Research Article Sleep Deprivation Influences Circadian ...

Research ArticleSleep Deprivation Influences Circadian GeneExpression in the Lateral Habenula

Beilin Zhang1 Yanxia Gao1 Yang Li1 Jing Yang1 and Hua Zhao12

1Department of Physiology College of Basic Medical Sciences Jilin University Changchun 130021 China2Neuroscience Research Center First Hospital of Jilin University Changchun 130021 China

Correspondence should be addressed to Hua Zhao zhuajlueducn

Received 23 March 2016 Accepted 22 May 2016

Academic Editor Barbara Picconi

Copyright copy 2016 Beilin Zhang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Sleep is governed by homeostasis and the circadian clock Clock genes play an important role in the generation andmaintenance ofcircadian rhythms but are also involved in regulating sleep homeostasis The lateral habenular nucleus (LHb) has been implicatedin sleep-wake regulation since LHb gene expression demonstrates circadian oscillation characteristics This study focuses on theparticipation of LHb clock genes in regulating sleep homeostasis as the nature of their involvement is unclear In this study weobserved changes in sleep pattern following sleep deprivation in LHb-lesioned rats using EEG recording techniques And then thechanges of clock gene expression (Per1 Per2 and Bmal1) in the LHb after 6 hours of sleep deprivation were detected by using real-time quantitative PCR (qPCR)We found that sleep deprivation increased the length of Non-Rapid EyeMovement Sleep (NREMS)and decreased wakefulness LHb-lesioning decreased the amplitude of reduced wake time and increased NREMS following sleepdeprivation in rats qPCR results demonstrated that Per2 expression was elevated after sleep deprivation while the other two geneswere unaffected Following sleep recovery Per2 expression was comparable to the control group This study provides the basis forfurther research on the role of LHb Per2 gene in the regulation of sleep homeostasis

1 Introduction

Clock genes are a class of molecules essential for the gen-eration and maintenance of circadian rhythms The proteinproducts of clock genes collaborate to establish a networkof autoregulatory feedback loops Such loops are thoughtto be exclusively involved in the maintenance of a sim24 hoscillation cycle and the regulation of physiological functionsThe core loop includes a brain andmuscle ARNT-like protein1circadian locomotor output cycles kaput (Bmal1clock)heterodimer that binds to Enhancer Box (E-box) containingelements on the promoters of the core clock genes period(Per1 Per2 Per3) and cryptochrome (Cry1 Cry2) [1 2]Knockout or mutation of circadian genes can cause analteration in rhythm such as in Bmal1 knockout mice whichdemonstrate loss of activity-based rhythmicity [3] Similarlythe absence of Per [4ndash6] or Cry gene [7] expression inmice can result in a shorter circadian period Furthermoretransgenic clock gene-deficient mice demonstrate robustcircadian rhythms when compared to wild-type animals [8]

These studies suggest that clock genes play an important rolein the generation and maintenance of circadian rhythms inmammals

Sleep is one of the most renown functions influencedby circadian rhythms and is modulated by both the circa-dian clock and sleep homeostasis [9 10] The influence ofgenes associated with the circadian clock on sleep patternand duration has been well characterized in recent yearsFor example the knockout of NPAS2 in mice produces areduction in Non-Rapid Eye Movement Sleep (NREMS) anddecreased NREMS delta power following sleep deprivation[11ndash13] Knockout of Cry12 reportedly decreased the numberof awakenings and increased NREMS delta power during thefirst hour after sleep deprivation [14] The Per gene mutationmice was observed to have increases in wakefulness timeThe NREMS delta power was enhanced in the Per genemutation mice after sleep deprivation compared with wide-type mice [15 16] Additionally both Per1 and Per2 levelsin cerebral cortex and forebrain were increased and DpbmRNAswere decreased by using qPCR after enforcedwaking

Hindawi Publishing CorporationBehavioural NeurologyVolume 2016 Article ID 7919534 8 pageshttpdxdoiorg10115520167919534

2 Behavioural Neurology

and these reverted to control levels within 2 h of recoverysleep [17 18] However the mRNA levels of Cry Bmal1and other clock genes demonstrated no significant changesfollowing sleep deprivation and recovery time [18] In situhybridization showed that sleep deprivation enhanced Per1expression in the forebrain cerebral cortex cerebellum [17]striatum and olfactory bulb [14] These studies suggest thatPer expression is likely dependent on the regionrsquos involvementin sleep homeostasis

A primary region implicated in circadian rhythm regu-lation and sleep homeostasis is the habenula (Hb) The Hbfeatures a semiautonomous circadian oscillator localized toits lateral region the LHb Circadian oscillators reportedlyhave the independent capacity to generate circadian rhythmsindependently of the suprachiasmatic nucleus (SCN) pace-maker [19 20] The LHb in particular has been linked tothe production of circadian functions due to vasopressin-ergic connections with the SCN which conveys circadianinformation to relevant regions [21] Additionally the LHbhas been reported to generate circadian firing-rate rhythmsfor at least two cycles in vitro [19] and to initiate circadianrhythms of clock gene expression [22] It is reported thatelectrical stimulation of the LHb increases NREMS in cats[23]These results suggest a role for the LHb in the regulationof sleep homeostasis and circadian rhythms However therelative involvement of clock gene expression in regulationof sleep homeostasis is still unknown In the current studywe examined the effect of LHb lesions on the sleep reboundinduced by a 6 h sleep deprivation And then we furtherdetected the changes of clock gene (Per1 Per2 and Bmal1)expression in the LHb after 6-hour sleep deprivation usingqPCR

