The Role And Mechanism Of Medial Prefrontal Somatostatin Neurons In Itch Perception

[Background]Itching is defined as an unpleasant feeling and emotional experience that causes the desire to scratch,while chronic itching can cause skin damage accompanied by anxiety,depression and other emotional disorders.These negative emotions will further aggravate itching,forming a vicious cycle and seriously affecting the physical and mental health and quality of life of patients.In order to provide experimental basis and new target for the clinical treatment of chronic itching,it is urgent to further study the mechanism of central pruritus.Somatostatin(SST)is a kind of neuropeptide.As an endogenous inhibitory regulator of different cell functions,Somatostatin is involved in development,proliferation,metabolism,secretion and nerve regulation.Some articles have found that SST neurons from dorsal root ganglia and spinal dorsal horn neurons mainly show "itching and analgesic" effects.The number of scratching caused by itching substances such as Histamine(His)and Chloroquine(CQ)can be significantly reduced after the conditioned removal of SST neurons in dorsal root ganglia and spinal cord.However,the role of SST neurons in the transmission of itching information in the upper spinal cord center is still unclear.As an important higher level brain function region,Medial prefrontal cortex(mPFC)plays an important role in higher functions such as learning,decision making and emotion,as well as in pain regulation and negative emotions associated with chronic pain.Functional imaging studies have found that mPFC activity is significantly enhanced in pruritus state,but the role of SST neurons in the mPFC in itch sensation information transmission remains unclear.In this study,SST Cre transgenic mice will be used to establish acute itching model induced by His and CQ and chronic itching model induced by carpotriol in Atopic Dermatitis(AD).Further combined with optogenetics,chemogenetics,behavioral pharmacology and in vivo fiber recording,mPFC will be used as the target area.The function and mechanism of SST neurons in mPFC were systematically studied from cell,synapse,neural circuit to the whole behavior.[Methods]1.Acute itching model was established by injecting His or CQ into the skin of the neck and back of mice.The chronic itching model of atopic dermatitis was established by applying carpotriol solution on both sides of both ears respectively.The itching behavior of mice was detected by using the itching video tracking system,and the scratching times of mice within 30 minutes were counted.2.Open field and elevated cross maze techniques were used to detect the anxiety of chronic itching mice with atopic dermatitis.3.The distribution of FOS positive cells in mPFC of acute and chronic itching mice was observed by immunofluorescence staining.The activation of SST neurons in mPFC of acute and chronic itching mice was observed.4.After SST Cre transgenic mice were used in combination with stereotactic injection technique to make SST-positive neurons in mPFC express GCa MP6 s specifically,the changes of calcium activity of SST-positive neurons in mPFC under pruritus condition were observed by in vivo optical fiber recording method.5.Using SST Cre transgenic mice,combined with stereotactic injection technique,chemogenetics and photogenetics,SST neurons in mPFC were activated or inhibited to observe the effect on itch behavior,and to study the role and mechanism of SST-positive neurons in mPFC in itch sensation.6.In SST Cre transgenic mice,auxiliary virus and RV-Env A-ΔGds Red were injected into the mPFC of SST Cre mice by stereolocalization technique,and the single synaptic afferent connections were observed.DIO-EGFP virus was injected into the mPFC of SST Cre mice by stereolocalization technique and its efferent association was observed.[Results]1.Acute and chronic itching models were successfully established.Scratching times were counted within 30 minutes after intradermal injection of pruritic agent.Compared with the control group(11.83 ± 7.02),the number of scratching in His group was(91.33 ± 14.00).The number of scratching in CQ group was(138.16 ± 12.38),and the number of scratching in model group was significantly higher than that in control group.The chronic itching model was established successfully.On the 21 st day,the itching behavior video was performed.The scratching times of mice in 30 min were counted,compared with the control group(14.16 ± 7.54).The number of scratching times in 30 min of atopic dermatitis chronic itching model group was(125.50 ± 32.77),which was significantly higher than that of control group.2.The open field test and elevated cross maze test were performed on chronic itching mice.The open field test results indicated that mice in the model group spent less time in the central area than those in the control group.The results of the elevated cross maze experiment indicated that,compared with the control group,the duration of mice in the model group on the open arm was lower than that in the control group,suggesting that the chronic itching model group mice had anxiety-like behavior.3.Immunofluorescence staining was used to observe the activation of neurons in the mPFC of acute itching model mice.The results showed that FOS positive cells in His and CQ model groups were significantly increased compared with the control group.The number of FOS positive cells in mPFC of atopic dermatitis chronic itching model mice was significantly higher than that of control group.4.Immunofluorescence staining was used to observe the activation of SST-positive neurons in the mPFC of acute itch model mice.The results showed that the SST/FOS double-labeled neurons in the mPFC of control group accounted for(15.00 ± 5.00)%of SST-positive neurons.SST/FOS double-labeled neurons in mPFC of His induced acute itch model accounted for(36.66 ± 11.93)% of SST-positive neurons.In CQinduced acute itching model,SST/FOS double-labeled neurons accounted for(60.00 ±19.07)% of SST-positive neurons,and the statistical results showed that the proportion of double-labeled cells in the model group increased significantly.SST/FOS doublelabeled neurons accounted for(60.33 ± 5.68)% of SST-positive neurons in the mPFC of the chronic itching model group,and SST/FOS double-labeled neurons accounted for(15.33 ± 5.50)% of SST-positive neurons in the control group.Compared with the control group,the proportion of double-labeled cells increased significantly.5.In vivo optical fiber recording results showed that the calcium signal of SSTpositive neurons in mPFC increased under acute and chronic itching conditions.The number of scratching induced by histamine decreased and the number of scratching induced by CQ increased when the SST-positive neurons in mPFC were specifically activated by optogenetics.Specifically inhibited SST-positive neurons in mPFC,histamine-dependent itch sensitivity increased,histamine-independent itch sensitivity decreased.Chemogenetics suggested that the activation of SST-positive neurons in mPFC showed no statistical difference in chronic itch sensitivity.Inhibition of SSTpositive neurons in mPFC reduced chronic itch sensitivity.6.Using retrograde transsynaptic technique,the afferent connection regions of SST neurons in mPFC were observed as follows: Basolateral amygdala(BLA),Central lateral thalamic nucleus(CL),Hippocampus(HIP),Insular cortex,IC),Thalamic nucleus reunions(Re),etc.In addition,the Lateral habenula(LH),Ventral tegmental area(VTA)and other efferent contact areas of SST neurons in mPFC were observed using the lateral virus tracing technique.[Conclusion]Scratching times of acute itching and atopic dermatitis chronic itching mice increased significantly,chronic itching mice showed anxiety-like behavior.In vivo optical fiber recording of SST neurons activation in mPFC of acute and chronic itching mice showed that the calcium signal of SST neurons in mPFC increased under acute and chronic itching conditions.The results of optogenetics and chemogenetics suggested that SST-positive neurons in mPFC were involved in the regulation of itching information.Sst-positive neurons in mPFC positively regulated histamine-independent itching and chronic itching in atopic dermatitis.Negatively regulates histaminedependent itching.The projective relationship between different regions of SSTpositive neurons in mPFC provides morphological basis for further study of the loop mechanism of SST neurons in itch sensation.