| Sleep is an evolutionarily conserved behavior that has been observed in both vertebrates and invertebrates.It accounts one third in one's lifetime.It is reported that sleep plays a very important role in memory formation,physiological regulation,growth and development during their whole life.Scientists have gradually discovered that the maintenance and switch of sleep and wakefulness are controlled by the neurotransmitters and signaling molecules.Although a lot of progress has been gained in the area of sleep research,however the underlying mechanism still remains to be investigated.At present,it is widely accepted that sleep is regulated by both circadian and homeostasis system.In mammals,the central center of the circadian system is the suprachiasmatic nucleus(SCN).After receiving external light signals,the SCN regulates CLOCK-Bmall,CRY-PER and other clock gene to keep its oscillation to be consistent with the external environment.It is also reported that creatures can not only adapted to the L:D(12h:12h)environment,but also can adapt to L:D(8h:16h)condition,which helps the animals live in high latitudes area.More interestingly,clock proteins can even adapt to conditions where the circadian cycle which is not 24h,such as L:D(11h:11h)environment.Moreover,the homeostasis system also plays an important role in sleep regulation.It is widely accepted that the ventral lateral preoptic area(VLPO)is the main center of the homeostatic system in the sleep-wake system.In VLPO removed rats,sleep duration was significantly reduced.Moreover,brain tissue staining also confirmed that the expression of c-fos in VLPO neurons was positively correlated with sleep status.Other studies have reported that VLPO could inhibit the arousal system,such as the locus coeruleus(LC),dorsal raphe nucleus(DRN)and tuberomammillary nucleus(TMN).In the meanwhile,VLPO and SCN are identified to have dense nerve projection,and the circadian system and homeostasis system works together to regulate sleep.NMDA receptor is identified as a glutamate receptor which is widely distributed in neurons.NMDA receptor is involved in virious biological processes such as neurodevelopment,synaptic plasticity,learning and memory formation,and is associated with a variety of neurogenic and psychiatric disease.Recently,more and more studies suggest that NMDAR are involved in the regulation of sleep.For example,it has been reported that the expression of NMDAR1 in the R2 brain region,the sleep-regulating center of Drosophila,is significantly up-regulated in the night compared with the daytime.The expression of NMDAR1 in R2 brain region is at higher level during sleep,and significantly decreased in the wake station.In addition,NMDAR is also identified to be related with psychiatric diseases.The NMDAR injury hypothesis is widely accepted in schizophrenia research.Further,sleep disorder is the earliest syndrome of schizophrenia,suggesting that NMDAR may play an important role in sleep regulation.Therefore,we selected NMDA receptor blocker Dezocilpine(MK-801)to build a NMDAR dysfunction model to investigate the mechanisms of sleep regulation.NMDA receptor has high permeability to calcium ions.Recent studies suggested that calcium is identified as the core ion participated in a variety of biological processes in the organisms.A lot of studies report that calcium ion signal participated in cardiac pacing,muscle contraction,immune response,thrombocyte coagulation,neurotransmitter release,and neuron plasticity mediation.In neuron,Ca2+binds calmodulin(CaM),and the activation of protein kinases helps the physiology activity in the neuron.The CaM kinase is a big family which contributes to learning,memory formation and synaptic potentiation.Among them,CaMKII is the most famous member,it is reported that CaMK?? and CaMK?? works together to finish E-T coupling by submitting information within CaMKVI channels.Further studies reported that CaMK? and CaMKK are expressed in nucleus.Neuronal activity drive CaMKK to phosphorylate and active CaMK? in nuclear,which phosphorylates CREB and CBP.CREB is cAMP response element binding protein.The mechanism by which CREB activation represses the certain genes expression is well understood.Other studies have shown that the expression of CaMKII in dorsal raphe nucleus(DRN)is closely related to the sleep-wake transition,and the mice showed obvious sleep difficulties in CaMKII down regulated models.CREB in the DRN brain has also been shown to regulate sleep through Orexin,and DR also plays an important role in sleep regulation.Recent studies have found that Ca2+ in cerebrospinal is also involved in the regulation of sleep and wake.As an important environment for the survival of nerve cells,the Ca2+ in CSF is involved in the regulation and switch in sleep and wakeness,when the Ca2+in CSF was in the low level,mice are in the awake station,while increasing Ca2+ level,the mice switched to sleep.In addition,adenosine is an important neurohumoral factor in sleep homeostasis regulation.However,whether Ca2+signaling is involved in sleep and circadian rhythm abnormal after NMDAR dysfunction still needs to be further elucidated.Melatonin is an endogenous hormone produced in the pineal gland.Its secretion has a significant circadian rhythm,with more secretion at night and almost no secretion in the daytime.Studies