Rhythmic Expression Of Per2 Gene In The Lateral Habenular Nucleus(LHb) And Its Effect And Mechanism On Depressive-like Behavior In Rat

Circadian pacemaker lie in the suprachiasmatic nucleus(SCN),which can receive the light signals from the retina,then regulate the melatonin(MT)secretion through retina—SCN—pineal gland(PG)tract,dominating the circadian rhythm activities.Habenular nucleus(Hb),which act as one of the SCN output pathway projecting targets,is considered to be the secondary regulating site of circadian rhythm,but also to accept projection fibers from the PG in round trips.Data indicates that there are MT receptors in the lateral habenular nucleus(LHb),so the LHb is double modulated by SCN and PG.In addition,the LHb,as an important point connecting limbic forebrain to brainstem,plays an important role in regulating many circadian activities and disease showing circadian rhythmicity,one of which is depression.The most powerful hypothesis in the pathogenesis of the disease is the decline of 5-Hydroxytryptamine(5-HT)content in the central and peripheral nervous system,and one of the main nucleus secreting 5-HT is the dorsal raphe nucleus(DRN).The LHb output fibers project to the DRN and regulate the 5-HT secretion in the DRN.Study on the rhythm of LHb can provide the theoretical support for the exploration of pathogenesis of depression and clinical treatment.In this study,we firstly revealed Period2(Per2)gene oscillation pattern in LHb and media habenular nucleus(MHb)both in LD(Light:dark =12 hours:12 hours)and DD(Dark:dark = 12 hours:12 hours)environment at molecular level,to investigate if the rhythm of Per2 gene expression in the Hb is consistent with the SCN or PG,trying to reveal the effect of SCN and PG to the Per2 gene rhythmic expression in the LHb.Then we selected 4 representative ZT(Zeitgeber time)time points(ZTO,ZT6,ZT12,ZT18)to detect the Per2 gene expression in LHb of normal rats and depression rats,to survey the Per2 gene expression difference between these two groups of rats at above time points.Combining with the wheel running behavior change,we speculated the effect of Per2 gene expression on neuronal activity in the Hb.After using the RNAi method to inhibit the Per2 gene expression in LHb,we determine the change of escaping time and immobility time between before and after administration in rats by forced swimming test(FST).After that,we detected the BMAL1 and PER2 protein expression in LHb of both RNAi rats and control rats by immunochemistry method,then the 5-HT content in DRN and Corpus striatum(CPU)of above two groups of rats was detected through HPLC(High performance liquid chromatography)method,to explore the possible mechanisms of Per2 gene expression in Hb affecting the depression incidence.The results showed that:1.In LD condition,Per2 gene expression in LHb showed diurnal rhythm,the peak appeared at ZT10 and a trough was on ZT22.Per2 gene expression in MHb in day was also higher than that at night.Under the DD environment,the Per2 gene expression in the LHb and MHb exhibits a periodic variation,with lower expression in subjective day and higher expression in subjective night,the peak appeared in the CT18,and the trough appeared in CT2(LHb).In MHb,the Per2 mRNA expression exibited a though at CT2,while the trough of PER2 expression appeared at CT6.2.24 hours average total wheel running counts of depression rats was lower than that of normal rats,and the ratio of daytime wheel running counts to 24 hours total wheel running counts was higher than that of normal rats,while the ratio of night wheel running counts to 24 hours total wheel running counts was lower than that of normal rats(P<0.05).Under the LD condition,Per2 gene expression in LHb of depression rat at ZT6 time point is lower than that of normal rats(P ＜ 0.05),while in ZTO,ZT12,ZT1 8 time points,the Per2 gene expression in LHb of depression rats was lower than that of normal rats,but the difference was not statistically significant(P>0.05).3.After Bmall siRNA was injected to the LHb of rats,in the FST,the escaping time is significantly shortened and the immobility time was significantly prolonged(P<0.05).The immunochemistry results showed that the BMAL1 and PER2 protein expression in LHb of RNAi rats was significantly lower than that of control rats(P<0.05).The 5-HT content in DRN and CPU of siRNA injected rats was significantly lower than that of control rats(P<0.05).Conclusion:1.Both in the LD and DD environment,the Per2 gene expression in LHb of the rat showed diurnal or circadian rhythms.Under LD environment,Per2 gene expression in LHb synchronized with the SCN,and in the DD environment,Per2 gene expression in LHb and MHb maintained synchronization with the PG.2.Per2 gene expression in LHb of depression rat at ZT6 time point was significantly lower than that in control group rats.3.Inhibiting the Per2 gene expression in LHb can promote effect on the incidence of depression behaviour,and the possible mechanisms may be associated with the decline of 5-HT content in DRN.