Cell proliferation in the dentate gyrus of the adult rat fluctuates with the light–dark cycle

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Cell proliferation in the dentate gyrus of the adult rat fluctuates with the light–dark cycle
  Neuroscience Letters 422 (2007) 198–201 Cell proliferation in the dentate gyrus of the adult rat fluctuateswith the light–dark cycle Ruben Guzman-Marin a , b , Natalia Suntsova a , b , d , Tariq Bashir a ,Ronald Szymusiak  a , c , Dennis McGinty a , b , ∗ a  Research Service, V.A. Greater Los Angeles Healthcare System, North Hills, CA 91343, USA b  Department of Psychology, University of California, Los Angeles, CA 90024, USA c  Department of Medicine, University of California, Los Angeles, CA 90024, USA d  A.B. Kogan Research Institute for Neurocybernetics, Rostov State University, Rostov-on-Don, Russia Received 22 May 2007; received in revised form 9 June 2007; accepted 12 June 2007 Abstract This study measured cell proliferation in the hippocampal dentate gyrus in the adult rat at different times within a 12:12h light–dark cycle. Theexperiments were conducted in animals living in either a complex environment or in standard lab cages. A single dose of the thymidine analog5-Bromo-2  -deoxyuridine (BrdU) was injected 2h before animals were sacrificed either 4, 11, 16, or 23h after the beginning of the light phaseof the light–dark cycle (designated ZT0). In both studies, we found a significant increase in the number of BrdU-positive cells in the subgranularcell layer (SGZ) following BrdU administration at ZT9 and sacrifice at ZT11, compared to other circadian times examined. BrdU administrationat ZT9 was timed to primarily identify proliferating cells that were in the S phase of the cell cycle during the light phase. Our results suggest thatcell proliferation is enhanced either by sleep or by other variables coupled to the light phase of the circadian cycle.Published by Elsevier Ireland Ltd. Keywords:  Cell proliferation; Dentate gyrus; Hippocampus; Circadian; BrdU The proliferation and maturation of new neurons in the dentategyrus (DG) in adult mammals has been shown to play a rolein certain hippocampal-dependent cognitive capabilities [13].For this reason, the identification of physiological and experi-ential processes that act as positive or negative modulators of cell proliferation and maturation is of importance. Sustainedsleep deprivation and sleep fragmentation have been shown tobe among the potent negative modulators of proliferation [5,7].However, there have been two interpretations of these findings.Ononehand,ithasbeenarguedthatsleep-relatedprocesseshavepro-proliferativeeffects,relatedtootheranabolicandplasticity-related functions associated with sleep [6]. Alternatively, it has been argued that application of sleep deprivation proceduresmay be stressful, and that the effects of sleep deprivation canbe explained as in terms of stress [15]. One type of data that is relevant to this issue in the distribution proliferation within the ∗ Correspondingauthorat:ResearchService(151A3),V.A.GreaterLosAnge-les Healthcare System, 16111 Plummer Street, North Hills, CA 91343, USA.Tel.: +1 818 891 7711x7579; fax: +1 818 895 9575.  E-mail address:  dmcginty@ucla.edu (D. McGinty). 