2 Materials and Methods

21 Animals and EEG Recording Six male Wistar rats (210ndash230 g) obtained from the Center for Experimental AnimalsJilin University (Certification number SCXK (Ji) 2007-0003)were surgically prepared for EEG and electromyographic(EMG) recording Rats were maintained under a 12 h light-dark cycle (lights on at 0800 am) while under anesthesiawith 10 chloral hydrate (035mL100 g) stainless steel jew-elry screws were implanted into regions of the skull overlyingthe frontal and parietal cortices for EEG recording Twostainless steel electrodes were placed into the dorsal neckmuscles to record EMGdata In addition a guide cannula wasstereotaxically implanted above the LHb (coordinates poste-rior 36mm lateral 06mm ventral 42mm frombregma anddura Paxinos andWatson 1986)The guide cannulawas fixedonto the skull with dental cement All electrodes were placedin a six-pin plastic plug and secured onto the skull usingdental cement In the study the difference between sleep andwake cycle following 6 h sleep deprivation before and afterLHb lesions in the same group rats was analyzed to determinethe effect of LHb lesions on the sleep rebound induced by a 6 hsleep deprivation After allowing 7 days for surgical recoverythe first time of EEG recording for 24 hours was performedat 1400 pm The second time of EEG recording followed bya 6 h period of sleep deprivation starting at 1400 pm was

performed for 18 h starting at 2000 pm and ending at thesecond day at 1400 pm We repeated the same experimentprocedure as mentioned above seven days after LHb lesionsin the same group of rats Sleep deprivation in all experimentswas achieved by gentle handling

In order to damage the LHb 02120583L kainic acid (15 120583g120583L)was slowly injected to the LHb (over 2min) using a needleconnected with a guide cannula its tip extended 05mmbelow the cannula end implanted

The recording system consisted of a computer with ananalog-to-digital converter (6036E NI company) and aGrassModel 12 Neurodata Amplifier system (Grass InstrumentDivision of Astro-Med Inc West Warwick RI) The signalswere amplified by 12A5 amplifiers and acquired by a computerprogram (gammar Grass Instrument Division of Astro-MedInc West Warwick RI) The EEG data recordings werescored manually by Sleepsign software (KISSEI NaganoJapan) in 10 s epochs for wakefulness NREMS and RapidEye Movement Sleep (REMS) according to the standardcriteria [24] The wake state was identified by the presenceof desynchronized EEG (6ndash30Hz) and high EMG activityA high-amplitude slow wave (075ndash4Hz) together with alow EMG tone relative to awake is NREM sleep REM sleepconsisted of regular 120579 activity (6ndash10Hz) coupled with lowEMG relative to NREM sleep The typical EEG patterns wereseen in Figure 2(a)

22 qPCR Study In this part 128 male Wistar rats (190ndash210 g) were maintained in 12 h light-dark cycles (light onat 700 am) with food and water available ad libitum priorto experimentation Rats were divided into four groups asleep deprivation group control for sleep deprivation grouprecovery group and a control for recovery group Drawingfrom the initial sleep deprivation experiment rats were sleep-deprived for 6 h starting at 700 am by gentle handlingand sacrificed at 100 pm together with non-sleep-deprivedcontrols The recovery group was allowed 2 h of recoverysleep following 6 h of sleep deprivation and sacrificed at 300pm together with non-sleep-deprived controls

The brains were rapidly removed and two 500 120583m thickcoronal slices containing LHb were collected by vibrationcutting machine (ZQP-86 Shanghai Five-Phase InstrumentCo Ltd Shanghai China) from Bregma minus312ndashminus420mmThemedial habenula consistsmostly of dense cells and lateralhabenula consists of more loosely arranged cells [25] Theboundary between medial and lateral habenula is visualizedWe dissected the LHb tissue with needle along the outeredge of the boundary to avoid the pollution of the medialhabenular tissue However it is difficult to extract selectivelylateral part of the LHb because there were no obviousboundaries visualized between lateral andmedial parts of theLHb Thus the complete LHb was targeted in the study

The LHb tissue from 3 rats was put into one Eppendorf(EP) tube as one sample There are 24 rats in each grouptherefore each group had 8 samples which were frozen atminus80∘CThe samples of rat LHbwere homogenized using TRI-zol Reagent (Invitrogen) and then separated into three phasesby chloroform a lower red phenol-chloroform phase aninterphase and a colorless upper aqueous phase RNA exists

Behavioural Neurology 3

Table 1 RT-qPCR primers used for the analysis of clock geneexpression

Gene Primers (51015840 to 31015840) Temperature(∘C)

Per1 Forward (51015840-31015840) gaggagccagagaggaaagagtReverse (51015840-31015840) caggaagaggaggcacatttac 63

Per2 Forward (51015840-31015840) agcaacaccacctttcacaaReverse (51015840-31015840) cgtaggcttagaccaccatc 63

Bmal1 Forward (51015840-31015840) caacccatacacagaagcaaacReverse (51015840-31015840) acagattcggagacaaagagga 60

120573-actin Forward (51015840-31015840) agccatgtacgtagccatccReverse (51015840-31015840) ctctcagctgtggtggtgaa 6063

exclusively in the aqueous phase After precipitating the RNAfrom the aqueous phase by mixing with isopropyl alcohol(Beijing Chemical Works Beijing China) the RNA pelletwas washed with 75 ethanol (Beijing Chemical WorksBeijing China) and dried at room temperature Finally RNApellets were redissolved in RNase-free double distilled waterThe concentration as well as purity of RNA was detectedby a microplate reader (Biotech Company) to achieve RNAquantification

qPCR was performed using SYBR Premix Ex Taq II 2x(Tli RNaseH Plus TAKARA) Prior to cDNA synthesis RNAsamples were treated with gDNA eraser (TAKARA ambion)to remove genomic DNA contamination and 400 ng of totalRNA was converted into first strand cDNA with Oligo(dT)and Random 6 mers (TAKARA ambion) Primers for threecircadian genes (Per1 Per2 and Bmal1 Table 1) were designedand synthesized by Sangon Biotech Company (ShanghaiChina) and checked by primer-BLAST at NCBI Quantitativefluorescence PCR was performed using an Mx3000P qPCRSystem This incorporated several steps 95∘C denaturationfor 30 s 40 cycles of 95∘C denaturation for 5 s and annealingand extension at 6063∘C for 20 s (Table 1) Data wereanalysed by MxPro QPCR software Ct value for Per1 Per2and Bmal1 was normalized to 120573-actin Ct value The relativeexpression level of each clock gene sample was calculated bythe 2minusΔΔCt methodThemean and SEM of relative expressionlevel were analyzed

