Chronic visceral pain is one of the primary symptoms of patients with irritable bowel syndrome (IBS),which affects up to 15％ of the population world-wide.The detailed mechanisms of visceral pain remain largely unclear.Our previous studies have shown that neonatal maternal deprivation (NMD) followed by adult multiple stress (AMS) advances the occurrence of visceral pain,likely due to enhanced norepinephrine (NE)-β2 adrenergic signaling.This study was designed to explore the roles of P2X3 receptors (P2X3Rs) in the chronic visceral pain induced by combined stress.Here,we showed that P2X3Rs were co-expressed in β2 adrenergic receptor (β2-AR)-positive dorsal root ganglion neurons and that NE significantly enhanced ATP-induced Ca2+ signals.NMD and AMS not only significantly increased the protein expression of P2X3Rs,but also greatly enhanced the ATP-evoked current density,number of action potentials,and intracellular Ca2+ concentration of colon-related DRG neurons.Intrathecal injection of the P2X3R inhibitor A317491 greatly attenuated the visceral pain and the ATP-induced Ca2+ signals in NMD and AMS rats.Furthermore,the β2-AR antagonist butoxamine significantly reversed the expression of P2X3Rs,the ATP-induced current density,and the number of action potentials of DRG neurons.Overall,our data demonstrate that NMD followed by AMS leads to P2X3R activation,which is most likely mediated by upregulation of β2 adrenergic signaling in primary sensory neurons,thus contributing to visceral hypersensitivity.