24h light/dark cycle. Since the rat and mouse species used formoststudieshavemoresleepoccurringduringthelightphaseof the 24h day, a hypothesis that sleep has pro-proliferative effectsleads to a prediction that more proliferation would occur in thelight phase. However, three studies have provided evidence thatthereisnocircadianpatternofproliferation[1,9,17]andafourth found increased proliferation during the light phase only in thehilus of the DG [10]. We have re-examined this issue, consider- ing that only one previous study was done in rats, which wereused in most sleep deprivation studies. We initially studied ratsin which we attempted to reinforce circadian rhythmicity byproviding an enriched environment to stimulate nocturnal activ-ity, and places to provide dark shelters during the light. Whenwe observed circadian rhythmicity in proliferation under theseconditions, we also studied standard cage conditions.AllprotocolswereconductedinaccordancewiththeNationalResearch Council Guide for the Care and Use of LaboratoryAnimals and were reviewed and approved by the Animal Careand Use Committee at the V.A.G.L.A.H.S.MaleSprague–Dawleyratsofapproximately2monthsofage( ∼ 300g) were used in this study. Animals were kept in pairs 0304-3940/$ – see front matter. Published by Elsevier Ireland Ltd.doi:10.1016/j.neulet.2007.06.022   R. Guzman-Marin et al. / Neuroscience Letters 422 (2007) 198–201  199 in a sound proof chamber at 23 ◦ C under artificial 12h light(rest period) and 12h dark (activity period) cycle with lights onat 6 a.m. (designated ZT0, zeitgeber time, in accordance withcircadian conventions), and with food and water available  ad libitum . These conditions were maintained for 2 weeks beforethe experiments were performed, in order to ensure that the ani-mals were entrained to the light–dark cycle. The cages whereanimalswherehousedwereservicedatthebeginningofthedark period.  Experiment 1 : Light–dark cycle variations of cell prolifera-tioninratslivinginenrichedenvironment.Atotalof20animalswereusedinthisexperiment.Rats,twopercage,werehousedina large cage (54cm × 58cm × 60cm). This cage was arrangedwith nesting material, plastic tubes, which provided dark nestsites, and soft plastic toys. A new toy was introduced in the cageat the beginning of the dark period every day for 7 days. Stan-dard rat chow was supplemented with wheat-based cereal (fruitloops). The thymidine analog, 5-bromo-2-deoxyuridine (BrdU,Sigma–Aldrich, 300mg/kg) was injected i.p. 2h before animalswere sacrificed either 4, 11, 16, or 23h after the beginning of the light phase.  Experiment 2 : Light–dark cycle differences in cell prolifera-tion in rats living in standard cages. A total of 19 rats were usedin this experiment. In this experiment animals were housed, twoper cage, in Plexiglas cages (27cm × 29cm × 30cm) withoutenrichment. Standard food was introduced at the beginning of the dark period and animals were sacrificed at ZT11 and ZT23,2h after administration of BrdU (300mg/Kg).Subjects from all groups were deeply anesthetized (Nembu-tal 100mg/kg), perfused transcardially with PB 0.1M followedby ice cold paraformaldehyde (4%); brains were removed andstored in 10% and 30% sucrose at 4 ◦ C until they sank. Brainswere cut in 40  m coronal sections. Sections encompassingthe hippocampus were preserved in a cryoprotectant solutioncontainingsucrose,polyvinyl-pyrrolidone(PVP-40,Sigma)andethylene glycol dissolved in PB pH 7.2, which provides long-term protection of the tissue. Sections were processed for BrdUimmunohistochemistry.To visualize the expression of the BrdU we used the perox-idase method (ABC system, Vectastain, Vector Laboratories).Immunohistochemistry was performed simultaneously on sec-tions from different time points to maximize the reliability of comparisons across groups. Staining was performed on slide-mountedsections.Aone-in-sixseriesofsectionswaspretreatedfor BrdU immunostaining by DNA denaturation (2M HCl at37 ◦ C for 30min) followed by 10min in borate buffer (pH8.5). Tissue was rinsed in TBS 0.1M. Sections were then incu-bated with a mouse anti-BrdU (BD Biosciences 1:400) primaryantibody for 48h. Tissue from both groups was treated withaliquots from the same batch of antibodies. Sections were sub-sequently incubated with a biotinylated horse anti-mouse IgG(1:200, Vector Laboratories), then reacted