<p>Depression stands as a widespread psychiatric illness, yet existing therapeutic approaches are constrained by delayed effectiveness and adverse reactions. Scopolamine, as a M-AChRs antagonist, has shown significant antidepressant-like effects in preclinical studies, but its mechanisms remain unclear. The present study is designed to explore the molecular mechanisms that underpin the antidepressant-like actions of scopolamine. Four-week chronic restraint stress (CRS) was employed to induce depression-like behaviors, while behavioral assessments—including TST (the tail suspension test) and FST (the forced swimming test)—were utilized to evaluate the behavioral effects of scopolamine. Additionally, RT-qPCR (quantitative real-time PCR), Golgi-Cox staining, and electrophysiological recordings were applied to explore the molecular mechanisms underlying these behavioral effects of scopolamine. Our behavioral results revealed that 24 h after drug injection, scopolamine (0.3 mg/kg, i.p.) notably reversed CRS-induced increases in the immobility time in the TST and FST. Notably, further RT-qPCR analyses revealed that CRS led to a significant reduction in the mRNA levels of glutamatergic pyramidal neuron markers in the prefrontal cortex (PFC) and hippocampus, and this downregulation was fully reversed by scopolamine administration.​ Moreover, our electrophysiological measurements showed that scopolamine notably counteracted the CRS-induced drops in the firing frequencies of glutamatergic pyramidal neurons situated in the medial prefrontal cortex (mPFC); our Golgi-Cox results suggested that scopolamine improved the CRS-induced impairments in the synaptic plasticity in the mPFC and hippocampus. These observations implied that the synaptic plasticity of pyramidal neurons might be involved in regulating the antidepressant-like behavioral effects of scopolamine, providing mechanistic insights for the development of next-generation antidepressant medications.</p> Graphical Abstract <p></p>