Download Entrainment of the Fetal Circadian Clock by Temperature Cycles

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.