Effects And Possible Mechanisms Of The Habenular Nucleus On The Circadian Rhythm Of Locomotor Activity In Rats

Circadian rhythm exists in life course of all organisms. It is the basic feature in the physiological and behavioral processes. It has been confirmed that hypothalamic suprachiasmatic nucleus (SCN), as the master circadian pacemaker, can output the signals and control the most of the physiological and behavioral rhythms. SCN has the ability to produce a near-24 h rhythm. It has been proved that SCN cells are able to display a circadian rhythm in neural activity, with the highest firing rates observed in the mid-subjective day. The habenular nucleus is located in the dorsal thalamus. It has been found that the lateral habenular nucleus cells show higher spontaneous firing in the subjected day vs. the night in vitro. This contrasts with electrically rhythmic extra-SCN brain regions, most of which display the highest firing rates in the subjective night.Retina-SCN-pineal pathway is an important and clear pathway in the circadian system. SCN can also accept and integrate light information by the retina-SCN-pineal pathway, so that endogenous rhythm can synchronizate with the circadian rhythm of the Earth` rotation. SCN and pineal gland, two important structures in regulation of the circadian rhythm, interact and affect each other. Not only may SCN directly affect the activities of the pineal gland further to control its synthesis and release of melatonin (Mel), but also Mel can affect SCN function and be involved in regulation of circadian rhythm. Except for the close connection with the habenular nucleus and SCN, the habenular nucleus and the pineal gland, another important structure in the circadian rhythm system, also show a special and unique relationship anatomically. It has also been called"peduncle of the pineal gland". It has been proved to have close relationships between the habenular nucleus and the pineal gland histologically, functionally and molecular biologically. The habenular nucleus can project to the pineal gland and also receive projections from the pineal gland. Electrical stimulation of the habenular nucleus can excite a lot of pineal gland cells. It has been found that there is the expression of Mel receptors in the habenular nucleus. These evidences directly or indirectly suggest that the habenular nucleus maybe plays an important role in the regulation of circadian system. It deserves to be investigated from multi-point of view. Thus, the present study was focused on the habenular nucleus and tried to reveal the effects and possible mechanisms of the habenular nucleus on the regulation of circadian rhythm of locomotor activity from the view of behavior, neurochemistry, histology and electrophysiology.In the present experiments, the running-wheel recording was firstly applied and effects of habenular lesion on the circadian rhythm of locomotor activity of rats were observed in constant dark (DD) and light dark cycle (LD) conditions. Subsequently, dopamine (DA) and serotonin (5-HT) concentrations in the striatum of rats at different time points zeitgeber time 4(ZT4)/circadian time (CT4) and ZT16/CT16 in different conditions were detected by using high-performance liquid chromatography (HPLC) after habenular lesion to further reveal the mechanisms of the effects of habenular lesion on locomotor activity in rats. At the same time, the changes of Mel levels in the pineal gland at different time points (ZT6/CT6 and ZT18/CT18) under different conditions were detected by the application of HPLC after habenular lesion. Furthermore, the ultrastructure of the pineal gland under LD conditions was also observed by transmission electron microscopy. At the same time, the response of habenular nucleus on different doses of Mel was also observed in vitro by using electrophysiological techniques, so that further to approach the effect and mechanism of the habenular nucleus in the regulatory process of circadian rhythm of locomotor activity. The results were shown as the following:1. In DD conditions, habenular lesions did not change the free-running period, however, the distribution of running-wheel activities were significantly changed during the free-running period. The specific performance was that the radio of activities at subjective night was decreased markedly and it was increased significantly at subjective day. In LD conditions, habenular lesion speed up the reentrainment to delayed 8 h light, but did not affect entainment to the original LD conditions and reentrainment to advanced 8 h light.2. In DD conditions, habenular lesion caused DA content in rat striatum increase significantly at CT16 and decrease