23 Histology Examination Histological staining with CresylViolet acetate was used to evaluate extent of the LHb lesionNylon body a characteristic structure of the neurons couldbe stained by Cresyl Violet acetate The areas of the lesionwere not stained with Cresyl Violet acetate (1 Cresyl Violetacetate in PBS pH = 38) because of the loss of Nylon bodyTherefore we can estimate the range of lesion based on thestaining under microscope (Figure 1) The data of damagedarea outside of the LHb are not included in the statistics

24 Statistics Data from 18 h EEG recording which followsleep deprivation before and after the LHb lesion wereanalyzed by paired 119905-test (SPSS110 software) and presentedas means plusmn SEM EEG data plotted per 6 h for a 24 hperiod following deprivation before and after lesion wereassessed by repeated measures one-way analysis of variance

(ANOVA) with two-way ANOVA process to achieve twocomparisons between sleep and wake times before and aftersleep deprivation at each time point Unpaired 119905-tests wereused to analyze qPCR results

3 Results

31 Effects of LHb Lesion on the Sleep-Wake Cycle in Ratsfollowing Sleep Deprivation In this study we compared thedifference between sleep and wake cycle following 6 h sleepdeprivation before and after LHb lesions in the same grouprats to determine the effect of LHb lesions on the sleeprebound induced by a 6 h sleep deprivation The first periodof sleep deprivation was initiated prior to lesioning whereinthe mean wake time significantly decreased from 57731 plusmn2967min to 46114plusmn1815min (119905 = 6459 df = 5119875 = 00013Figure 2(b)(B1)) Additionally the duration of NREMS wasincreased significantly from 43817 plusmn 212 to 50136 plusmn1423min (119905 = 5102 df = 5 119875 = 00038 Figure 2(c)(C1))The duration of REMS also increased (119905 = 4602 df = 5 119875 =00058 Figure 2(d)(D1)) Therefore the initial experimentalphase demonstrated that sleep deprivation could elicit a sleeprebound effect in ratsThe second period of sleep deprivationwas performed 7 days after lesioning of the LHb At thisstage the wake time was significantly reduced from 63931 plusmn1822min to 54044plusmn2732min (119905 = 3788 df = 5119875 = 00128Figure 2(b)(B1)) and NREMS was significantly increasedfrom 38414 plusmn 1722min to 44958 plusmn 939min (119905 = 3204df = 5 119875 = 00239 Figure 2(c)(C1)) Further analysisdemonstrated that the duration of wakefulness following thesleep deprivation was longer after LHb lesion than beforeLHb lesion (54044 plusmn 6691min versus 46114 plusmn 4446min119905 = 5787 df = 5 119875 = 00022 Figure 2(b)(B1)) and NREMSwas shorter after LHb lesion than before LHb lesion (43969plusmn3129min versus 49314 plusmn 5107min 119905 = 4335 df = 5119875 = 00075 Figure 2(c)(C1)) Figures 2(b)(B2) 2(c)(C2)and 2(d)(D2) showed sleep-stage distributions following 6 hsleep deprivation before and after LHb lesion In the first6 h after SD the LHb lesion decreased the amplitude ofincreased REMS and reducedwake time by sleep deprivationNo significantly differencewas found between sleep andwaketimes before and aftermicroinjected 02120583L vehicle to the LHbin sham surgery group

32 Effects of Sleep Deprivation on Circadian Gene Expressionin the LHb ThemRNA levels for Per1 Per2 and Bmal1 werequantified using qPCR for two brain regions the SCN andLHbThemRNA levels of Per2 in the LHb increased following6 h of sleep deprivation wherein Per2 expression in the sleepdeprivation group (125plusmn011 119899 = 8) was significantly higherthan control group (094 plusmn 008 119899 = 8 119905 = 2326 df = 14 119875 lt005) Comparatively sleep deprivation did not affect Per1 orBmal1 expression in the LHb (Figure 3(a)) The mRNA levelsof these three genes in the SCN showed no alterations after6 h of sleep deprivation (119899 = 8 Figure 3(b))

33 CircadianGene Expression Changes following 2 h RecoverySleep Per1 Per2 and Bmal1 mRNA levels were not affected


(a) (b)

Figure 1 A crystal violet staining photomicrograph of LHb lesion (a) showed the normal Hb and (b) showed the lesion of LHb The singlearrow identifies the MHb whereas the double arrows identify the LHb Scale bars represent 100 120583m

following 2 hours of recovery after sleep deprivation ineither the LHb or SCN compared with the control group(119899 = 8each group Figures 4(a) and 4(b)) No significantdifferences were observed in the LHb for Per1 expressionfor the recovery group (109 plusmn 026) when compared withthe control group (134 plusmn 009 119905 = minus0896 119875 gt 005) Asimilar pattern was observed for Bmal1 expression betweenthe control group (083plusmn008) and the recovery group (100plusmn003 119905 = 2 119875 gt 005) Likewise there was no difference inPer2 expression between the control group (142 plusmn 011) andthe recovery group (135 plusmn 013 119905 = minus044 119875 gt 005) Asimilar pattern was observed for these genes in the SCN

