The Mechanism Of Anterior Cingulate Cortex Neurons In Itch Sensation Regulation

Itch sensation is an unpleasant feeling and emotional experience that causes scratching desire,often accompanied by scratching behavior.Most skin diseases such as Atopic dermatitis(AD)and xeroderma are often accompanied by severe itching symptoms.As China enters the aging society,the incidence of chronic itch is increasing year by year.Chronic itch can cause anxiety,depression and other emotional disorders,and seriously affect the physical and mental health and quality of life of patients.However,there is no effective treatment for most pruritus with low side effects.Although important advances have been made in understanding the molecular mechanisms of itch sensation,most of the research has focused on peripheral and spinal cord levels.The research on how itch sensation information is transmitted and regulated at the supraspinal level is very limited.It is urgent to carry out in-depth research on the neural mechanism and intervention strategies of central itch sensation,which may provide experimental basis and new targets for the clinical treatment of chronic itch.Anterior cingulate cortex(ACC)is an important structure of the limbic system,which is involved in the regulation of pain sensation and emotion.Functional imaging studies found that ACC activity was significantly enhanced under pruritus,suggesting that ACC may be involved in the regulation of itch sensation information.Studies have shown that ACC and DMS(dorsomedial striatum)are activated during histamine-induced acute itch.ACC-DMS projections can promote histamine-induced acute itching.The ACC contains mainly glutamatergic neurons andγ-aminobutiric acid(GABA)ergic neurons.PV-and SSTexpressing interneurons account for a large majority of GABAergic interneurons in ACC.However,the specific role of different types of neurons in ACC in the occurrence and development of itch sensation is still unclear.In this study,ACC was used as the target brain regiona.Glutamate decarboxylase 2(GAD2)Cre,Parvalbumin(PV)Cre,Somatostatin,SST Cre and other transgenic mice were used.To study the role and mechanism of different types of neurons in itch transmission and regulation,acute itch model was established.It will provide experimental basis for further study on the changes of various neuron plasticity and local microcircuits,and finally provide a new intervention target for the development of pruritus prevention and treatment drugs.Part Ⅰ: The role of ACC in itch processingObjective: To observe whether ACC brain region was activated under acute itch stimulation and to clarify the role of ACC in the regulation of itch sensation.Methods: 1)Acute itch model was established by intradermal injection of histamine or chloroquine into the neck of mice,and itch behavior was detected by itch video tracking system.2)The distribution of c-Fos positive cells in ACC and the activation of different types of neurons in histamine or chloroquine itch models were detected by using immunofluorescence staining.3)To determine the overall contribution of ACC to itch processing,bilateral microinjection of quinolinic acid(QA)into bilateral ACC were performed.Main results: 1)Compared with the control group(saline),intradermal injection of histamine or chloroquine induced intense scratching behavior in mice.2)Compared with the control group(saline),the number of c-Fos positive cells in ACC increased significantly in histamine and chloroquine-induced itch models,in which glutamate neurons were mainly activated by itch stimuli.3)Bilateral ACC lesion significantly attenuated scratching evoked by intradermal injection of histamine and chloroquine,while vehicle injection did not produce any changes in both types of acute itch stimuli.Conclusion: ACC neurons were activated under acute itch conditions.Bilateral ACC lesion significantly attenuated scratching evoked by intradermal injection of histamine and chloroquine,suggesting the importance of ACC in the transmission or regulation of acute itch.So we further studied different types of neurons.Part Ⅱ: The role of excitatory and inhibitory neurons in ACC in the regulation of itch sensationObjective: To observe the activity changes of excitatory and inhibitory neurons in ACC under itch stimuli,and the specific role of excitatory and inhibitory neurons in the regulation of itch sensation.Methods: 1)After specific expression of GCaMP6 s in target neurons by stereotactic brain injection,the calcium activity in excitatory neurons and inhibitory neurons under itch stimulation were observed by in vivo fiber optic recording technique.2)The modulatory roles of excitatory and inhibitory neurons in scratching behavior evoked by both histaminergic and non-histaminergic itch were performed by pharmocogenetic and optogenetic manipulationsMain results: 1)Fiber photometry data showed that the calcium activity of ACC glutamatergic neurons was increased in both histamine and chloroquine itch models.Histamine stimuli resulted in robust increase in GABAergic neuronal activity.With nape injection of chloroquine,GABAergic neurons displayed biphasic responses,increases in the pre-scratching period and later decreases.2)Pharmacogenetic or optogenetic activation of ACC glutamatergic neurons greatly attenuated mouse scratching behavior induced by intradermal injection of histamine or chloroquin,while inhibition of ACC glutamatergic neurons did not influence scratching number induced by histamine or chloroquine stimulation.3)Chemogenetic or optogenetic activation of ACC GABAergic neurons significantly enhanced scratching related to chloroquine but not histamine,while inactivation of these neurons greatly attenuated both histamine and chloroquine evoked scratching behaviors.Conclusion: 1)The activation of ACC glutamatergic neurons was seen under both acute itch stimuli,while the activity of GABAergic neurons differed with time phase and itch modality.For chloroquine induced itch,a transient increase in Ca2+ signals was observed in the pre-scratching period,followed by sustaining decreased activity after scratching.2)ACC glutamatergic and GABAergic neurons exhibited opposite effects on itch processing.The activation of excitatory neurons,or the inactivation of GABAergic neurons,resulted in decreased acute itch-induced scratching.GABAergic neurons are divided into different subgroups,and we further studied different subgroups of neurons.Part Ⅲ: The functional role of different subsets of GABAergic neurons in ACC in itch processingObjective: To examine the ctivity of PV and SST interneurons in ACC under itch stimulationand the role of them in itch processing.Methods: 1)In vivo optical fiber recording was used to observe the calcium activity in PV and SST neurons under itch stimulation.2)The roles of PV and SST neurons in scratching behavior evoked by both histaminergic and non-histaminergic itch were performed by pharmocogenetic and optogenetic manipulations.Main results: 1)Fiber photometry results indicated that the calcium signal of PV neurons did not change in histamine-dependent itch model,but increased in SST neurons.Upon chloroquine stimuli,the calcium signal of PV and SST neurons changed significantly.2)Neither optical activation nor inactivation of PV-INs had significant effects on histaminedependent itch.In the context of chloroquine stimuli,optogenetic activation of PV neurons resulted in significant decreased scratching behavior,while deactivation induced a robust increase in scratching behavior.3)No change in histamine-evoked scratching was observed with the up-regulation or down-regulation of SST neuronal activities.CQ-evoked scratching was potentiated by optical activation of SST neurons,but suppressed by inactivation of them.Conclusions: Both PV and SST interneurons play a facilitatory role in histamineindependent itch processing.ACC PV neurons are deactivated in the post-scratching period only upon histamine-independent itch.ACC SST neurons are activated with differential temporal patterns in different itch modalities,which exhibited elevated neural activity across the scratching period with histamine stimuli,but only in the pre-scratching period with chloroquine stimuli.PV and SST neurons play different roles in the regulation of histamine-dependent and histamine-independent itch sensation.