Microglial Engulfment Regulates Pain-Induced Anxiety-Like Behaviors In The Ventral Zona Incerta

Clinically,patients suffering from pain have a higher possibility of emerging anxiety,creating comorbid anxiety symptoms in pain(CASP).Due to its complex pathogenesis and unclear mechanism,it is challenging to have an efficient treatment.Recently,neuroinflammation is emerging as an essential target for the comorbidity of neurological disorders.Microglial cells are suggested as the brain’s resident immune cells.Although growing evidence shows that microglia activation is critical in developing brain disorders,their role in neuroinflammation and CASP is still vague.Recent works suggest that microglia activation mediates immune responses.Therefore,studying the microglia activation states during CASP and its effect on neuronal activity is vital.The zona incerta(ZI)is involved in nociceptive processing.Previous studies show that the spinothalamic tract is connected to the ZI and is involved in regulating cognition,mood disorders,and pain.In addition,the ZI mainly contains inhibitory GABAergic neurons,which can exert effective tonic inhibition on the posterior thalamic nucleus neurons,which are involved in processing the nociceptive information.Therefore,ZI may be a capable novel target for pain modulation and anxiety comorbidity.However,under pain conditions,does GABAergic neuron activity in ZI alter,and how does this neuronal alteration affect microglia activation in ZI?How do these neuronal alterations and microglial changes interact to regulate the comorbid anxiety symptoms in acute pain?It is still unknown and will be the focus of this study.In this study,an animal model of acute inflammatory pain and anxiety-like behavior was established by complete Freund’s adjuvant(CFA)injection into the left hind paw of mice one day before experiments(CFA-1D).Using electrophysiological recording,animal behavior models,chemogenetic,qPCR,three-dimensional reconstruction of microglia,and pharmacological techniques,we explored the role of ventral ZI(ZIv)microglial cells(ZIvMicroglia)and the ZIv GABAergic neurons(ZIvGABA)in acute pain,and role of maladaptive alterations ZIvMicroglia in regulating ZIvGABA activity during CASP development.In this study,we used the elevated plus-maze and open field tests to find that CFA-1D mice exhibited significant anxiety-like behaviors;however,it disappeared seven days post-CFA injection(CFA-7D).The excitatory and synaptic transmission activities of ZIvGABA were visualized and recorded in GADtdTOM transgenic mice.Operating electrophysiological recordings,we observed decreased neuronal activity in ZIvGABA and reduced frequency of the miniature excitatory postsynaptic currents in CFA-1D mice compared with the control mice.Chemogenetic activation of ZIvGABA significantly relieved anxiety-like behaviors in CFA-1D mice.Conversely,in naive mice,chemogenetic inhibition of ZIvGABA induced anxiety-like behaviors with decreasing neural hyperactivity in ZIvGABA.Moreover,we found that ZIv microglial cells(ZIvMicroglia)notably activated in CFA-1D;qPCR detection found a significant increase in inflammatory factors such as interleukin-1β(IL-1β)and tumor necrosis factor(TNFα)in ZIv.Immunofluorescence assay found the extent of microglial engulfment spines and upregulation of surface markers MHC-II and CD68,accompanied by a significant increase in the postsynaptic density protein95(PSD95).Furthermore,after intra-ZIv minocycline injection,a microglia inhibitor,the ZIvGABA excitability in CFA-1D mice reversed,and anxiety-like behaviors and pain sensitization alleviated.In conclusion,by establishing an acute inflammatory pain model,combined with three-dimensional reconstruction of microglia,electrophysiological,pharmacological and chemogenetical techniques,we demonstrate alterations in the state of microglial cells in the ZIv,leading to decreased ZIvGABA excitability.This microglia activation promotes excessive engulfment of spines that decreases ZIvGABA activity,promoting anxiety-like behaviors in pain.Our result provides novel insights into the interaction of microglial cells and neurons in an inflammatory condition:microglia activation encodes maladaptation of ZIvGABA,thus promoting the development of comorbid anxiety symptoms in pain.Also,our study provides a possible outline for understanding the cellular basis for how inflammatory proceedings manifest as comorbid anxiety symptoms in pain.