have found that melatonin can promote sleep.In clinical treatment,melatonin has been used as a sleep aid in patients in various types of sleep disorders.In people who suffering jet-lag after fly far distances,melatonin is also used to help them adapt to the new time zone,suggesting that melatonin also plays an important role in rhythm regulation.Studies found that melatonin could advance the sleep lantency phase and accelerate the wake-sleep switch process in mice.Recent investigation confirmed that melatonin can improve the oscillatory level of ROR,a circadian rhythm-related protein,to manipulate the biological circadian rhythms.Melatonin receptors are identified to have three different subtypes:MT1,MT2 and MT3,while MT1 and MT2 receptor are distributed in all brain regions.The distribution of melatonin receptors in the basal forebrain has been preliminarily confirmed.MT1 receptors are the dominant receptors in the suprachiasmatic nucleus(SCN)which is identified as the circadian center while MT2 receptor expressed only a little in the region.In the VLPO,MT2 receptor was in dominant.MT3 receptor is thought to be quinone only existed in plants and has not been found in animals.The above studies indicate that melatonin can regulate sleep from the two process:circadian system and homeostasis system.However,whether or not melatonin has a regulatory effect on sleep abnormalities caused by NMDA receptor dysfunction,and its underlying mechanism needs to be further illustrated.Based on the above background,we designed the following research.First,MK-801 was administered to build the NMDAR dysfunction model,and detect the circadian rhythm of mice through wheel running behavior in standard station and constant darkness condition,melatonin intervening group is also tested.We found that melatonin can rescue the period length prolonged by MK-801 in constant darkness mainly through homeostasis system instead of circadian system,and Ca2+-CaMKII-p-CREB pathway in VLPO is crucial in the process.Finally,EEG and MT2 receptor antagonist are used to further investigate the mechanism underlying the process.Our study provides a new perspective for understanding the mechanism of sleep and a theoretical basis for the clinical application of melatonin.Part ?:SCN does not involve in period length changed by MK-801 and melatonin in constant darknessFirst,voluntary wheel running experiment was designed to detect the circadian rhythm in mice.The data showed that in the light:darkness(12h:12h)environment,the circadian rhythm is 24h stably.Then the mice are placed in continuous darkness environment,to show their own circadian rhythm,the result turns out to be that the mice exhibited a nearly 23.6 hours period in constant darkness which is accordance with the precious study,showing that our running wheel detection is accurate and effective.Then,we examined whether the circadian system of mice was affected by MK-801 or melatonin.The animals are distributed into three groups and placed in L:D(12h:12h)condition for 7 days.The results showed all the groups exhibited a 24hour rhythm in standard environment.After that,we delayed or advanced the phase for 4hours to simulate the jet-lag syndrome.It is generally believed that the ability to regulate jet lag is mainly controlled by the circadian system:firstly,the light-dark time phase was shifted forward for 4h.The data showed that there was no difference in adapting to the phase advanced condition between the three groups.Then,the phase was delayed by 4h,and it was also found that the mice in the three groups spent 3-5d to adapt the new environment indicating that the circadian system of the mice was not significantly affected.To further confirm this conclusion,we extracted circadian center SCN,and detected the expression levels of clock protein PER1 and CLOCK at CTO-1 and CT12-13 periods,and detect the transcriptional levels of miR-219 at CT6-7.The results showed that the PER1 and CLOCK in MK-801 group were not significantly different from those in the control group at CTO-1 and CT12-13,and melatonin intervention also had no significant effect on the expression levels of the above molecules.Meanwhile,there was no statistical difference in the transcription level of miR-219 between the three groups.These results indicated that inhibition of NMDAR prolonged the period of in constant darkness,and melatonin almost restored the behavior,whereas the circadian system was not involved in the process.Part ?:Melatonin recovers the prolonged period length in constant darkness through VLPO in miceBased on the previous experiments,we focused on the homeostasis system,and designed the following experiments to test whether the VLPO was involved in this process.Sleep homeostasis is crucial for sleep and wake state maintaining and switch.To determine whether NMDA receptor is involved in sleep homeostasis regulation,we detect whether the expression of NMDA receptor is correlated with sleep drive.First,we extracted VLPO tissues of control group in different sleep drive conditon respectively,and detect the transcriptional level of NMDARI.The results showed that the expression of NMDAR1 in VLPO brain region was positively correlated with sleep drive.During CT12-13,mice are in the lowest sleep drive station,and the expression of NMDAR1 was also at a lower level.At