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Undergraduate Category:PhysicalandLifeSciences DegreeLevel:BachelorofScience AbstractID#:1060 EntrainmentoftheFetalCircadianClockbyTemperatureCycles KaterinaA.ToroTorres,ShaneRoesemann,NoelleCas6lla-Ojo,MeganCavanough,RyanMargolin,VictoriaKing,andFredDavis DepartmentofBiology,CollegeofScience Abstract: Fig.3.Eachfetushas2SCN(onefromeachsideofthebrain) thatareinsynchronywhenexposedtothesamecondi6ons. TherhythmsbelowwereexpressedbySCNfromthesame fetusandexposedtothesameconstanttemperature condi6onsimmediatelyaUerdissec6on. Circadianrhythmsare24-hourrhythmsinbehavior,physiology,andbiochemistryaffec6ngprocessesfrom geneexpression,tothe6mingofsleepandwakefulness.Inthecellsofhumans,andotheranimals, circadianrhythmsaregeneratedbyinterac6onsamongafewkeygenesandtheirproteinproducts.While thesemolecular“clocks”areineverycell,aspecializedregionofthebrain,theSuprachiasma6cNucleus (SCN),isessen6alforcoordina6ngrhythmsincellsand6ssuesthroughoutthebody.Thiscoordinated 6mingisoUenreferredtoas“theCircadianSystem.”Disrup6onsoftheCircadianSystem,asaresultof shiUwork,forexample,arecorrelatedwitharangeofdetrimentalcondi6ons,fromsleepdisorders,toan increasedriskofcancer,andcertainmetabolicdiseases. Ourprojectisrelatedtothegeneralhypothesisthatduringdevelopment,amammal,suchasthehuman fetus,isacomponentofthemother’scircadiansystem.Morespecifically,wetestedthehypothesisthat thefetalcircadianclockisentrainedbytemperaturecycles,acuethoughttobeimportantformaintaining circadianorganiza6oninadults.WeisolatedtheSCNoffetalmiceandmonitoredmolecularrhythms,while exposingtheSCNtodifferenttemperaturecycles.Theresultsshowedthatthefetalcircadianclock,atan ageequivalenttomid-gesta6oninhumans,isstronglyinfluencedbytemperaturecyclessimilartohuman bodytemperaturerhythms.Ourresultssupportthehypothesisthatfetalcircadianclocksarenormally entrainedbymaternalrhythms.Disrup6onofmaternalcircadianrhythmscouldhavenega6ve consequencesfornormaldevelopmentandpregnancy. Goal: Ourgoalistotestthehypothesisthat24-hourtemperaturecyclesentrainthefetalcircadianclock(the SCN).Ourlonger-termgoalistodetermineifthedevelopmentofcircadianrhythmsisaffectedby prenatalcondi6ons,includingrhythmsinmaternalphysiology,suchasbodytemperaturerhythms. Fig.4.FetalSCNareentrainedby24-hourtemperature cycles(12hoursat35.5°Cand12hoursat39.5°C).The rhythmsbelowwereexpressedbytwoSCNfromthesame fetusexposedtodifferenttemperaturecyclesfor5days beforerecording.Therhythmsshowoppositephasessimilar tothe12-hourdifferencebetweentemperaturecycles. Methods: Suprachiasma6cnucleus(SCN)wereremovedfromfetalknock-inper2::lucmice. Per2::lucmiceexpressareportergeneforthebiologicalclock.Thiscyclingcanbe detected(withaLumiCycle)byquan6fica6onoftheexpressionofthe bioluminescentreporterprotein(Luciferin).Luciferasepresenceisrequiredfor proteinexpression.Thisbioluminescencewasquan6fiedandfurtheranalyzed.All 6ssuesampleswerekeptinnutrientrichmediafortheen6retyoftheexperiment andreplacedwithmediawithLuciferasebeforeinputintotheLumiCycle. 1. TheSCNFetalmicesacrificedday18ofgesta6onweredissectedandplacedintwo differentincubatorsofpre-settemperatures,highandlow,withaone-degree difference. 2. TheSCNweresubjectedtoeachcondi6on,alterna6ngevery12hoursforthree subsequentdaysaUerdissec6on.OnesetofSCNwereplacedinhightemperature duringtheday,whiletheotherathightemperatureduringthenight.Thisallowed fortemperatureentrainmentofthebiologicalclock. 3. Followingentrainment,thesampleswereplacedintheLumi-Cyclefor bioluminescencequan6fica6on,datacollec6on,andfurtheranalysis. 