The Role Of Glutamatergic Projections From The Midcingulate Cortex To Zona Incerta In Neuropathic Pain

Chronic pain is the primary cause of long-term disability globally,and poses a significant health-related burden on society.Neuropathic pain is one of the most common and notorious chronic pain,affecting 7%-10%of population worldwide.At present,the drugs recommended by the clinical guidelines of neuropathic pain management are only effective for part of patients,and many of them discontinue medication because of intolerance of side effects.Therefore,the treatment of neuropathic pain is still a clinical difficulty,which is largely due to that the pathogenesis is not clear.More research is urgently needed to explore the pathogenesis of neuropathic pain in order to find effective interventions and drug targets.The changes in cerebral structures after nerve injury and the corresponding contributions to neuropathic pain have not been well understood yet.Here,we took advanges of in vivo single unit recordings,calcium signal fiber photometry,optogenetics,chemogenetics,retrograde trans-monosynaptic virus tracing and classical pharmacological approaches to investigate the role of glutamatergic projections from the area 2 of midcingulate cortex(MCC Cg2^Glu)to zona incerta?ZI?in neuropathic pain.Firstly,we determined whether MCC Cg2^Glu are related to pain.In vivo single unit recordings and calcium signal fiber photometry found that the majority of MCC Cg2^Glulu were inhibited by painful stimulation,indicating MCC Cg2^Glu participate in pain processing.We then used optogenetic approach to selectively manipulate the activity of MCC Cg2^Glu and found that activation of MCC Cg2^Glu inhibited pain,while inhibition of MCC Cg2^Glu aggravated pain,indicating that these neurons are tonically involved in inhibitory modulation of nociception.We further explored the neural circuits of pain inhibitory role of MCC Cg2^Glu.We found that MCC Cg2^Glu sent dense projections to ZI and optical activation of MCC Cg2^Glu terminals in the ZI inhibited nociception in a glutamate-dependent manner.These results demonstrate that projections to ZI mediate the pain modulatory role of MCC Cg2^Glu.Retrograde trans-monosynaptic tracing of GABAergic neurons in ZI(ZI^GABA)revealed that Cg2^Glu formed synaptic connection with ZI^GABA.Single unit recordings found that the majority of ZI neurons were activated by optical activation of Cg2^Glu terminals in ZI.Chemogenetic inhibition of ZI^GABA abrogated the pain inhibitory effect of activating MCC Cg2^Glu.These results demonstrate that the excitatory innervation of ZI^GABA by MCC Cg2^Glu plays a pain inhibitory effect.We further investigated the role of MCC Cg2^Glu in neuropathic pain.Firstly,we found MCC Cg2^Glu became hypoactive after nerve injury by using in vivo single unit recordings.The hypoactivity of MCC Cg2^Glu should impair the pain inhibitory role and contribute to neuropathic hypersensitivity.Although a brief activation of the MCC Cg2^Glulu to ZI^GABA circuit was able to relieve the aversiveness associated with spontaneous ongoing pain,consecutive daily activation of the circuit through postoperative days was required to alleviate neuropathic allodynia.In contrast,glutamatergic neurons in the area1 of midcingulate cortex play opposite roles in pain modulation.They became hyperactive after nerve injury and only consecutive inhibition of their activity relieved allodynia.These results demonstrate that MCC Cg2^Glu constitutes a component of intrinsic pain inhibitory circuitry and their hypoactivity underlies neuropathic pain.We propose that selective and persistent activation of the MCC Cg2^Glu to ZI^GABA circuit may serve as a potential therapeutic strategy for this disease,either by physical or chemical approaches.