CTO-1,mice had normal sleep demand and NMDAR1 transcriptional level also up-regulated.NMDAR1 expression in VLPO was further increased after 24h of sleep deprivation treatment.In the meanwhile,NMDAR1 transcriptional level in PFC brain regions showed no correlation with sleep drive.This indicates that the expression of NMDAR1 in VLPO brain region is closely related to sleep regulation.Next,Western blot were used to detect NMDAR1 level in VLPO and the data showed that NMDAR1 and CaMK? expression was also positively correlated with sleep drive.Then,we detected the underlying mechanism of MK-801 and melatonin in the process.Firstly,we detected the CaMK? and p-CREB in VLPO.The results showed that MK-801 could reduce the expression of CaMK? and p-CREB,while melatonin could effectively restore their expression.Finally,we measured the evoked potentials in the VLPO brain region using the brain slice clamp technique.The results showed that MK-801 significantly reduced the discharge frequency of evoked potential and the discharge amplitude.But melatonin can only restore the discharge frequency.The data showed that the NMDAR1 in VLPO was closely related to the sleep drive,and MK-801 could affect the function of VLPO by blocking the NMDAR1,thus affecting its sleep regulation,and becoming a potential cause of the prolonged period length in constant darkness conditions.In this part of the experiment,Ca2+indicator pAAV-Gcamp6-dtomato virus was first transfected into three groups of mice.After transfection for 21d,MK-801 and melatonin were administered to mice.Mice were sacrificed and brain tissue was frozen into sections,and the calcium level in VLPO was detected by confocal microscopy.The results showed that MK-801 could significantly reduce the calcium level in VLPO.Melatonin could significantly improve the Ca2+ level in VLPO decreased by MK-801.In order to further verify this conclusion,newborn mice(P7-P10)were were used to process calcium time course detection.The results showed that the fluorescence intensity of calcium ions in brain tablets decreased significantly in MK-801 group compared with the control group,while melatonin could also effectively improve the Ca2+level.Part ?:Melatonin recovers the sleep latency through MT2 receptor in VLPO detecting by EEG/EMGIn order to further investigate the underlying mechanism in the process.Labchart devices are used to detect the EEG and EMG in mice.The results showed that under the L:D(12h:12h)condition,the sleep latency in MK-801 treated group delayed nearly 4-7min compared to control group,while the melatonin intervention group significantly recovered the sleep latency delayed by MK-801.These results further confirmed that the homeostasis system was obviously affected by MK-801 and melatonin.It has been reported that MT2 receptor is mainly expressed in VLPO brain region which is mainly responsible for homeostatic regulation,while MT1 receptor is mainly expressed in SCN region.In addition,other literatures have confirmed that after MT2 knockout,the NREM sleep of mice is significantly reduced,while MT1 receptor mainly affects the duration of REM.Based on the above studiess,we hypothesized that MT2 receptor plays a more important role in the process.Therefore,we used MT2 receptor blocker 4P-PDOT to further testify our hypothesis.The results showed that 4P-PDOT prolonged the period length in constant darkness,apparently blocking the intervention effect of melatonin.In addition,the expressions of CaMK? and p-CREB in VLPO brain regions were also found to be down-regulated after 4P-PDOT administration.Moreover,CaMK? and p-CREB levels in mice treated with MK-801 and 4P-PDOT alone were also detected,and the results showed that the expression levels of the above two molecules were also significantly down-regulated.This further confirmed that MT2 receptor in VLPO played an important role in this process.Recent studies reported that melatonin can increase the adenosine level in the brain,and the accumulation of adenosine which can promote sleep.Therefore,we used the adenosine receptor agonist NECA to simulate the melatonin effect.The wheel running experiment confirmed that NECA can effectively simulate the effect of melatonin in wheel running detection.Furthermore,using primary cortical neuron culture,we also found that NECA inproved the intracellular Ca2+decreased by MK-801.Finally,EEG detection also showed that NECA improved the sleep latency delayed by MK-801.The above experiments proved that adenosine pathway can simulate the effect of melatonin,which can be considered as a potential mechanism in the process.ConclusionBased on the above,the main conclusions might be summarized as follows:1)MK-801 could prolong mice's period length in constant darkness,while melatonin could intervene the process,and the circaidian system did not participate in this process.2)Melatonin can intervene the effect of MK-801 through homeostasis system instead of circadian system,and Ca2+-CamK?-p-CREB pathway plays an important role in this process.3)EEG detection showed that MK-801 could delay the sleep latency that can be intervened by melatonin.MT2 receptor plays an important role in this process.Ca2+is identified to be the crucial element in the process.Our job serves a new sight in sleep regulation. |
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