can be significantly changed by peripheral inflammatory pain state that also caused parallel long-term behavioral responses.The correlation between the cortical neural ensemble responses and behavioral responses were analyzed.Conclusion The long-term stability of unitary recordings and neural ensemble responses to the various nociceptive stimuli applied to the hindpaws of behaving rats were successfully achieved by combination of the Plexon MAP and Video Tracker system.The results showed advantages of the technique in study of pain in the cerebral cortex that (1) allows simultaneous recordings of many units from multiple sites that avoid bias of single unit recording, (2) allows simultaneous analysis of different patterns of neuronal responses, (3) allows simultaneous analysis of neuronal interactions, (4) allows analysis of correlation between neuronal activities and behaviors, with equal high spatial and temporal resolutions.