The Role And Underlying Mechanisms Of The Dorsal Medial Prefrontal Cortex In The Descending Modulation Of Itch

Objective:Itch is an unpleasant sensation that causes a strong desire to scratch.Itch is one of the common clinical symptoms of most skin diseases and some systemic diseases.Intractable pathological itch not only aggravate the disease but also seriously affect the quality of life of patients,and is difficult to treat.The main reason is that our understanding of the generation,transmission pathway and regulation mechanisms underlying itch information,especially the inhibition regulation mechanism of itch in the brain is still very limited.In recent years,many progresses have been made in the neural mechanism underlying facilitation regulation of itch,including the molecular,cellular,brain region and circuit level analysis of itch facilitation regulation,but so far,there is little understanding of the key brain regions and circuits involved in the inhibition regulation of itch,especially the inhibitory regulation of excitatory neurons.The balance or imbalance between facilitation regulation and inhibition regulation in the brain may be one of the key factors to maintain the formation of physiological or pathological itch.It is well known that dorsomedial prefrontal cortex（dm PFC）,including prelimbic cortex（Pr L）and rostral anterior cingulate cortex（r ACC）,is involved in the inhibitory regulation of pain,learning and memory,but its role in the regulation of itch sensation remains unclear.Therefore,in this study,a variety of technical methods were comprehensively used to investigate the role of the Pr L and r ACC in the regulation of itch and the underlying mechanisms of cellular and neural circuits.Methods:In this project,we used a combination of advanced techniques,including animal behaviors,optogenetics,chemogenetics,c-Fos tet-off labeling system,in vivo fiber photometry recording,neurotransmitter fluorescent probe,neural circuit tracing and histological method to investigate the role of Pr L and r ACC in the regulation of itch and the underlying mechanisms of cellular and neural circuits at the neurotransmitter,cellular,neural circuit,behavioral level.Results:（1）After itch,the number of c-Fos positive neurons in Pr L brain area increased significantly.The fiber photometry recording results also showed that the activity of Pr L neurons increased significantly during the itch-scratching cycle.Moreover,chemogenetic and optogenetic inhibition of Pr L neurons significantly decreased scratching behavior induced by both histaminergic and nonhistaminergic itch.（2）The fiber photometry recording results showed that the activity of Pr L itch-responsive neurons increased significantly during the itch-scratching cycle.In addition,chemogenetic inhibition of Pr L itch-responsive neurons significantly reduced the scratching behavior induced by itch.（3）Double immunofluorescence staining indicated that the number of r ACC pyramidal neurons(r ACC^PN)with c-Fos positive increased significantly after acute itch.The proportion of c-Fos positive pyramidal neurons in the r ACC of the 5-HT high responder group was significantly lower than that of the 5-HT low responder group.Moreover,the number of activated pyramidal neurons was negatively correlated with the number of scratching bouts.Additionally,in vivo fiber photometry recording also showed that the activity of r ACC pyramidal neurons was significantly inhibited during itch-scratching（the decrease of fluorescence signals was slightly earlier than the scratching onset）.（4）Chemogenetic inhibition of r ACC pyramidal neurons was found to significantly increase itch-induced scratching behavior.In contrast,Chemogenetic and photogenetic stimulation of the r ACC pyramidal neurons was found to significantly reduce itch-induced scratching behavior.（5）After itch,the number of c-Fos positive r ACC GABAergic(r ACC^GABA)neurons increased significantly.The proportion of c-Fos positive r ACC^GABA neurons in 5-HT high responder group was significantly higher than that in the 5-HT low responder group.Moreover,the number of activated r ACC^GABAneurons was positively correlated with the number of scratching bouts.Additionally,in vivo fiber photometry recording also showed that r ACC^GABA neurons significantly increased activity during itch-scratching（the increase of fluorescence signals was slightly earlier than the scratching onset）.（6）Chemogenetic inhibition of r ACC^GABA neurons significantly reduced itch-induced scratching behavior.In contrast,chemogenetic inhibition activation of r ACC^GABA neurons significantly increased itch-induced scratching behavior.（7）The results of the neurotransmitter fluorescent probe with fiber photometry showed that ACC pyramidal neurons received significantly more inhibitory neurotransmitter GABA during itch processing.（8）The activity of the r ACC itch-responsive pyramidal neurons were significantly inhibited during scratching induced by itch.Chemogenetic inhibition of the r ACC itch-responsive pyramidal neurons significantly increased itch-induced scratching behavior.In contrast,chemogenetic activation of the r ACC itch-responsive pyramidal neurons significantly inhibited itch-induced scratching behavior.（9）The r ACC pyramidal neurons send glutamatergic projections to PAG.The pyramidal neurons expressing vesicular glutamate transporter 1（VGLUT1）in r ACC mainly project to the rostral dorsolateral periaqueductal gray（dl PAG）,lateral periaqueductal gray（l PAG）,and ventrolateral periaqueductal gray（vl PAG）,and send monosynaptic projections to PAG^GABAand PAG^Glu neurons.The r ACC pyramidal neurons projecting to PAG were mainly distributed in layerⅤof cortex.（10）The PAG-projecting r ACC pyramidal neurons were significantly activated during itch-induced scratching behavior.Chemogenetic inhibition of PAG-projecting r ACC pyramidal neurons could significantly increase itch-induced scratching behavior,while activation of PAG-projecting r ACC pyramidal neurons could significantly decrease itch-induced scratching behavior.（11）Chemogenetic inhibition of PAG-projecting r ACC itch-responsive pyramidal neurons significantly increased itch-induced scratching behavior,while activation of PAG-projecting r ACC itch-responsive pyramidal neurons had a significant inhibitory effect on itch-induced scratching behavior.（12）The fiber photometry recording results showed that the activity of PAG^GABAneurons decreased significantly itch-induced scratching behavior（the decrease of fluorescence signals was slightly earlier than the scratching onset）,while the activity of PAG^Glu neurons increased significantly itch-induced scratching behavior（the increase of fluorescence signals was slightly earlier than the scratching onset）.（13）The results of the neurotransmitter fluorescent probe with fiber photometry showed that PAG^GABA neurons received significantly less excitatory neurotransmitter Glu during itch-induced scratching behavior.（14）Optogenetic activation of r ACC itch-responsive pyramidal neurons significantly increased the activity of PAG^GABA neurons and significantly decreased the activity of PAG^Gluneurons.（15）Chemogenetic inhibition of PAG^GABA neurons postsynaptic to r ACC pyramidal neurons significantly increased itch-induced scratching behavior,while activation of PAG^GABA neurons postsynaptic to r ACC pyramidal neurons significantly inhibited itch-induced scratching behavior.Conclusion:The Pr L neurons are involved in the facilitation regulation of itch,while the r ACC pyramidal neurons are involved in inhibition regulation of itch.The r ACC pyramidal neurons to PAG^GABA neurons projections are a key neural circuit in the descending inhibition of itch.This study helps to reveal the neurobiological basis of itch sensation,enrich our understanding of the descending inhibitory systems of itch information,and provides an important theoretical basis for the search for potential therapeutic targets of intractable itch.