The Effect Of Chronic Sleep Deprivation On Dopamine D₁Receptor Signaling Pathway In Rats’ Prefrontal Cortex

Sleep deprivation （SD） is a common phenomenon in society, which can seriouslyaffect the behavior, study and work. Compared to the short-term acute sleep deprivation（ASD）, long-termed chronic sleep deprivation （CSD） has more severe effect on workefficiencies and higher neural function, including learning and memory, cognition, anddecision making. Previous studies demonstrated that the prefrontal cortex （PFC） plays avery important role in the neuroethological changes induced by CSD. In our previousworks, it was found that CSD can lead to the reduction of locomotor, learning and memoryand the damage of PFC deep neuronal ultrastructure, CSD can also decrease the content ofdopamine （DA） and the expression of D₁receptor （D₁R）, while SKF38393, which is a D₁Ragonist, improved this phenomenon. Those studis showed that DA and D₁R in PFC maymodulate the process which CSD caused the reduction of learning and memory capability.As a G-protein-couple receptor, D₁R can regulate the expression of cAMP responseelement binding protein （CREB）, which is a nuclear transcription factor, through severalpathways such as the AC-cAMP-PKA pathway, phosphoinositol pathway and mitogenactivated protein kinase （MAPK） pathway, thereby modulate the functions of nerve cells.However, during the process of CSD-caused dysfunction in PFC and the improvement ofdamage by activation of D₁R, which pathway and key molecule paly a main role in theregulation of D₁R? Whether there are other ways make efforts to the damage of PFCinduced by CSD? All this needs further research. Therefore, we used the microarray todetect the all levels of gene transcription in rats PFC, to find the new pathway and keymolecule in dysfunction of PFC during CSD. And then through the real time PCR andwestern blot, the genetic and proteidic expression of key molecule in D₁R signalingpathway were verified and measured, to find the main pathway and key molecule in D₁Rsignaling pathway in PFC during CSD.CHAPTER ONE Effect of chronic sleep deprivation on widegenetic expression in prefrontal cortexObjective: To observe the effect of CSD on the level of genetic transcription inPFC, and search new functional approaches and modulator during the structural andfunctional impairment of PFC induced by CSD.Materials and methods:45of60healthy male Sprague Dawle rats was screened out by weight, Morris water maze and platform training, and then randomlydivided into three groups: treatment control （TC） group, chronic sleep deprivation （CSD）group and agonist SKF38393adminstrated （SKF） group,15samples in each group. TCrats were treated in the same water environment by the large platform control box to ensuretheir normal sleep, while the other two groups were deprived rapid eye movement sleep（REM） through the modified multi-platform method （MMPM） for18h per day （16:00-10:00）, this process continued21days. From the15thday, TC and CSD rats wereadministrated daily （10:00-11:00） by1mL PBS control solution （peritoneal injection） forconsecutive7days, at the same time, the SKF rats were injected1mL SKF38393（1mg/kg）which is a dopamine D₁R specific agonist. At the end of CSD, bilateral PFC were quicklydisserted on the ice and keeped in the-80℃.4samples were randomly selected in pergroup （3for detection and1for spare）, genetic expression in PFC were detected by genechip （Affymetrix Rat Genome230Arrays, Bo Hao, Shanghai）. Then we chosed19genesand used the real time quantitative PCR （RT-PCR/qPCR） to validate the results of chip.Results: We got the qualified chip verification with r2=0.85. After screening, theresults of the significant difference （p＜0.05and FC≥1.5） gene showed that:Campared to TC group, there are208differentially expressed genes in CSD group, inwhich88genes were down-regulated and120genes were up-regulated. Through GeneOntology enrichment analysis （p＜0.05）, there are29third functional classifications and5second functional classifications between the two groups. Through signaling pathwayenrichment analysis：there are22meanful signaling pathway between the two groups,including immunomodulatory, metabolic regulation, circadian rhythm regulation andMAPK pathway.Compared with CSD group, there are115differentially expressed genes in SKF group,in which57were down-regulated and58were up-regulated. Compared with TC group,there are126differentially expressed genes in SKF group, in which59were down-regulated and67were up-regulated.In addition, compared with CSD, TC group and SKF group had14same trenddifferential genes including Hspb1, S100a9and Homer1. Compared with TC, CSD groupand SKF group had15same trend differential genes including