A Preliminary Study Of Neurotransmitter Disorder In The Entorhinal Cortex Of Mice With Chronic Restraint Stress-induced Depression

BackgroundMajor depressive disorder(MDD)is a mental illness with sustained mood despondency and anhedonia as the main clinical manifestations.Its high morbidity and mortality seriously affect physical and mental health in human,and bring heavy economic burden to society.Although hundreds of millions of people suffer from MDD,the specific pathogenesis is not clear,and antidepressants are limited,which have brought huge difficulties to the prevention and treatment of depression.Hypotheses about the pathogenesis of MDD have been put forward,like decreased monoamine neurotransmitters,disorder of neurogenesis,disturbance of hypothalamicpituitary-adrenal and loss of brain-derived neurotrophic factor.Among them,the neurotransmitter dysfunction has been widely found in MDD patients,which not only contributes to the diagnosis of depression,but also provides important targets for the development of antidepressants.The entorhinal cortex(EC)is the main nerve afferent input of the hippocampus.Previous studies have confirmed that the neural circuit from EC to hippocampus plays an important role in regulating learning and memory.However,it is recently reported that glutamatergic neurons in the EC can affect neurogenesis in the dentate gyrus(DG)of hippocampus through nerve endings projections,regulating depressive-like behaviors in mice.However,it is still unclear whether the EC is affected by neurotransmitter disorders in a depressed state,and how altered neurotransmitter metabolites affect the function in EC.As an important brain region recently reported to be involved in the regulation of MDD,EC indicates a new direction for depression-related research.Therefore,exploring of the role of EC in MDD and its specific molecular mechanisms,and using this as a target for the development of antidepressants will greatly promote our understanding of MDD.Objectives1.Based on a stable mouse model of depression,quantitative detection was performed to clarify the neurotransmitter disorder in the EC of mice treated by chronic restraint stress(CRS),and to explore the connection among differential neurotransmitters-related metabolic pathway.2.Direct molecular evidence leading to neurotransmitter disorders in EC provides a basis for exploring drug targets in antidepressant therapy.Methods1.We adopted liquid chromatography-coupled tandem mass spectrometry(LC-MS/MS)method to detect neurotransmitter levels in the EC of mice exposed to CRS.Differential neurotransmitters were then screened.2.The pathways of differential neurotransmitters were analyzed by Kyoto gene and genome encyclopedia(KEGG).RT-qPCR and Western bolt methods were used to verify key enzymes and transporters in the pathways from the gene and protein levels.Results1.In this study,after 14 consecutive days of CRS on mice in the experimental group,we evaluated the depression-related behaviors of the mice by the sucrose preference test(SPT)and forced swimming test(FST).The results showed that compared with the control group,the mice in the experimental group had significantly different depressive-like phenotypes.2.Comparing neurotransmitter levels in both groups,we found significant upregulation of glutamate(Glu),ornithine(Orn),aspartic acid(Asp),5-hydroxytryptophan(5-HTP),L-tyrosine(L-Tyr)and norepinephrine(NE)in CRS group,accompanied with downregulation of homovanillic acid(HVA).3.In view of these differential neurotransmitters,we further screened key enzymes and transporters of corresponding molecular pathways by KEGG analysis,and verified them at the transcription and translation levels,respectively.We verified gene levels of glutamate decarboxylase 1(GAD1),glutamine synthetase(GLUL)and sodium-coupled neutral amino acid transporter 1(SNAT1)were increased with application of RT-qPCR.The upregulation of sodium-dependent serotonin transporter(SERT)and excitatory amino acid transporter 2(EAAT2)in protein levels were also validated with Western blot.ConclusionsOur results for the first time confirmed the alteration of neurotransmitter metabolites and related molecules in the EC of mice with CRS-induced depression,implying the underlying pathophysiologic mechanism of MDD and providing new ideas for antidepressant treatment.