with avidin–biotincomplex(1:100,VectorElite)anddevelopedwithdiaminobenzi-dine tetrahydrochloride (DAB, Sigma). Absence of the primaryantibody resulted in an absence of specific nuclear staining.BrdU-positive cells were counted using a 40 ×  objective(Nikon E600) throughout the rostrocaudal extent of the DGgranule cell layer. The optical fractionator method was used forcounting as previously described [5]. Stereo Investigator soft- ware was used to estimate the total number of BrdU-positivecells per DG by utilizing the optical fractionator formula.To study distribution of newborn cells, their numbers werecountedseparatelyperhippocampalsubregions,i.e.thegranulecelllayer(GCL)/subgranularzone(SGZ)andthehilus.TheSGZwas defined as a two-cell thick layer along the inner border of the GCL and the hilus.Differencesincellcountsbetweengroupswereassessedwithone-way ANOVA followed by Fisher LSD post hoc test or Stu-dents’  t  -test for a two-group comparison. A  P  value of <0.05was adopted for significance.  Experiment 1 : We first quantified the number of labeled cellswithinSGZ/GCLfollowingBrdUexposureatdifferentcircadiantimes in rats living in an enriched environment. The num-ber of BrdU+ cells significantly varied across circadian times( F  (3,16)=6.3,  P <0.01, one-way ANOVA). As it is shown inFig. 1, cell proliferation was the highest in animals sacrificedat ZT11 (the end of the light phase) and at its minimum in ani-malssacrificed at ZT23 (theend of thedark phase). At ZT11 thenumber of BrdU+ cells was significantly higher than at the restof circadian times tested ( P =0.001 for ZT23,  P <0.01 for ZT16and  P =0.01 for ZT 4, Fisher LSDtest). The remaining pairwisecomparisons did not reveal statistically significant differences.In the hilus (Fig. 1), the numbers of BrdU+ cells were not significantly different among the circadian times examined( F  (3,16)=1.6,  P =0.2).  Experiment 2 : We quantified the rate of proliferation in stan-dard cage conditions after BrdU exposure at two time points,ZT11 and ZT23, where the highest and lowest number of BrdU+ cells were seen under enriched conditions (Fig. 2). We found a higher number of BrdU+ cells at ZT11 in both theGCL/SGZ ( P <0.01,  t  =3.0, d.f.=17) and the hilus ( P <0.05, t  =2.3, d.f.=17).Our study was designed to measure proliferation at timesof maximum and minimum amounts of sleep within the 24hperiod, considering the concept that BrdU labels proliferatingcells in the S-phase, which is estimated to last 9h in the adultrat, and BrdU exposure after administration is limited to 2h[3]. BrdU is available for uptake by cells in the S phase of thecell cycle for 2h following IP administration [16]. Moreover, Fig. 1. In rats living in an enriched environment, the numbers of proliferatingcells in the SGZ and GCL identified by BrdU labeling was dependent on thetime of BrdU administration within the light–dark cycle. Labeled cell countswere significantly higher after BrdU administration at ZT9 and animals weresacrificed at ZT11, at the end of the light phase of the light–dark cycle. Therewas no significant effect of circadian time on labeling in the hilus. **,  P <0.01.  200  R. Guzman-Marin et al. / Neuroscience Letters 422 (2007) 198–201 Fig. 2. In rats living in standard cage conditions, the numbers of proliferatingcells identified by BrdU labeling were significantly higher in both SGZ/GLCand hilus when animals were injected with BrdU at ZT9 and sacrificed at ZT11compared to ZT21/23 schedule. *,  P <0.05. recently it has been shown that minimum time of BrdU avail-abilityforincorporationintoDNAislessthan15min[14].Thus, BrdU labeled cell counts obtained after administration at ZT9andsacrificeatZT11identifiesproliferationduringthefirst11hofthelightcycle,andBrdUadministrationatZT21andsacrificeat ZT23 identifies