4 Discussion

Sleep deprivation tends to produce a correlated increasein sleep rebound an effect that was demonstrated in thecurrent study through an increased duration of both NREMand REM sleep This suggests that sleep homeostasis mayplay an important role in the regulation of sleep pattern[9 26] In this experiment we determined the effect of LHblesions on the sleep rebound induced by 6 h sleep deprivationCompared to prelesion data the postlesion sleep recoverytime induced by sleep deprivation was reduced as shown bya decrease in NREMS and an increase in wake time Whenthe difference between sleep and wake time before and afterLHb lesions in the same group rats was analyzed we foundthat the LHb lesion could induce a decrease in NREMS timewhich is consistent with the results of Goldsteinrsquos studiesshowing that stimulation the LHb could increase NREMStime [23] It is considered that the role of the LHb lesionon sleep rebound evoked by sleep deprivation may relateto its role in promoting sleep Previous studies have alsosuggested that the LHbhas a close functional connectionwithseveral structures implicated in sleep homeostasis includingthe preoptic hypothalamus the raphe nuclei and the pinealgland [27ndash29] The LHb also regulates the release of various

neurotransmitters linked to homeostatic regulation such asadenosine melatonin and monoamine neurotransmitters[30ndash32] These studies provide further evidence to suggest arole for the LHb in the regulation of sleep homeostasis

The sleep-wake cycle features its own distinct circadianrhythm which contributes to maintenance of sleep-wakecycle [10 33] It has been well-established that clock genes arenot only the basis of the generation of circadian rhythm butalso related to the regulation of sleep homeostasis [34 35]They were reported to have an increase in the NREMS deltapower in the Per gene mutation mice after sleep deprivationcompared with wide-type mice [15 16] Additional studyshowed that the Per gene induction was found in thebasal forebrain and cerebral cortex of mice following sleepdeprivation [17 18] The results of the current study showedthat sleep deprivation induces increase of Per2 expressionin the LHb whereas the increase returns to basal levelsfollowing sleep recovery Drawing from the data obtained inthe current study sleep not only is affected by the activityof clock genes but is also able to regulate their expressionThis suggests that clock genes are involved in the regulationof sleep homeostasis

There is a link between clock gene expression and sleep-related processes however the underlying regions supportingsleep homeostasis have received relatively limited focusNumerous studies have provided evidence to suggest theimportance of the SCN as the mammalian circadian pace-maker but in recent years increasing studies have demon-strated a growing role for the LHb which is thought tocontribute significantly to the production of higher brainfunctions and may be implicated in the pathology of neu-rological disease [32 36ndash39] The LHb is considered tofeature a semiautonomous circadian oscillator similar tothose identified in the SCN retina and olfactory bulb [20] Asa function of this the ability of the LHb to influence circadianrhythm production and maintenance has garnered interestin recent years The LHb neurons have been shown to have


EEG

EMG

Wake NREM REM

(a)

Normal sleepSleep deprivation


After lesionBefore lesion

After lesionBefore lesionlowastlowastlowast

WakeWakeWake

8 amndash14 pm2 amndash8 am20 pmndash2 am14 pmndash20 pm

(B1) (B2)


8 amndash14 pm2 amndash8 am20 pmndash2 am14 pmndash20 pm0

200

400

600

800

Tim

e (m

in)

0

100

200

300

400

Tim

e (m

in)

0

100

200

300

400

Tim

e (m

in)lowast lowast

(b)



lowastlowastlowast

(C1)

0

200

400

600

Tim

e (m

in)


Before lesion After lesionNREM NREM NREM


(C2)

0

100

200

300

Tim

e (m

in)

0

100

200

300

Tim

e (m

in)



(c)

REM REMREMAfter lesion



lowastlowast

(D1)

0

50

100

150

Tim

e (m

in)


Before lesion


(D2)

0

20

40

60

Tim

e (m

in)

0

20

40

60

Tim

e (m

in)



lowast

(d)

Figure 2The effect of LHb lesion on sleep-wake times following sleep deprivation in rats (a) indicates the typical EEG and EMG recordingrespectively for wakeNREMS andREMS timesThewake state was identified by the presence of desynchronized EEG and high EMGactivityThe NREMS consisted of high-amplitude slow waves together with a low EMG relative to awake The REMS was identified by the presenceof regular 120579 activity coupled with low EMG relative to NREMS (B1) (C1) and (D1) respectively show the wake NREMS and REMS timesfollowing sleep deprivation before and after LHb lesion Data from 18 h EEG recording were analyzed and presented as means plusmn SEM (119899 = 6)lowast

119875 lt 005 lowastlowast119875 lt 001 compared respectively difference between the wake NREMS and REMS times before and after sleep deprivation119875 lt 005 119875 lt 001 compared respectively difference between the wake and NREMS times following deprivation before and after LHblesion (B2) (C2) and (D2) show respectively the graphs of wake NREMS and REMS plotted per 6 h for a 24 h period before and afterlesion lowast119875 lt 005 compared with sleep deprivation at the same time point


Per2 Per1 Bmal1

NormalSD

mRN

A ex

pres

sion

relat

ive t

o120573

-act

in20

15

10

05

00

LHb

lowast

(a)

Per2 Per1 Bmal1

mRN

A ex

pres

sion

relat

ive t

o120573

-act

in

25

20

15

10

05

00

SCN

NormalSD

(b)

Figure 3 Sleep deprivation alters mRNA levels of clock genes qPCR analysis of the expression of three clock genes (Per1 Per2 and Bmal1)in the LHb (a) and SCN (b) of the rat Data was presented as mean plusmn SEM (119899 = 8each group) lowast119875 lt 005 compared with control group (byunpaired 119905-test)

Per2 Per1 Bmal1

RecoveryNormal

LHb

00

05

10

15

20

mRN

A ex

pres

sion

relat

ive t

o120573

-act

in

(a)

Per2 Per1 Bmal1

SCN

00

05

10

15

20

25

30

mRN

A ex

pres

sion

relat

ive t

o120573

-act

in

RecoveryNormal

(b)

Figure 4 qPCR analysis of Per1 Per2 and Bmal1 levels in the LHb following 2 hours of recovery after sleep deprivation Data was presentedas mean plusmn SEM (119899 = 8each group)

rhythmic electrical activity [19] whereas Per2 mRNA andprotein levels appear to oscillatewith circadian characteristics[22 40] This study supports an additional role for the LHbin sleep homeostasis in which lesions to the LHb suppressincreases in sleep rebound time and enhance wakefulnessfollowing sleep deprivation

