Studies On Dec Gene-involved Regulation Of Murine Sleep-Wake Cycle Mediated By Modafinil And Explorations Of Related Mechanisms

The circadian rhythm is one of most salient features in the mammalian sleep.Circadian rhythm refers to the behavioral and physiological oscillations of nearly24hoursgenerated by the internal biological clock (pacemaker or oscillator). In fact, most of themammalian behavioral and physiological rhythms are under the dual regulations bycircadian rhythm and sleep-wake. The performance of the individual on a particularmoment is the result of integration in the biological clock and sleep-wake. Clock genesusually express in many brain regions, which suggests that altered expression of clockgenes (independent of the SCN cycle) may be the basis of sleep regulation.As the fifth known clock gene following clock, period, bmal and cry, Dec has been amajor research focus in the biological clock regulatory mechanism among many species.Dec2plays a key role in the regulation of mammalian sleep length. When the gene ismutated, the sleep time will be shortened. However, whether these physiological andbehavioral abnormalities are owing to changes in the SCN or circadian rhythm signalsremains unclear. Different circadian rhythm phenotypes are attributed to different circadianrhythm gene, or the expression of different alleles of specific circadian clock genes. Andthe case may be universal rather than a special case.Modafinil is of a higher safety and few side effects compared with other centralstimulant, which has been used as a first-line drug for treatment of narcolepsy. Studies onits function mechanisms are generally focused in the epinephrine, dopamine, histamine andorexin neurons and their interactions. However, the function mechanisms of modafinil arewidely controversial due to its unknown targets and unclear roles in the neuroanatomicalsites.Therefore, studies on the spatial and temporal distribution of circadian clock geneexpression, particularly the relationship between outside-SCN areas and the sleep-wakecycle, and roles of drug intervention, are of great significance.Our project aims to explore the functions of circadian clock gene Dec inmodafinil-mediated sleep-wake and related mechanisms. This study mainly consists offour aspects with corresponding results as follows.Firstly, we have determined the expression of Dec in different murine brain regionson the normal circadian rhythm, and both changes of circadian rhythm and Dec expressionpost modafinil usage. In this study, immunohistochemistry, Western blot and real-timequantitative PCR techniques were adopted, with hypothalamus, striatum, hippocampus and piriform cortex as the target research areas. As results shown, in the four brain regions ofthe control group, expressions of Dec changed with a certain rhythm either at the level ofprotein or that of mRNA. In the experimental groups intervented by modafinil, all theclock genes Dec1Dec2and Ck1exhibited significant dose-dependent changes in theirexpression levels especially at certain time points. Therefore, Dec was thought toparticipate in the modafinil-mediated sleep regulation.Secondly, a rhythm disturbance model of the acute sleep deprivation for6hours(CT8-CT14) has been made to further investigate whether Dec still involves in themodafinil-mediated sleep regulation. As immunoblotting results shown, Dec expressionchanges in different brain regions post destruction of the circadian rhythm cycle, whichpossibly relate to both grades of the circadian system and the synchronization of theoscillation within the system. Dec expressions also showed different changes post theusage of modafinil, which further supported the participation of Dec in themodafinil-mediated sleep regulation.Thirdly, a rhythm disturbance model was made through repeated sleep deprivationand used to explore changes of clock gene expressions, functions of Dec2in circadianclock resetting and interventionist role of modafinil. Western blot analysis showed that thehippocampus, cortex and cerebellum are three brain regions with high-abundanceexpression of Dec2post sleep deprivation and modafinil medication, compared with thecontrol group with various degrees of change. Thus, Dec2involves in the circadian clockresetting process, and its expression in the hippocampus, changes suggestive of neuralplasticity, may be involved in spatial memory formation, consolidation and subsidedprocess. Morris water maze behavioral experiments have further verified the above results.The studies also suggested that repeated sleep deprivation may damage learning andmemory in cognitive ability, which can not resume from sleep by the compensation.Reversal platform showed that the re-learning ability of mice decreased post sleepdeprivation, and that the use of modafinil can improve the memory impairment. However,in a reversal platform experiment during the sleep recovery period, it did not differsignificantly between the treatment group and the tank control group. Interestingly, afterthe second sleep deprivation (ie, repeated sleep deprivation), there is an obvious relatedadvantage in the modafinil-treated group, which was also supported by space explorationexperiments. Therefore, the cognition improvement due to modafinil depends on theestablishment of the stressor. The improvement is not significant when without stressors or during the sleep recovery period. And, performances improved significantly inmodafinil-treated groups post repeated sleep deprivation. Both the spatial learning abilityand water maze performance are dependent on the interactions among different brainregions, together of which form a complete neural network. Therefore, the roles ofmodafinil may be related to stressors, building processes of memory and interactionfeedback from different brain areas.Finally, an in vitro cell model was established to further explore regulatorymechanisms of modafinil on the Dec expression. As results shown, at different time pointsafter modafinil stimulation, Dec2expression exhibited some nucleo-plasm transferringchanges. This finding has been confirmed by the laser confocal experiment and Westernblot analysis, which still further supported the involvement of Dec gene in themodafinil-mediated sleep regulation.In a word, Dec2as a transcription factor, is of unclear in its regulation mechanisms atboth translation and transcription levels. A deep exploration of the nucleation mechanismand adjustment mechanism of Dec gene is helpful for the in-depth understanding of sleepdisorders and circadian rhythm disorders at gene level, and even provides potential newtargets for related prevention and treatment.