proliferation during the dark phase. In con-trast to previous studies, our studies showed significantly higherproliferation in animals sacrificed at ZT11 under both standardand enriched caging conditions. This finding is congruent withthe hypothesis that proliferation is increased either during sleepor by other circadian variables associated with the light phase.Potential stress-inducing factors were minimized.In the first experiment we found a peak in proliferationonly when BrdU was administered at ZT9 (sacrifice at ZT11),although BrdU administration at ZT2 or ZT14 would also iden-tify proliferation during parts of the light phase. This findingsuggests that proliferation may peak during a restricted part of the light phase, but further work is needed to confirm this pos-sibility. Circadian variations in the bioavailability of BrdU foruptake by proliferating cells after IP administration have notbeen described, but we cannot rule out this possibility.Several factors could account for the differences between thepresentstudyandpreviousstudies.Threeofthefourstudiesusedpreviouslywereconductedinmice[9,10,17].Eitherspeciesdif- ferencesorothermethodologicaldetailscouldbeimportant.TheS-phase of the cell cycle duration has been shown to be shorterin mice [8] than in rats [3]. Thus, single BrdU injections will sample a more limited part of the circadian cycle. In one mousestudy, there was increased proliferation during the light phasein the hilus of the DG [10], a finding replicated in our standard housing study. Another mouse study was based on labeling bythe endogenous marker of cell proliferation, Ki-67 which labelsall phases of the cell cycle except G0 [17]. As the mouse DG proliferating cell cycle duration is 14h [14], this marker may fail to reveal shorter-term variations in proliferation, as notedpreviously [10]. Another mouse study [9] was based on daily BrdU injections for seven consecutive days before sacrifice, soproliferationcountscouldbeinfluencedbyfactorsaffectingsur-vival of proliferating cells over the 7-day study period. The ratstudy [1] also found no significant circadian variation in DGcell proliferation. However, this study used a 24h survival timepost-administration of BrdU, which makes the cohort of prolif-erating cells susceptible to other influences in the period afterBrdU injection.We also note that in the present study servicing was doneand new food was introduced into the cages at the beginning of the dark period. It has been shown that food presentation canalter circadian entrainment in rats [11]. Food availability was not restricted in our study.Circadianrhythmicityinproliferationmayresultfromeffectsof circadian gene expression on the cell cycle. The expressionof the circadian gene,  Per2 , is strongly expressed in the DG[12], and, was reported to have permissive inhibitory role oncell proliferation [4]. Indeed, it has been reported that increases incellproliferationintheDGwerecoincidentwithreductionsin Per2  expression in this region [2]. In adult rats the lowest point inthecircadianvariationofexpressionof  Per2 intheDGoccursat ZT10 [12], about the time we found maximal proliferation. Our study has methodological implications for studies mea-suring proliferation in the DG. First, measurement of the rateof proliferation is strongly dependent on the timing of BrdUadministration within the circadian cycle. Researchers typicallycarefully control the timing of light–dark exposure of experi-mental groups and match the circadian timing of sacrifice of experimental groups. However, animal servicing proceduresor experimental manipulations that differentially alter experi-mental and control groups with respect to entrainment to thelight–dark cycle could affect the outcome of experiments. Acknowledgements WegratefullyacknowledgeFengXuandKeng-TeeChewfortheir