Despite evidence of Per2 induction in the LHb we foundno change in the expression of Per2 in the SCN after sleepdeprivation which was consistent with the experimentalresults of a similar study [41] Sleep deprivation is a complexstimulus that invokes arousal and inducesmild stress Furtherto this previous studies have reported a link between Perexpression and the pattern and duration of stress [42] Sincethe LHb has demonstrated involvement in the production

of stress responses [32 43] the alteration of Per2 expressionobserved in the current experiment may be linked to stressfollowing exposure to sleep deprivation As of yet no studieshave reported the involvement of the SCN in the productionof stress which might explain the absence of Per2 geneinduction in the SCN following sleep deprivation

Our findings showed that LHb lesion significantly short-ens the sleep rebound time induced by sleep deprivationsuggesting an important role for the LHb in sleep homeosta-sis Furthermore Per2 expression in the LHb is significantlyincreased following sleep deprivation indicating that theaction of Per2 in the LHb may be related to the regulationof sleep homeostasis However further research is needed toprove that


Competing Interests

The authors declare no conflict of interests

Acknowledgments

This work was supported by National Natural Science Foun-dation of China (nos 81000570 and 81271465) andHealth andFamily Planning Commission of Jilin Province (2012Z004)The authors would like to thank Professor Juxiong Liu in theJilin University for technical assistance

References

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[2] S M Reppert and D R Weaver ldquoCoordination of circadiantiming in mammalsrdquo Nature vol 418 no 6901 pp 935ndash9412002

[3] M K Bunger L D Wilsbacher S M Moran et al ldquoMop3 isan essential component of the master circadian pacemaker inmammalsrdquo Cell vol 103 no 7 pp 1009ndash1017 2000

[4] B Zheng D W Larkin U Albrecht et al ldquoThe mPer2 geneencodes a functional component of the mammalian circadianclockrdquo Nature vol 400 no 6740 pp 169ndash173 1999

[5] B Zheng U Albrecht K Kaasik et al ldquoNonredundant roles ofthe mPer1 andmPer2 genes in the mammalian circadian clockrdquoCell vol 105 no 5 pp 683ndash694 2001

[6] L P Shearman X Jin C Lee S M Reppert and D RWeaver ldquoTargeted disruption of the mPer3 gene subtle effectson circadian clock functionrdquo Molecular and Cellular Biologyvol 20 no 17 pp 6269ndash6275 2000

[7] M H Vitaterna C P Selby T Todo et al ldquoDifferential regula-tion of mammalian period genes and circadian rhythmicity bycryptochromes 1 and 2rdquo Proceedings of the National Academy ofSciences of the United States of America vol 96 no 21 pp 12114ndash12119 1999

[8] J P Debruyne E Noton C M Lambert E S Maywood D RWeaver and S M Reppert ldquoA clock shock mouse CLOCK isnot required for circadian oscillator functionrdquo Neuron vol 50no 3 pp 465ndash477 2006

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[10] S Daan D G Beersma and A A Borbely ldquoTiming ofhuman sleep recovery process gated by a circadian pacemakerrdquoAmerican Journal of PhysiologymdashRegulatory Integrative andComparative Physiology vol 246 no 2 pp R161ndashR183 1984

[11] E Naylor B M Bergmann K Krauski et al ldquoThe circadianClock mutation alters sleep homeostasis in the mouserdquo Journalof Neuroscience vol 20 no 21 pp 8138ndash8143 2000

[12] C A Dudley C Erbel-Sieler S J Estill et al ldquoAltered patternsof sleep and behavioral adaptability in NPAS2-deficient micerdquoScience vol 301 no 5631 pp 379ndash383 2003

[13] P Franken C A Dudley S J Estill et al ldquoNPAS2 as a tran-scriptional regulator of non-rapid eye movement sleep geno-type and sex interactionsrdquo Proceedings of the National Academyof Sciences of the United States of America vol 103 no 18 pp7118ndash7123 2006

[14] J P Wisor R K Pasumarthi D Gerashchenko et al ldquoSleepdeprivation effects on circadian clock gene expression in thecerebral cortex parallel electroencephalographic differencesamong mouse strainsrdquo Journal of Neuroscience vol 28 no 31pp 7193ndash7201 2008

[15] P J Shiromani M Xu E M Winston S N ShiromaniD Gerashchenko and D R Weaver ldquoSleep rhythmicity andhomeostasis in mice with targeted disruption of mPeriodgenesrdquo American Journal of PhysiologymdashRegulatory Integrativeand Comparative Physiology vol 287 no 1 pp R47ndashR57 2004

[16] C Kopp U Albrecht B Zheng and I Tobler ldquoHomeostaticsleep regulation is preserved inmPer1 andmPer2mutant micerdquoEuropean Journal of Neuroscience vol 16 no 6 pp 1099ndash11062002

[17] P Franken R Thomason H C Craig and B F OrsquoHara ldquoAnon-circadian role for clock-genes in sleep homeostasis a straincomparisonrdquo BMC Neuroscience vol 8 article 87 2007

[18] J PWisor B F OrsquoHara A Terao et al ldquoA role of cryptochromesin sleep regulationrdquo BMC Neuroscience vol 3 article 20 2002

[19] H Zhao and B Rusak ldquoCircadian firing-rate rhythms and lightresponses of rat habenular nucleus neurons in vivo and in vitrordquoNeuroscience vol 132 no 2 pp 519ndash528 2005

[20] C Dibner U Schibler and U Albrecht ldquoThe mammaliancircadian timing system organization and coordination ofcentral and peripheral clocksrdquoAnnual Review of Physiology vol72 pp 517ndash549 2010

[21] G J De Vries R M Buds and D F Swaab ldquoOntogeny of thevasopressinergic neurons of the suprachiasmatic nucleus andtheir extrahypothalamic projections in the rat brain-presence ofa sex difference in the lateral septumrdquo Brain Research vol 218no 1-2 pp 67ndash78 1981

[22] C Guilding A T L Hughes and H D Piggins ldquoCircadianoscillators in the epithalamusrdquo Neuroscience vol 169 no 4 pp1630ndash1639 2010

