Mechanism Research On Depressive-like Behaviors Induced By Changing DNA Methylation In Lateral Habenula Of Rats

Depression is a kind of mood disorder and its pathogenesis is complex.It is generally believed that deficiency or insufficient of neurotransmitters,such as serotonin and dopamine,in the central nervous system is an important mechanism for depression.Epigenetics research confirmed that emotional disorders has a close contact with epigenetic modifications.In addition,impacts of lateral habenula(LHb)on mood disorder including depression have attracted much attention of researchers,because the LHb is a key connecter between the forebrain and midbrain.Many studies have shown that hyper-excitation of neurons in the LHb could induce depression,while inhibition of the activity could produce an effect of anti-depression.So far,it is unknown whether DNA methylation is associated with the excitatory change in the LHb and which genes have involved.In the earlier study,we confirmed that there were expressions of DNA methyltransferse(DNMTs)in the LHb and the behaviors associated with emotion in rats could be altered when the DNA methylation level in the LHb was changed by DNMTs inhibitor,however,it is not detected that which genes were impacted and how they were influenced in promoter regions of these genes.In this study,we investigated the behavioral effect of microinjection of5-aza D,a DNMTs inhibitor,into the LHb,and also detected the expressions of gene related to excitation,inhibition and stress in the LHb.And methylation DNA immunoprecipitation(Me DIP)method was used to detect methylation level in promoter region of genes in the LHb to explore the possible mechanism on depressive-like behaviors when the level of DNA methylation in the LHb was changed.Our previous study confirmed that microinjection of DNMTs inhibitor 5-aza D into the LHb of rats significantly reduced the methylation level of genomic DNA in the LHb,which reduced the exploring behavior of rats in the open field test andincreased the immobility time during forced swimming test.In this study,we used the same injection method and examined anhedonia through sucrose preference test in rats.In the sucrose preference test,microinjection of 5-aza D into the LHb had a significantly lower percent for sugar water than vehicle group,indicating that decreased of DNA methylation in the LHb could induce anhedonia.All of the results suggest that decreased of genomic DNA methylation level in the LHb of rats can induce depressive-like behaviors.After microinjection of DNMTs inhibitor 5-aza D or vehicle into the LHb of rats,m RNA expression of excitation-associated genes(?Ca MKII,and Glu R1),inhibition-associated genes(GABAA?1,GAD67 and GAD65)and stress-responsed gene(GR)in the LHb were detected by real-time PCR technique.The results showed that m RNA expressions of ?Ca MKII and Glu R1 were significantly increased,m RNA expression of GR was markedly reduced and m RNA expressions of ?Ca MKII,GABAA?1,GAD67 and GAD65 were not different in the LHb of 5-aza D group,when compared with vehicle group.Our results suggested that decreasing the level of genomic DNA methylation in the LHb can induce depressive-like behaviors through up-regulation of ?Ca MKII and Glu R1 expression to enhance the excitability of neurons in the LHb.Next,we detected the methylation status in the promoter region of Exon I of?Ca MKII gene(named Cam K2b)and GR gene(named NR3C1)through Me DIP-q PCR method.The results showed that there was no significant change in methylation degree at the promoter region of Exon I of Cam K2 b and NR3C1 gene after 5-aza D injection,indicating that the effects of 5-aza D on m RNA expression is not by the way of changing methylation at the promoter region of Exon I of Cam K2 b and NR3C1 gene.Further studies at others promoter regions are necessary to identify the mechanism by which 5-aza D exert their effects.In summary,microinjection of DNMTs inhibitor 5-aza D into the LHb could induce depressive-like behavior characterized by anhedonia in sucrose preference test,and the drug into the LHb increased m RNA expressions of ?Ca MKII and Glu R1 anddecreased expression of GR m RNA in the LHb.Moreover,the methylation level at the promoter region of Exon I of Cam K2 b and NR3C1 gene was not altered.This study explored the relationship between DNA methylation in the LHb and depressive-like behaviors by the way of epigenetics to more understand the pathogenesis of mood disorder and provide possible therapy strategy.