excellent assistance. This research was made possible bya grant from the American Sleep Medicine Foundation, a foun-dation of the American Academy of Sleep Medicine to RGM.Supported by the US Department of Veterans Affairs MedicalResearch service and US National Institutes of Health grantsNIMH 075076, HL 60296. References [1] P. Ambrogini, L. Orsini, C. Mancini, P. Ferri, I. Barbanti, R. Cuppini,Persistently high corticosterone levels but not normal circadian fluctua-tions of the hormone affect cell proliferation in the adult rat dentate gyrus,Neuroendocrinology 76 (2002) 366–372.[2] L. Borgs, B. Malgrange, L. Nguyen, G. Hans, J. Mangin, G. Moonen,P. Maquet, U. Albrecht, S. Belachew, The circadian gene  Per2  controlsprogenitor cell proliferation and neurogenesis in the adult hippocampusProgram No. 607.1. 2004 Abstract Viewer/Itinerary Planner, Society forNeuroscience, Washington, DC, 2004.[3] H.A. Cameron, R.D.G. McKay, Adult neurogenesis produces a large poolof new granule cells in the dentate gyrus, J. Comp. Neurol. 435 (2001)406–417.[4] L. Fu, H. Pelicano, J. Liu, P. Huang, C. Lee, The circadian gene Period2plyas an important role in tumor suppression and DNA damage responsein vivo, Cell 111 (2002) 41–50.[5] R. Guzman-Marin, N. Suntsova, D.R. Stewart, R. Szymusiak, D. McGinty,Sleep deprivation reduces proliferation of cells in the dentate gyrus of thehippocampus in rats, J. Physiol. 549 (2003) 563–571.[6] R. Guzman-Marin, D. McGinty, Sleep deprivation suppresses adult neuro-genesis: clues to the role of sleep in brain plasticity, Sleep Biol. Rhythms4 (2006) 27–34.[7] R. Guzman-Marin, T. Bashir, N. Suntsova, R. Szymusiak, D. McGinty,Adult hippocampal neurogenesis is reduced by sleep fragmentation in theadult rat, Neuroscience, in press.   R. Guzman-Marin et al. / Neuroscience Letters 422 (2007) 198–201  201[8] N.L. Hayes, R.S. Nowarkowski, Dynamics of cell proliferation in the adultdentategyrusoftwoinbredstrainsofmice,BrainRes.Dev.BrainRes.134(2002) 77–85.[9] M.M. Holmes, L.A. Galea, R.E. Mistlberger, G. Kempermann, Adulthippocampal neurogenesis and voluntary running activity: circadian anddose-dependent effects, J. Neurosci. Res. 76 (2004) 216–222.[10] L.J. Kochman, E.T. Weber, F.A. Fornal, B.L. Jacobs, Circadian variationin mouse hippocampal cell proliferation, Neurosci. Lett. 406 (2006) 256–259.[11] D.T. Krieger, H. Houser, Comparison of synchronization of circadian cor-ticosteroidrhythmsbyphotoperiodandfood,Proc.Natl.Acad.Sci.U.S.A.75 (1978) 1577–1581.[12] E.W. Lamont, B. Robinson, J. Stewart, S. Amir, The central and basolat-eral nuclei of the amygdala exhibit opposite diurnal rhythms of expressionof the clock protein Period2, Proc. Natl. Acad. Sci. U.S.A. 102 (2005)4180–4184.[13] P.M. Lledo, M. Alonso, M. Grubb, Adult neurogenesis and functionalplasticity in neuronal circuits, Nat. Rev. Neurosci. 7 (2006) 179–193.[14] C.D. Mandyam, G.C. Harburg, A.J. Eisch, Determination of key aspectsof precursor cell proliferation, cell cycle length and kinetics in the adultmouse subgranular zone, Neuroscience 146 (2007) 108–122.[15] C. Mirescu, J.D. Peters, L. Noiman, E. Gould, Sleep deprivation inhibitsadult neurogenesis in the hippocampus by elevating glucocorticoids, Proc.Natl. Acad. Sci. U.S.A. 103 (2006) 19170–19175.[16] R.S.Nowakowski,S.B.Lwein,M.W.Miller,Bromodeoxyuridineimmuno-histochemical determination of the lengths of the cell cycle and theDNA-synthetic phase for an anatomically defined population, J. Neuro-cytol. 18 (1989) 311–318.[17] K.VanderBorght,F.Ferrari,K.Klauke,V.Roman,R.Havekes,A.Sgoifo,E.A.vanderZee,P.Meerlo,Hippocampalcellproliferationacrosstheday:increase by running wheel activity, but no effect of sleep and wakefulness,Behav. Brain Res. 167 (2006) 36–41.
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