[23] R Goldstein ldquoA gabaergic habenulo-raphe pathway mediationof the hypnogenic effects of vasotocin in catrdquo Neuroscience vol10 no 3 pp 941ndash945 1983

[24] Z-L Huang W-M Qu N Eguchi et al ldquoAdenosine A2A butnot A1 receptors mediate the arousal effect of caffeinerdquo NatureNeuroscience vol 8 no 7 pp 858ndash859 2005

[25] K H Andres M von During and R W Veh ldquoSubnuclearorganization of the rat habenular complexesrdquo Journal of Com-parative Neurology vol 407 no 1 pp 130ndash150 1999

[26] L Friedman B M Bergmann and A Rechtschaffen ldquoEffects ofsleep deprivation on sleepiness sleep intensity and subsequentsleep in the ratrdquo Sleep vol 1 no 4 pp 369ndash391 1979

[27] A B Kowski S Geisler M Krauss and RW Veh ldquoDifferentialprojections from subfields in the lateral preoptic area to thelateral habenular complex of the ratrdquo Journal of ComparativeNeurology vol 507 no 4 pp 1465ndash1478 2008

[28] D A Pasquier C Anderson W B Forbes and P J Mor-gane ldquoHorseradish peroxidase tracing of the lateral habenular-midbrain raphe nuclei connections in the ratrdquo Brain ResearchBulletin vol 1 no 5 pp 443ndash451 1976

[29] V Guglielmotti and L Cristino ldquoThe interplay between thepineal complex and the habenular nuclei in lower vertebratesin the context of the evolution of cerebral asymmetryrdquo BrainResearch Bulletin vol 69 no 5 pp 475ndash488 2006

[30] W A Staines T Yamamoto K M Dewar P E Daddona J DGeiger and J I Nagy ldquoDistributionmorphology and habenularprojections of adenosine deaminase-containing neurons in theseptal area of ratrdquo Brain Research vol 455 no 1 pp 72ndash87 1988


[31] B Lacoste D Angeloni S Dominguez-Lopez et al ldquoAnatom-ical and cellular localization of melatonin MT1 and MT2receptors in the adult rat brainrdquo Journal of Pineal Research vol58 no 4 pp 397ndash417 2015

[32] L-M Yang B Hu Y-H Xia B-L Zhang andH Zhao ldquoLateralhabenula lesions improve the behavioral response in depressedrats via increasing the serotonin level in dorsal raphe nucleusrdquoBehavioural Brain Research vol 188 no 1 pp 84ndash90 2008

[33] A C Skeldon G Derks and D-J Dijk ldquoModelling changesin sleep timing and duration across the lifespan changes incircadian rhythmicity or sleep homeostasisrdquo Sleep MedicineReviews vol 28 pp 92ndash103 2016

[34] P L Lowrey and J S Takahashi ldquoMammalian circadian biol-ogy elucidating genome-wide levels of temporal organizationrdquoAnnual Review of Genomics and Human Genetics vol 5 pp407ndash441 2004

[35] P Franken and D-J Dijk ldquoCircadian clock genes and sleephomeostasisrdquo European Journal of Neuroscience vol 29 no 9pp 1820ndash1829 2009

[36] X Shen X Ruan and H Zhao ldquoStimulation of midbraindopaminergic structures modifies firing rates of rat lateralhabenula neuronsrdquo PLoS ONE vol 7 no 4 article e34323 2012

[37] L-M Yang L Yu H-J Jin and H Zhao ldquoSubstance P receptorantagonist in lateral habenula improves rat depression-likebehaviorrdquo Brain Research Bulletin vol 100 pp 22ndash28 2014

[38] X F Luo B L Zhang J C Li Y Y Yang Y F Sun and HZhao ldquoLateral habenula as a link between dopaminergic andserotonergic systems contributes to depressive symptoms inParkinsonrsquos diseaserdquo Brain Research Bulletin vol 110 pp 40ndash462015

[39] B Han H J Jin M Y Song TWang and H Zhao ldquoA potentialtarget for the treatment of Parkinsonrsquos disease effect of lateralhabenula lesionsrdquoParkinsonismampRelatedDisorders vol 20 no11 pp 1191ndash1195 2014

[40] Z Zhao H Xu Y Liu L Mu J Xiao and H Zhao ldquoDiurnalexpression of the per2 gene and protein in the lateral habenularnucleusrdquo International Journal of Molecular Sciences vol 16 no8 pp 16740ndash16749 2015

[41] T Curie S Maret Y Emmenegger and P Franken ldquoIn Vivoimaging of the central and peripheral effects of sleep deprivationand suprachiasmatic nuclei lesion on PERIOD-2 protein inmicerdquo Sleep vol 38 no 9 pp 1381ndash1394 2015

[42] W-G Jiang S-X Li S-J Zhou Y Sun J Shi and L LuldquoChronic unpredictable stress induces a reversible change ofPER2 rhythm in the suprachiasmatic nucleusrdquo Brain Researchvol 1399 pp 25ndash32 2011

[43] M M Mirrione D Schulz K A B Lapidus S Zhang WGoodman and F A Henn ldquoIncreased metabolic activity inthe septum and habenula during stress is linked to subse-quent expression of learned helplessness behaviorrdquo Frontiers inHuman Neuroscience vol 8 no 1 article 29 2014

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



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OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


and these reverted to control levels within 2 h of recoverysleep [17 18] However the mRNA levels of Cry Bmal1and other clock genes demonstrated no significant changesfollowing sleep deprivation and recovery time [18] In situhybridization showed that sleep deprivation enhanced Per1expression in the forebrain cerebral cortex cerebellum [17]striatum and olfactory bulb [14] These studies suggest thatPer expression is likely dependent on the regionrsquos involvementin sleep homeostasis

