Dissecting The Role And Neural Mechanism Of MPFC GHS-R1a Signaling In Regulating Stress-induced Anxiety Disorders

Previous studies have shown that growth hormone secretagogue receptor 1a(GHS-R1a),a seven-transmembrane G protein coupled receptor(GPCR),is abundantly expressed in the medial prefrontal cortex(m PFC),hypothalamus,ventral hippocampus,amygdala and other brain regions related to anxiety.In recent years,several studies have suggested that GHS-R1a signaling not only plays an important role in controlling food intake and energy metabolism,in regulating the cardiovascular system and motivation and reward,it also participates in regulating anxiety and depression.Previously,our lab reported that the expression of endogenous GHS-R1a in the m PFC was significantly up-regulated after repeated restraint stress(RRS)exposure,suggesting its involvement in stress-induced anxiety.However,the underlying mechanism remains uncertain.Objective:Dissecting the role and mechanism of GHS-R1a signaling in the m PFC in regulating anxiety and stress-induced anxiety disorders.Methods:Mice were exposed to RRS stress,and their anxiety-related behaviors were evaluated in elevated plus maze(EPM)and tail suspension test(TST).Immunofluorescent staining was used to check co-localization of the Fos protein and theαCa MKII protein in m PFC in order to identify neurons activated by stress exposure.The activity ofαCa MKII~+neurons in the m PFC region,with and without viral-mediated Ghsr1a overexpression,was chemogenetically manipulated to explore the role of this population of neurons in regulating anxiety-related behaviors both at basal state and after stress exposure.Optogenetic stimulation was also applied to activate theαCa MKII~+neurons in the m PFC of behavioral mice to confirm the effect of activatingαCa MKII~+neurons in the m PFC on anxiety-related behaviors.Results:1.αCa MKII~+neurons in the m PFC were activated after repeated restraint stress,suggesting involvement ofαCa MKII~+neurons in regulating anxiety-related behaviors.2.Chemogenetic activation of αCa MKII~+neurons in the m PFC alleviates anxiety-related behaviors at the baseline state and anxiety disorder induced by RRS exposure.The TST immobility time of h M3Dq virus-transfected mice was less after intraperitoneal injection of CNO than after NS injection.Meanwhile after RRS,the EPM open arm duration of h M3Dq virustransfected mice were longer after intraperitoneal injection of CNO than after NS injection.3.Optogenetic activation of αCa MKII~+neurons in the m PFC inhibits anxiety-related behaviors at the baseline state.The EPM open-arm duration of the Ch R2 virus-transfected mice was significantly increased with the blue light on compared to that with the light off.4.Optogenetic activation of αCa MKII~+neurons in the m PFC could not alleviate anxiety disorder induced by RRS exposure.After RRS,the EPM open-arm exploration time of Ch R2 virus-transfected mice was not altered with the blue light on compared to that with the blue light off.5.Optogenetic activation ofαCa MKII~+neurons in the m PFC had no effect on real time place preference.Specifically,mice spent similar time exploring the inside(stimulated side)arena as the outside(non-stimulated side)arena.6.Chemogenetic inhibition ofαCa MKII~+neurons in the m PFC had no effect on anxiety-related behaviors at the baseline state and after RRS exposure.In the EPM and TST tests,basal anxiety-related behaviors of h M4Di virustransfected mice was not altered after CNO injection compared to NS injection.7.Chemogenetic activation ofαCa MKII~+neurons with Ghsr1a overexpression in the m PFC could not alleviate anxiety-related behaviors at baseline state and after RRS exposure.In both EPM and TST tests,anxiety-related behaviors of h M3Dq-Ghsr1a virus-transfected mice were not altered after CNO injection compared to NS injection.Conclusion:Our study demonstrate thatαCa MKII~+neurons in the m PFC are involved in regulating anxiety-related behaviors both at baseline state and after RRS exposure.ActivatingαCa MKII~+neurons in the m PFC reduces anxiety,and this effect is abolished by elevated GHS-R1a expression inαCa MKII~+neurons.Our findings thus suggest that stress-induced up-regulation of endogenous GHS-R1a in the m PFC contributes to stress-induced anxiety disorders by antagonizing the activity ofαCa MKII~+neurons.However,further studies are still required to reveal the underlying mechanism.