Per, Chm and Cm13.Discussion: there are most differential genes （208） between CSD and TC groups,by which CSD impairs PFC structure and induces the higher nervous dysfunction. Thesedifferential genes may be associated with the regulation of many functions or pathways, including development, cell killing, immune system and metabolism. Interestingly, theSKF38393reversed14genes influenced by CSD, such as Hspb1, S100a9and Homer. Thismay indicate that, during the improvement of PFC dysfunction by the activation of D₁R,the14genes may play a major role in regulation. These genes are associated with calciumion pathway, MAPK pathway, stress and apoptosis pathways, and these functionalpathways may be the new approaches influenced by SKF38393, apart from the foregoneD₁R signaling pathway.Furthermore, activated D₁R does not completely reversed CSD’s damage to the genesin PFC, such as Per2, Chm and Cm13. Consequently, there may be other damagemechanisms except for the D₁R system.CHAPTER TWO Effect of chronic sleep deprivation on D₁receptor signaling pathway in prefrontal cortexObjective: To study the effect of CSD on the signaling pathway mediated by D₁Rin PFC, demonstrate the main pathway and molecule by which D₁R regulates the damageof structure and function in PFC during CSD.Materials and methods: With the results of gene chip, we analyzed the changesof known genetic expression in the D₁R signaling pathway. mRNA were extracted from6samples per group, and then the level of selective17genes in D₁R signaling pathway werevalidated by RT-PCR. In addition, proteins were extracted from4samples per group, andthen the protein expression and phosphorylation of selective7molecules in D₁R signalingpathway were measured by Western blot.Results: The results of chips indicated that, within known signaling molecules inD₁R pathway, there are15differential genes （p＜0.05or FC≥1.5）, in which Camk4andCamk1g were notably differential （p＜0.05and FC≥1.5）. Compared with the CSD group,Rap1a、Rasgrf1and Creb1returned normal level in SKF group （FC≥1.5）.The results of RT-PCR showed that, compared with the TC group, the level of Plcb1,Camk4, Camk1g, Rap1a, Rap1b and Erk1were significantly decreased in CSD group, andcompared with the CSD group, the expression of Plcb1, Camk4, Drd1a, Prkaca, Darpp32,Pkcγ, Rap1a and Rap1b increased significantly in SKF group.The results of Western blot showed that, in the level of total protein, the expression ofPLCβand NMDAR1reduced significantly in CSD group, while the expression of PLCβ,CaMKⅣ and CREB increased notably in SKF group. In the level of phosphorylation, the expression of PLCγ, IP3R1, CaMKⅣ, NMDAR1and CREB reduced significantly inCSD group, while the expression of all measured7molecules increased notably in SKFgroup.Discussion: Our previous studies showed that, the decline of learning and memorycapacity induced by CSD related to the damage of PFC neuron which caused by theblockage of DA and D₁R regulation. The activation of D₁R modulates CREB, which is anuclear transcription factor, via three signaling pathways including AC-cAMP-PKA,MAPK and phosphoinositide pathway.The genetic detection of chip and PCR indicated that, in the level of geneticetranscription, CSD did not significantly change the gene expression of key molecules inPKA pathway, while the enrichment test of chip differential gene inferred that CSD havean important impact on the MAPK pathway in rats PFC, the detection speculated thatERK1/2pathway, regulated by small G proteins Rap and Ras in MAPK pathway, may playan important role in the regulation of change and recovery of PFC during CSD. The studyalso found that, in phosphoinositide pathway, CSD significantly changed the genetictranscription and protein phosphorylation levels of several molecules such as PLC, IP3Rand CaMK, which have a relationship with the regulation of intracellular calcium ionlevels, while this phenomenon was revered by the intervention of D₁R agonist SKF38393.In summary, it can be assumed that, during the process of damage of PFC induced by CSD,the MAPK pathway and the phosphoinositide pathway mediated by D₁R could play amajor regulatory role in PFC, rather than PKA pathway.It is noteworthy that, in addition to the release of intracellular calcium storesassociated with IP3R activation, the glutamate NMDAR channel and membrane calciumchannel are involved in the regulation of intracellular calcium level. This study found that,with the activation of D₁R, the level of NMDAR1phosphorylation was enhanced.Therefore, we supposed that, during CSD, the phosphoinositide pathway and NMDARpathway could coregulate the biological functions associated with the intracellular calciumlevels, and CaMKⅣ may play a key role in this process.