TNF Signaling Pathway-Mediated Microglial Activation In The PFC Underlies Acute Paradoxical Sleep Deprivation-Induced Anxiety-Like Behaviors In Mice

BackgroundSleep is an important physiological process,which helps to maintain homeostasis balance in mammals,and accounts for approximately one third of the human life time.Sleep plays an essential physiological role in the preservation,utilization and redistribution of energy,and maintaining cellular homeostasis,anabolism,immune function and normal neuroplasticity.Sleep deprivation consists of a variety of sleep disorders,which regulate metabolism and hormone secretion,seriously deteriorate life quality,produce psychological disorders,disease occurrence,cognitive dysfunction and so on.Thus,good sleep and adequate sleep time are of great importance to maintaining human health.However,with the pace of life and work stress in modern life accelerating,sleep deprivation is becoming a serious social health problem.Sleep deprivation disrupts the normal sleep-wake rhythms and causes changes in mood,cognition,and memory.Among them,anxiety disorder is one of the most common symptoms of sleep deprivation.The prefrontal cortex(PFC)area has extensive neural projection to the subcortical structures and the other cerebral cortex,and it is the center of emotion control and related to many advanced cognitive functions.The activity of PFC neuron is significantly decreased in anxiety disorders and individuals with increased anxiety.Therefore,the PFC is considered as an important brain region for sleep deprivation-induced anxiety-like behaviors.Microglia are the main neuroimmune cells in the central nervous system(CNS),of which the morphology shows high plasticity.Microglia is extremely sensitive to microenvironment change and can be activated by the inflammatory responses during pathogens invading.It can also efficiently clear apoptotic cells and neuronal debris.The production of inflammatory cytokines in brain tissue and peripheral blood during sleep deprivation could be related to the activation of microglia.To explore the underlying mechanisms between acute paradoxical sleep deprivation(PSD)-induced anxiety-like behaviors and microglia activation,PSD mice model were established in the current study.We found that 6 h acute PSD induced typical anxiety-like behaviors in mice.The bioinformatic analysis of gene expression omnibus(GEO)database showed that inflammation-related signaling pathways were involved in the regulation of sleep deprivation.The tumor necrosis factor(TNF)signaling pathway is a key pathway.Expression of core genes that enrich in this pathway were detected in the microglia cells of PFC by Western Blot,Quantitative Real-time PCR(q PCR),and immunofluorescence histochemistry.Microglia activation and expression of the core genes in PFC during PSD were inhibited by minocycline treatment,which clarified that TNF signaling pathway-mediated microglial activation participated in acute PSD-induced anxiety-like behaviors.This study revealed the biological mechanism of anxiety-like behaviors induced by acute PSD,and clarified the relationship among sleep deprivation,microglial activation,and inflammatory response,which provided new ideas and theoretical base for the clinical treatment of sleep disorders.Methods1)Establishment of PSD model:The PSD was established by using the modified multi-platform water environment method(MMPM).This method can deprive more than95%of paradoxical sleep(also named rapid eye movement sleep,REM),and about 40%of non-rapid eye movement sleep(NREM).2)Behavior tests of PSD mice:Open field test(OFT),light-dark box test,marble burying test,and elevated plus maze(EPM)were used to examine the anxiety-like behaviors of mice.The three-chamber test was used to examine the sociability of mice,and grooming behavior was used to examine stereotyped behavior in mice.3)Bioinformatic analysis:Gene expression data from GEO database were analyzed.Differential expression genes(DEGs)were identified through over-expression analysis(ORA).Gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment were performed.Protein-protein interaction(PPI)network was constructed.Then key genes were verified by Western blot and q PCR experiments.4)Activation of microglia:The cell morphology of the microglia was staining by Ionized calcium binding adaptor molecule 1(IBA1),and was reconstructed by three-dimensional software.The activation status of microglia were evaluated by Sholl analysis.The expression of key genes in microglia were detected by immunofluorescence histochemistry.5)Drug treatment:Mice were treated with minocycline by intraperitoneal injection,the activation of microglia and the expression of key genes were detected after acute PSD.And anxiety-like behaviors were re-evaluated after minocycline treatment.Results1)After 6 h acute PSD or 72 h chronic PSD,a battery of behavior tests were employed for evaluating the anxiety level of the PSD mice.We found that the time that PSD mice spent in the central area of OFT,the time spent in the open arms of EPM,the number of the central area and open arms entries,and the time in the bright box were significantly reduced compared with the control group.The number of marbles buried was also increased significantly in the PSD group.These results indicated that PSD caused typical anxiety-like behaviors.2)Three gene chips GSE77393,GSE6514 and GSE78215 from GEO database were analyzed by bioinformatic analysis,TNF signaling pathway and key genes Casp3、Ptgs2、Socs3、Fos,and Nfkbia were detected to be related to acute sleep deprivation.The expression level of Casp3、Ptgs2,and Socs3,which enriched in the TNF signaling pathway,were altered in PFC after acute PSD.3)After acute PSD,microglial cells in the PFC were activated with decreased branch structure,and the enlarged cell body area.The expression of PTGS2 in microglia was significantly up-regulated,and the expression of CASP3 was down-regulated.4)After being treated with minocycline,a tetracycline with anti-inflammatory properties,microglia activation was effectively inhibited with increased intersections and reduced cell body area.The number of PTGS2~+cells in the PFC was significantly reduced,and the anxiogenic effect was ameliorated.ConclusionIn the current study,we found that 6 h acute PSD was enough to induce typical anxiety-like behaviors in mice.Inflammatory-related signaling pathways,especially TNF signaling pathway,were involved in the process of sleep deprivation.Microglia of PFC were highly responsive during anxiety-like behaviors.Minocycline treatment could effectively inhibit the activation of microglia and ameliorate anxiety-like symptoms.This study revealed the relationship between acute sleep deprivation and the inflammatory responses of microglial activation,which provided a new experience and theoretical knowledge for the clinical treatment of sleep disorders.