A primary region implicated in circadian rhythm regu-lation and sleep homeostasis is the habenula (Hb) The Hbfeatures a semiautonomous circadian oscillator localized toits lateral region the LHb Circadian oscillators reportedlyhave the independent capacity to generate circadian rhythmsindependently of the suprachiasmatic nucleus (SCN) pace-maker [19 20] The LHb in particular has been linked tothe production of circadian functions due to vasopressin-ergic connections with the SCN which conveys circadianinformation to relevant regions [21] Additionally the LHbhas been reported to generate circadian firing-rate rhythmsfor at least two cycles in vitro [19] and to initiate circadianrhythms of clock gene expression [22] It is reported thatelectrical stimulation of the LHb increases NREMS in cats[23]These results suggest a role for the LHb in the regulationof sleep homeostasis and circadian rhythms However therelative involvement of clock gene expression in regulationof sleep homeostasis is still unknown In the current studywe examined the effect of LHb lesions on the sleep reboundinduced by a 6 h sleep deprivation And then we furtherdetected the changes of clock gene (Per1 Per2 and Bmal1)expression in the LHb after 6-hour sleep deprivation usingqPCR

2 Materials and Methods

21 Animals and EEG Recording Six male Wistar rats (210ndash230 g) obtained from the Center for Experimental AnimalsJilin University (Certification number SCXK (Ji) 2007-0003)were surgically prepared for EEG and electromyographic(EMG) recording Rats were maintained under a 12 h light-dark cycle (lights on at 0800 am) while under anesthesiawith 10 chloral hydrate (035mL100 g) stainless steel jew-elry screws were implanted into regions of the skull overlyingthe frontal and parietal cortices for EEG recording Twostainless steel electrodes were placed into the dorsal neckmuscles to record EMGdata In addition a guide cannula wasstereotaxically implanted above the LHb (coordinates poste-rior 36mm lateral 06mm ventral 42mm frombregma anddura Paxinos andWatson 1986)The guide cannulawas fixedonto the skull with dental cement All electrodes were placedin a six-pin plastic plug and secured onto the skull usingdental cement In the study the difference between sleep andwake cycle following 6 h sleep deprivation before and afterLHb lesions in the same group rats was analyzed to determinethe effect of LHb lesions on the sleep rebound induced by a 6 hsleep deprivation After allowing 7 days for surgical recoverythe first time of EEG recording for 24 hours was performedat 1400 pm The second time of EEG recording followed bya 6 h period of sleep deprivation starting at 1400 pm was

performed for 18 h starting at 2000 pm and ending at thesecond day at 1400 pm We repeated the same experimentprocedure as mentioned above seven days after LHb lesionsin the same group of rats Sleep deprivation in all experimentswas achieved by gentle handling

In order to damage the LHb 02120583L kainic acid (15 120583g120583L)was slowly injected to the LHb (over 2min) using a needleconnected with a guide cannula its tip extended 05mmbelow the cannula end implanted

The recording system consisted of a computer with ananalog-to-digital converter (6036E NI company) and aGrassModel 12 Neurodata Amplifier system (Grass InstrumentDivision of Astro-Med Inc West Warwick RI) The signalswere amplified by 12A5 amplifiers and acquired by a computerprogram (gammar Grass Instrument Division of Astro-MedInc West Warwick RI) The EEG data recordings werescored manually by Sleepsign software (KISSEI NaganoJapan) in 10 s epochs for wakefulness NREMS and RapidEye Movement Sleep (REMS) according to the standardcriteria [24] The wake state was identified by the presenceof desynchronized EEG (6ndash30Hz) and high EMG activityA high-amplitude slow wave (075ndash4Hz) together with alow EMG tone relative to awake is NREM sleep REM sleepconsisted of regular 120579 activity (6ndash10Hz) coupled with lowEMG relative to NREM sleep The typical EEG patterns wereseen in Figure 2(a)

22 qPCR Study In this part 128 male Wistar rats (190ndash210 g) were maintained in 12 h light-dark cycles (light onat 700 am) with food and water available ad libitum priorto experimentation Rats were divided into four groups asleep deprivation group control for sleep deprivation grouprecovery group and a control for recovery group Drawingfrom the initial sleep deprivation experiment rats were sleep-deprived for 6 h starting at 700 am by gentle handlingand sacrificed at 100 pm together with non-sleep-deprivedcontrols The recovery group was allowed 2 h of recoverysleep following 6 h of sleep deprivation and sacrificed at 300pm together with non-sleep-deprived controls

The brains were rapidly removed and two 500 120583m thickcoronal slices containing LHb were collected by vibrationcutting machine (ZQP-86 Shanghai Five-Phase InstrumentCo Ltd Shanghai China) from Bregma minus312ndashminus420mmThemedial habenula consistsmostly of dense cells and lateralhabenula consists of more loosely arranged cells [25] Theboundary between medial and lateral habenula is visualizedWe dissected the LHb tissue with needle along the outeredge of the boundary to avoid the pollution of the medialhabenular tissue However it is difficult to extract selectivelylateral part of the LHb because there were no obviousboundaries visualized between lateral andmedial parts of theLHb Thus the complete LHb was targeted in the study

The LHb tissue from 3 rats was put into one Eppendorf(EP) tube as one sample There are 24 rats in each grouptherefore each group had 8 samples which were frozen atminus80∘CThe samples of rat LHbwere homogenized using TRI-zol Reagent (Invitrogen) and then separated into three phasesby chloroform a lower red phenol-chloroform phase aninterphase and a colorless upper aqueous phase RNA exists













3 Results





(a) (b)



4 Discussion






EEG

EMG

Wake NREM REM

(a)





WakeWakeWake


(B1) (B2)



200

400

600

800

Tim

e (m

in)

0

100

200

300

400

Tim

e (m

in)

0

100

200

300

400

Tim

e (m

in)lowast lowast

(b)



lowastlowastlowast

(C1)

0

200

400

600

Tim

e (m

in)




(C2)

0

100

200

300

Tim

e (m

in)

0

100

200

300

Tim

e (m

in)



(c)




lowastlowast

(D1)

0

50

100

150

Tim

e (m

in)


Before lesion


(D2)

0

20

40

60

Tim

e (m

in)

