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Objective To investigate the discharge profiles of different types of neurons in the medial prefrontal cortex (mPFC) during associative motor learning and their correlation with learning behaviors. Methods Trace eyeblink conditioning (tEBC) was used as the behavioral model of associative motor learning. Freely-moving mice (n=4) were trained to acquire tEBC for 5 consecutive days. During daily tEBC training, the mice were received 100 paired presentations of the conditioned stimulus (CS, 150-ms blue LED pulse) and unconditioned stimulus (US, 100-ms corneal airpuff). Tungsten tetrodes were utilized to record the discharge activity of mPFC neurons. The mPFC neurons were categorized into putative pyramidal cells (n=174) and putative interneurons (n=40) according to the discharge rate and width of the mean spike waveform from trough to peak for each neuron. The correlation between the activity of distinct mPFC neurons and the performance of conditioned eyeblink responses (CR) was analyzed in either the early- or the late-learning stage. Results After 5 days' CS-US paired training, the mice exhibited increased CR incidence from (38.1±8.2)% on the 1st day to (66.7±10.0)% on the 5th day (P < 0.001). The statistical analysis revealed that the CS-evoked discharge activity of putative mPFC pyramidal cells at the late-leaning stage was significantly weaker than the early-learning stage (P < 0.05). In contrast, the CS-evoked discharge activity of putative interneurons at the late-learning stage was significantly greater than that at the early-learning stage (P < 0.05). Consistent with this, the proportion of CS-evoked increased firing interneurons was significantly greater at the late-learning stage (66.7%, 14/21) than the early-learning stage (25.0%, 4/16, P < 0.05). Moreover, there was no significant difference in the CS-evoked activity of mPFC pyramidal cells between the CR and the non-CR states (P>0.05), whereas the CS-evoked activity of mPFC interneurons in the presence of CRs was stronger than in the lack of CRs (P < 0.05). Conclusion During the acquisition of tEBC, the change of CS-evoked firing activities in mPFC interneurons is opposite to that in mPFC pyramidal cells, suggesting that these 2 types of mPFC neurons might participate in associative motor learning in different ways. In particular, the CS-evoked firing activity of mPFC interneurons is closely correlated with the CR performance.