| Circadian rhythm is the rhythm with 24h periodic variations which is a basic vital phenomena. Disruption of circadian rhythm will lead to functional disorder even disease. The suprachiasmatic nucleus of the hypothalamus (SCN) is the master circadian pacemaker or clock in the mammalian brain. A key feature of a circadian pacemaker is its ability to entrain to changing environmental cues. The principal entraining stimulus in mammals is the LD cycle, with photic information being conveyed to the SCN via the direct pathway-glutamatergic retinohypothalamic tract (RHT) or other indirect pathways to coordinate the whole circadian system and control the most activities of the bodies such as physiological, metabolic and behavioural. Habenular nucleus(Hb)which locates epithalamus is functionally interposed between limbic and striatal systems through the involvement of the midbrain structures. Hb is the cross point of cardiovascular activity, sleep-wake cycle, production, body temperature and behabior which all show daily rhythmicity. Our previous study showed that the discharge of habenula neurons had obvious circadian rhythm. Cells in the Hb had higher baseline firing rates during the day than during the night in vivo and vitro, while photic responses were of significantly larger amplitude among LHb cells duringthe projected night than during the projected day. Mahboubeh found the Number of c-FOS were higher during the subjective night-time than that during the subjectve day-time in Hamsters LHbM in constant darkness (DD). So the habenula may be one important component in linking circadian and motivational systems and may contribute to the regulation of these systems, as well as to the rhythmicity of their function. Yet it is not reported about the molecular mechanism of daily rhythmicity in the Hb till now. So many experiments found that the subtypes of Period(Per) family: Period1(Per1) and Period2(Per2) play the fundamental effects in creating circadian rhythm. Particularly Per2 plalys more important roles. Methods:Hb was removed on 13: 00 and 1: 00 and LHb and MHb were separated on 13: 00, 17: 00, 21: 00, 1: 00, 5: 00 and 9: 00 from normal rats and SCN lesioned rats. Immunohistochemical staining was used to detect the expression of FOS protein and Per2 and c-fos mRNA expression was determined with RT-PCR method on the time point as shown above.Results:1. The expression of Per2 and c-fos mRNA and FOS protein in the Hb:1)The expression of Per2 and c-fos mRNA and FOS protein in the Hb showed the rhyhmicity with higher expression during the projective day than night in LD cycle(P<0.05). And this rhythmicity will disappear when SCN was lesioned(P>0.05)。2)The expression of Per2 and c-fos mRNA and FOS protein in the Hb showed the rhyhmicity with higher expression during the subjective night than the subjective day in DD cycle when the photic stimulus from the environment was abolished(P<0.05), which was antipodal to the change in LD cycle. When SCN was lesioned, this rhythmicity could be maintained in the Hb(P<0.05)。2. The expression of Per2 and c-fos mRNA and FOS protein in the LHb and MHb:1)The expression of Per2 and c-fos mRNA and FOS protein in the LHb showed the rhyhmicity with higher expression during the projective day than night in LD cycle as in the Hb:The expression of Per2 mRNA was higher on 9: 00 and 13: 00 than that on 1: 00(P<0.05); The expression of c-fos mRNA and FOS protein was higher on 13: 00 and 17: 00 than that on 1: 00(P<0.05); The expression of Per2 and c-fos mRNA and FOS protein in the MHb had no rhythmicity(P>0.05). The rhythmic expression of Per2 and c-fos mRNA and FOS protein lost after SCN was lesioned(P>0.05).2)The expression of Per2 and c-fos mRNA and FOS protein in the LHb and MHb showed the rhyhmicity with higher expression during the subjective night than day in DD cycle when the photic stimulus from the environment was abolished as in the Hb: The expression of Per2 mRNA was lower on 9: 00 and 13: 00 than that on 1: 00(P<0.05)in the LHb(P<0.05) and the expression was also lower on 13: 00 than that on 21: 00(P<0.05); The expression of c-fos mRNA and FOS protein was higher on 1: 00 than that on any time point during the whole 24h in the LHb(P<0.05); The expression of Per2 was higher on 1: 00 than that on any time point during the whole 24h in the MHb(P<0.05); and c-fos mRNA and FOS protein in the MHb had no rhythmicity(P<0.05); The expression of c-fos mRNA and FOS protein was higher on 1: 00 than that on 13: 00 and 17: 00 in the MHb(P<0.05).The expression of Per2 and c-fos mRNA and FOS protein in the LHb could be maintained the rhyhmicity with higher expression during the subjective night than day in DD cycle after SCN was lesioned: The expression was higher om 21: 00 and 1: 00 than that on 1: 00(P<0.05). The rhythmic expression of Per2 and c-fos mRNA and FOS protein lost after SCN was lesioned in the MHb(P>0.05).Discussion:The expression of Per2 and c-fos mRNA and FOS protein in the LHb and Hb showed the rhyhmicity with higher expression during the projective day than night, while there was no obvious rhythnicity in the MHb on different tine point in LD cycle. This rhythmic oscillation was coincident with the results from Prof. Zhao Hua who showed that cells in the LHb showed higher baseline firing rates during the day than during the night and cells in the MHb showed no rhythmic oscillation in vitro slice preparation. And in Zhao's experiment of cells recorded in the LHb, 42% were tonically activated or suppressed by retinal illumination, while significantly fewer cells recorded in the MHb responded to retinal illumination(19%). It was reported that photosignal from the environment could project to the Hb directly and indirectly which made Hb receive the effect of photosignal. The rhythmic expression of Per2 and c-fos mRNA and FOS protein lost after SCN was lesioned in the LHb and Hb and there was still no rhythmicity in the MHb. Therefore, we can conclude that SCN is very significant to the Hb to maintain its rhythmicity in LD cycle; And because the rhythmic expression of Per2 and c-fos mRNA and FOS protein oscillate in the LHb coincident to the Hb, the rhythmicity of the LHb master that of the whole Hb.The expression of Per2 and c-fos mRNA and FOS protein showed the rhyhmicity with lower expression during the subjective day than the subjective night no matter in the LHb, MHb or in the Hb on different time point in DD cycle. Of all our results, there was no significance between 17: 00 and 21: 00(2 h before lights off and after lights off)about the expression of FOS protein which was identical with the story from Mahboubeh. Although Mahboubeh also found that the FOS protein expressed low on the subjective day and high on the subjective night in LHbM of hamster in DD cycle, the mechanism is excluded. The expression of Per2 and c-fos mRNA and FOS protein in the LHb could be maintained the rhyhmicity with higher expression during the subjective night than the subjective day in DD cycle after SCN was lesioned. Although the expressive difference abolished among some time points, the difference between 13: 00 and 1: 00 still existed in the LHb; The rhythmic expression of Per2 and c-fos mRNA and FOS protein lost in the MHb after SCN was lesioned. Thus, we can conclude that SCN is not indispensable to the Hb in DD; And the results futher support the phenomena: the rhythmicity of the LHb master that of the whole Hb.Conclusion:On the whole, there is a rhythmic oscillation in the LHb, MHb and Hb. Hb must rely on SCN in LD cycle, and SCN is very important. Although the expressive difference abolished among some time points in DD cycle, the expression of Per2 and c-fos mRNA and FOS protein still maintain the rhyhmicity with lower expression during day than the subjective night in the LHb, MHb or in the Hb. Hb can oscillates independent of SCN in DD cycle, but it is not a fully automic oscillation. Hb may participate the regulation of circadian rhythm system on the body and may play a different role from SCN in this system. |
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