Acupuncture at Lianquan (CV23) acupoint has been shown to improve swallowing function in post-stroke dysphagia (PSD). This improvement is supposed to be associated with the regulation of neuronal activity in the contralateral primary motor cortex (M1), while the underlying mechanism still needs to be elucidated. Perineuronal nets (PNNs) are well-known to be involved in the regulation of neuronal activity. Thus, we here aimed to detect the role of PNNs in the contralateral M1 hemisphere in the electroacupuncture (EA)-mediated effect in male mice. The results were obtained from a combination of methods, including in vitro slice electrophysiological recording, in vivo electrophysiological recording, and immunofluorescent staining in male mice. These results showed a decrease of the excitatory postsynaptic currents (sEPSCs) and no alteration of the inhibitory postsynaptic currents (sIPSCs) in the GABAergic neurons and the tonic inhibition in the excitatory neurons in the contralateral M1 after stroke induction, and EA recovered the impaired sEPSCs in the GABAergic neurons. We further found that the effect of EA-induced increase of c-Fos expression, enhancement of spike firing, potentiation of sEPSCs in the excitatory neurons, and improvement of swallowing function were all blocked by the removal of PNNs in the contralateral M1. In conclusion, the PNNs in the contralateral M1 was suggested to be participated in stroke pathogenesis and might be associated with the EA-mediated swallowing function rehabilitation of PSD in male mice. Our study provides insight into how PNNs might be involved in the mechanism of EA treatment for stroke rehabilitation.Significance StatementIn the present study, we concluded that the effect of EA-mediated the regulation of neuronal activity and swallowing function was absent after PNNs degradation in the contralateral M1. The PNNs in the contralateral M1 was associated with the stroke pathogenesis and might be participated in the EA-mediated rehabilitation of PSD mice, and whether PNNs involved in the regulation of EA-mediated swallowing function in PSD mice still needs to be explored. Our study provides insight into how the PNNs might be involved in the mechanism of EA treatment for stroke rehabilitation.Copyright © 2023 Yuan et al.