0

20

40

60

Tim

e (m

in)



lowast

(d)




Per2 Per1 Bmal1

NormalSD

mRN

A ex

pres

sion

relat

ive t

o120573

-act

in20

15

10

05

00

LHb

lowast

(a)

Per2 Per1 Bmal1

mRN

A ex

pres

sion

relat

ive t

o120573

-act

in

25

20

15

10

05

00

SCN

NormalSD

(b)


Per2 Per1 Bmal1

RecoveryNormal

LHb

00

05

10

15

20

mRN

A ex

pres

sion

relat

ive t

o120573

-act

in

(a)

Per2 Per1 Bmal1

SCN

00

05

10

15

20

25

30

mRN

A ex

pres

sion

relat

ive t

o120573

-act

in

RecoveryNormal

(b)







Competing Interests


Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























3 Results





(a) (b)



4 Discussion






EEG

EMG

Wake NREM REM

(a)





WakeWakeWake


(B1) (B2)



200

400

600

800

Tim

e (m

in)

0

100

200

300

400

Tim

e (m

in)

0

100

200

300

400

Tim

e (m

in)lowast lowast

(b)



lowastlowastlowast

(C1)

0

200

400

600

Tim

e (m

in)




(C2)

0

100

200

300

Tim

e (m

in)

0

100

200

300

Tim

e (m

in)



(c)




lowastlowast

(D1)

0

50

100

150

Tim

e (m

in)


Before lesion


(D2)

0

20

40

60

Tim

e (m

in)

0

20

40

60

Tim

e (m

in)



lowast

(d)




Per2 Per1 Bmal1

NormalSD

mRN

A ex

pres

sion

relat

ive t

o120573

-act

in20

15

10

05

00

LHb

lowast

(a)

Per2 Per1 Bmal1

mRN

A ex

pres

sion

relat

ive t

o120573

-act

in

25

20

15

10

05

00

SCN

NormalSD

(b)


Per2 Per1 Bmal1

RecoveryNormal

LHb

00

05

10

15

20

mRN

A ex

pres

sion

relat

ive t

o120573

-act

in

(a)

Per2 Per1 Bmal1

SCN

00

05

10

15

20

25

30

mRN

A ex

pres

sion

relat

ive t

o120573

-act

in

RecoveryNormal

(b)







Competing Interests


Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)



4 Discussion






EEG

EMG

Wake NREM REM

(a)





WakeWakeWake


(B1) (B2)



200

400

600

800

Tim

e (m

in)

0

100

200

300

400

Tim

e (m

in)

0

100

200

300

400

Tim

e (m

in)lowast lowast

(b)



lowastlowastlowast

(C1)

0

200

400

600

Tim

e (m

in)




(C2)

0

100

200

300

Tim

e (m

in)

0

100

200

300

Tim

e (m

in)



(c)




lowastlowast

(D1)

0

50

100

150

Tim

e (m

in)


Before lesion


(D2)

0

20

40

60

Tim

e (m

in)

0

20

40

60

Tim

e (m

in)



lowast

(d)




Per2 Per1 Bmal1

NormalSD

mRN

A ex

pres

sion

relat

ive t

o120573

-act

in20

15

10

05

00

LHb

lowast

(a)

Per2 Per1 Bmal1

mRN

A ex

pres

sion

relat

ive t

o120573

-act

in

25

20

15

10

05

00

SCN

NormalSD

(b)


Per2 Per1 Bmal1

RecoveryNormal

LHb

00

05

10

15

20

mRN

A ex

pres

sion

relat

ive t

o120573

-act

in

(a)

Per2 Per1 Bmal1

SCN

00

05

10

15

20

25

30

mRN

A ex

pres

sion

relat

ive t

o120573

-act

in

RecoveryNormal

(b)







Competing Interests


Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















EEG

EMG

Wake NREM REM

(a)





WakeWakeWake


(B1) (B2)



200

400

600

800

Tim

e (m

in)

0

100

200

300

400

Tim

e (m

in)

0

100

200

300

400

Tim

e (m

in)lowast lowast

(b)



lowastlowastlowast

(C1)

0

200

400

600

Tim

e (m

in)




(C2)

0

100

200

300

Tim

e (m

in)

0

100

200

300

Tim

e (m

in)



(c)




lowastlowast

(D1)

0

50

100

150

Tim

e (m

in)


Before lesion


(D2)

0

20

40

60

Tim

e (m

in)

0

20

40

60

Tim

e (m

in)



lowast

(d)




Per2 Per1 Bmal1

NormalSD

mRN

A ex

pres

sion

relat

ive t

o120573

-act

in20

15

10

05

00

LHb

lowast

(a)

Per2 Per1 Bmal1

mRN

A ex

pres

sion

relat

ive t

o120573

-act

in

25

20

15

10

05

00

SCN

NormalSD

(b)


Per2 Per1 Bmal1

RecoveryNormal

LHb

00

05

10

15

20

mRN

A ex

pres

sion

relat

ive t

o120573

-act

in

(a)

Per2 Per1 Bmal1

SCN

00

05

10

15

20

25

30

mRN

A ex

pres

sion

relat

ive t

o120573

-act

in

RecoveryNormal

(b)







Competing Interests


Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Per2 Per1 Bmal1

NormalSD

mRN

A ex

pres

sion

relat

ive t

o120573

-act

in20

15

10

05

00

LHb

lowast

(a)

Per2 Per1 Bmal1

mRN

A ex

pres

sion

relat

ive t

o120573

-act

in

25

20

15

10

05

00

SCN

NormalSD

(b)


Per2 Per1 Bmal1

RecoveryNormal

LHb

00

05

10

15

20

mRN

A ex

pres

sion

relat

ive t

o120573

-act

in

(a)

Per2 Per1 Bmal1

SCN

00

05

10

15

20

25

30

mRN

A ex

pres

sion

relat

ive t

o120573

-act

in

RecoveryNormal

(b)







Competing Interests


Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Competing Interests


Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Sleep Deprivation Influences Circadian ...

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