Microglial CR3-mediated synaptic pruning in the dmPFC promotes the generation and maintenance of chronic muscle pain via glutamatergic dysfunction.

Chronic muscle pain (CMP) is highly prevalent, frequently comorbid with emotional disorders and characterized by a high risk of recurrence. Yet, the complex mechanisms underlying the generation and maintenance of CMP remain unclear, limiting the development of therapy. Here we identified suppressed glutamatergic neuronal excitability and reduced synaptic plasticity in the dorsomedial prefrontal cortex (dmPFC) of CMP rats using fiber photometry, patch-clamp, in vivo recording of field potentials and other techniques. The optochemical genetical activation of dmPFC glutamatergic neurons alleviated pain and anxiety-like behaviors. Single-cell RNA sequencing revealed a marked upregulation of proinflammatory microglia and complement receptor 3 (CR3) in the dmPFC, which correlated with reduced neuronal excitability and synaptic function. Flow cytometry and immunofluorescence further showed that hyperactive glutamatergic neurons induced microglial activation, proliferation, polarization and chemotaxis. Notably, the inhibition of microglia or knockdown of microglial CR3 restored dmPFC glutamatergic neuronal excitability and synaptic plasticity, thereby alleviating hyperalgesia and anxiety-like behaviors. This study demonstrates that microglial CR3-dependent synaptic pruning underlies suppressed glutamatergic neuronal excitability and reduced synaptic plasticity, playing a pivotal role in CMP generation and maintenance. These findings uncover novel microglia-neuron interactions and offer promising therapeutic targets for CMP and its emotional comorbid disorders. Microglial CR3-mediated synaptic pruning in the dmPFC suppresses glutamatergic neuronal excitability, contributing to chronic muscle pain generation and maintenance and represents a promising therapeutic target.