We found that spinal nerve ligation (SNL) significantly increased the level of P2Y12 receptors specifically in the microglia of the ipsilateral dorsal horn

We found that spinal nerve ligation (SNL) significantly increased the level of P2Y12 receptors specifically in the microglia of the ipsilateral dorsal horn. response to thermal stimuli on the ipsilateral side without affecting the basal threshold on the contralateral side. These effects on pain behaviors were replicated in P2Y12 knockout mice. Patch-clamp recordings further revealed that partial sciatic nerve ligation (PSNL)-induced excessive miniature excitatory postsynaptic currents (mEPSCs) were significantly attenuated in P2Y12 knockout mice. Moreover, we found that SNL activates the GTP-RhoA/ROCK2 signaling pathway and elevates the level of phosphorylated p38 mitogen-activated protein kinase (MAPK), which was inhibited by the P2Y12 antagonist. The phosphorylation of p38 MAPK was inhibited by Timp1 a ROCK inhibitor, but not vice versa, suggesting that p38 MAPK is downstream of ROCK activation. Our findings suggest that nerve injury engages the P2Y12 receptor-dependent GTP-RhoA/ROCK2 signaling pathway to upregulate excitatory synaptic transmission in the MG149 dorsal horn. This cross-talk ultimately participates in the manifestation of nociceptive allodynia, implicating P2Y12 receptor as a potential target for alleviating neuropathic pain. Introduction Nerve injury-induced neuropathic pain involves painful responses evoked by normally innocuous tactile stimuli, and it is one of the most challenging clinical problems1. However, the currently available therapeutics for this pathological pain are relatively limited. Microglia play an important role in the process of pathological pain. As potent stimulators of microglia, extracellular nucleotides caught our attention2. They play MG149 roles in various functions by activating purinergic receptors expressed in microglia3. In the pathological course of nerve injury, MG149 ATP can be released or leaked from a variety of sources, such as primary afferent terminals, dorsal horn neurons, and spinal astrocytes4. The release or leakage of ATP after nerve injury can then activate the neighboring microglia. Increasing evidence has emphasized the importance of P2 receptors for spinal microglia. These receptors, such as P2X45 and P2X76, have important roles in chronic pain. Among them, P2Y12, a P2Y metabotropic G-protein-coupled purinergic receptors, has become a new focus7. Research shows that P2Y12 is constitutively involved in cancer pain8, synaptic plasticity in the mouse visual cortex9 and ATP-induced membrane ruffling and chemotaxis10,11. P2Y12 is restrictively expressed on microglia in the central nervous system12. Once microglia are activated, neurotransmitters and inflammatory cytokines are released, which regulate neuronal function13, but whether P2Y12 is involved in the changes in neuronal function has never been reported before. Neuropathic pain is thought to be initiated by a series of changes in the sensory processing system, such as the functional reorganization of sensory transmission or aberrant development of neural plasticity. Our focus is on the superficial dorsal horn, especially the substantia gelatinosa (SG) area, which is highly involved in modulating nociceptive transmission14. In a previous study, whole-cell patch-clamp techniques were adapted to SG MG149 neurons in a spinal cord slice with an attached dorsal root to investigate synaptic responses to peripheral nerve stimulation15. However, our method involved stimulating the SG neurons directly and then assessing the miniature excitatory postsynaptic current (mEPSC) changes. Furthermore, the shape of EPSCs is determined by many factors, such as the amount of presynaptically released glutamate, the properties of postsynaptic glutamate receptors and the time course of glutamate clearance from the synaptic cleft16. MG149 Antagonists of P2Y12 have been reported to attenuate inflammatory and neuropathic pain17,18. In our study, we confirmed that P2Y12 is involved in the pathological activation of microglia, a process that is presumably involved in synapse remodeling and neural plasticity. We also confirmed the underlying molecular signaling pathway between P2Y12 and neuropathic pain, involving p38 mitogen-activated protein kinase (MAPK) and GTP-RhoA/Rho-associated coiled-coil-forming protein serine/threonine kinase 2 (ROCK2). Our data demonstrate that P2Y12 antagonists can potently inhibit the activation of microglia and the classic signaling pathway of microglia. Most important of all, P2Y12 knockout mice showed lower mEPSC increases after nerve injury than wild-type (WT) mice. Results Spinal nerve ligation increased the expression of P2Y12 The western blot analyses were used to determine the effect of spinal nerve ligation (SNL) surgery on P2Y12 expression in the spinal cord.