4. SuccessfulentrainmentofthefetalSCNresultsinoppositecyclesinthetwogroups (high-temperatureday,hightemperaturenight).ControlSCNwereplacedinthe machine(6mezero)tomonitorrhythmspre-temperatureentrainment. Fig.2.Circadianrhythmsintheabsence ofalight-darkcycle,expressingtheir innate“circa”24-hourperiod.Alsoknown as“free-running”rhythms. Fig.1.TheSuprachiasma6cNucleus(SCN). Background: Fig.5.FetalSCNarepar6allyentrainedbyweak24-hour temperaturecycles(12hoursat35.75°Cand12hoursat 36.75°C).TherhythmsbelowwereexpressedbytwoSCN fromthesamefetusexposedtodifferenttemperaturecycles for3daysbeforerecording.TherhythmshadnotyetshiUed tomatchthethe12-hourdifferencebetweentemperature cycles. TheCircadianSystem Circadianrhythmsare24-hourrhythmsinbehavior,physiology,andbiochemistrywhichmanifestinfunc6onsfromgeneexpressiontothe6ming ofsleepandwakefulness.Inhumansandotheranimals,circadianrhythmsaregeneratedthroughinterac6onsamongseveralkeygenes,andtheir proteinproducts,incellsand6ssuesthroughoutthebody.Circadian6mingisnowrecognizedasamajorregulatorofnormalcellularhomeostasis. Althoughmolecularclocksareineverycell,aspecializedregionofthebrain,theSuprachiasma6cnucleus(SCN),isessen6alforthecoordina6on amongrhythmsinother6ssuesandorgans(Fig.1).Thiscoordinated6mingisoUenreferredtoas“theCircadianSystem.”Disrup6onsofthe CircadianSystem,asaresultofshiUworkforexample,arecorrelatedwitharangeofdetrimentaleffectsfromsleepdisorderstoincreasedriskof cancerandmetabolicdisease. Thelight/darkcycleisthepredominantexternalentrainingsignalforbehavioralrhythms,ac6ngthroughthere6naanddirectneuralconnec6ons totheSCN.Intheabsenceoflight/darkcycles,circadianrhythms“free-run”,expressingtheinnatecirca-24-hourperiodoftheclockwithinthe SCN(Fig.2).Lessisknownabouttheinternalsignalsthatmediateentrainmentofthecircadiansystem.Thenervousandendocrinesystemsare probablyinvolved,butthebodytemperaturerhythmisalsothoughttobeimportant. Conclusions: • The dominant circadian clock in the brain, the Suprachiasmatic nucleus (SCN), begins to generate rhythms during prenatal development • Development of the circadian clock in mice suggests that the circadian clock also develops several weeks before before birth in humans • The SCN from each side of the fetal brain have similar properties • The fetal SCN of mice are entrained by temperature cycles in vitro • Entrainment by temperature cycles in vitro indicate that the body temperature rhythm of the mother would be sufficient to entrain the fetal clock • Entrainment of the fetal clock in vitro indicates that the maternal and fetal circadian would be in synchrony during development (see Fig. 7) • The human fetus is likely to have functioning circadian clocks that are integrated (entrained) into the maternal circadian system Development Fig.6.Thesmallcirclesandtrianglesindicatethe6mingoffetalSCNrhythmsrela6veto temperaturecyclesexperiencedinvitro.Duringthecycles,temperaturewashighatnight (8PMto8AM)orduringtheday(8AMto8PM).Eachsymbolrepresentsthe6mingfora singlefetalSCN.AstrongtemperaturecyclecausedcompleteseparaEonofrhythms,while aweakcycleonlyparEallyresettherhythms. Fig.7.IntheadultSCN,PER2expression risesduringthecoolphaseofthethebody temperaturerhythm.Whenentrainedbya temperaturerhythm,PER2expressioninthe fetalSCNalsorisesduringthecoolphase. Muchlessisknownaboutthecircadiansystemduringdevelopment.When,forexample,dorhythmsdevelopandwhen,orhow,aretheyfirst entrained?EvidenceindicatesthattheSCN,andpossiblyotherclocks,begintofunc6onbeforebirth.Ifso,thenitmaybeappropriatetothinkof themotherandfetusaspartofthesamecircadiansystem.WehavebeenstudyingthedevelopmentoftheSCNinmicetodeterminewhenit beginstogeneraterhythmsandif,aUertheyappear,theyareentrainedbythemother’scircadiansystem.Inpar6cular,wearetes6ngthe hypothesisthattemperaturecycles(mimickingthebodytemperaturerhythm)canentrainthefetalSCN.Iffetalcircadianrhythmsarepartofthe maternalcircadiansystem,thendisrup6onsofthecircadiansystemand/orthesignalsthatkeepitcoordinatedcouldhavedetrimentaleffectson pregnancyanddevelopment,astheydoforadults. References: Bae,K.,etal.(2001)."Differen6alfunc6onsofmPer1,mPer2,andmPer3intheSCNcircadianclock."Neuron30(2):525-536.