at CT4. In LD conditions, habenular lesion after 3 weeks induced DA content increase in the striatum both at ZT16 and at ZT4. But habenular lesion after 8 weeks made striatal DA content significantly reduced at ZT4 and no change at ZT16. At the same time, the phenomenon that Striatal DA contents were high in day and low at night disappears.3. Both in LD and DD conditions, it disappeared after habenular lesion that the difference of 5-HT levels in rat striatum between at ZT4/CT4 and at ZT16/CT16. In DD condition and LD conditions, 5-HT content in striatum were significantly increased in CT16/ZT16 at 3 weeks after habenular lesion; but in LD conditions, the striatum 5-HT content in the ZT4 point decreased at 8 weeks after habenula lesion.4. In LD conditions, Mel contents in pineal gland were significantly decrease either at 3 weeks or at 8 weeks after habenular lesion; in DD conditions, contents of Mel in the pineal gland had no significant change induced by habenular lesion. However, under any condition detected, habenular lesion did not change the phenomenon that Mel content in the pineal gland appeared low in the day and high at night.5. In LD conditions, body weights of the pineal gland were significantly reduced at the ZT18 after 3 weeks habenular lesion. By using the transmission electron microscope, the changes of the pinealocytes were observed after habenular lesion. The pinealocytes became smaller and denser. Cytoplasmic organelles decreased and were obviously changed, such as the Golgi apparatus shinked, mitochondrial swelling, crest fracture, shrinkage, etc. In addition, lysosomes were significantly increased and large debris was formed, including multivesicular body and myeloid body. Clear vesicles and synaptic ribbon almost disappeared in the pinealocytes after habenular lesion.6. The electrophysiological experiments showed that different doses of Mel caused markedly different responses on habenular neurons in brain slices. High concentrations of Mel (such as 10-4 - 10-6 mol/L) caused obviously inhibitory reactions on habenular neurons, while 10-7 mol/L Mel induced firstly obviously excitatory and then inhibitory response on the habenular cells. There was no significant regional difference in the response of MHb and LHb on infusion 10-7 mol/L Mel.From the all above results, the following conclusion can be safely drawn out:1. In DD conditions, habenular lesion had no effect on free-running period; however, it caused significantly changes in the distribution of running wheel activities during the free-running period, suggesting that the habenular nucleus may reduce the intensity (amplitude) of free-running rhythm. In LD conditions, habenular lesion speed up the reentrainment to delayed 8 h light. Thus, these results indicate that the habenular nucleus maybe play an important role in the regulatory process of locomotor activity circadian rhythm.2. Habenular lesion induced the changes of DA and 5-HT concentrations in the striatum during the DD and LD conditions. These showed that DA and 5-HT system may be involved in the mechanisms of effects of habenular nucleus on circadian rhythms of locomotor activity.3. The habenular nucleus may affect the function of the pineal gland and further control the synthesis and secretion of Mel in the pineal gland. At the same time, the habenular nucleus can be as a target for Mel in the brain. Thus, the habenular nucleus can take part in the regulation of circadian system through the interaction with the pineal gland.In summary, the study is the first time to investigate the role of the habenular nucleus in the circadian rhythm system from the level of behavioral study. It clearly explains the effects of habenular lesion on the circadian rhythm of running-wheel activity under DD and LD conditions, further confirmed that these effects may be closely related to the DA and 5-HT system. These results provide important behavioral evidence to reveal the biological significance of circadian rhythm of habenular neural activity. They further demonstrate that there is a relationship between the habenular nucleus and the two monoaminergic systems (DA and 5-HT system) in the brain from the view of circadian regulation. In addition, the study has also identified the involvement of habenular nucleus in the retina-SCN-pineal neural pathway of circadian rhythm. It is confirmed not only that the habenular nucleus can affect the synthesis and secretion of Mel throughout the regulation of the pineal gland, but also that the habenular nucleus is a target on Mel in the brain. The habenular nucleus can carry out its role on the regulation of circadian system through the interaction with the pineal gland.