Probing arousal to better understand sleep defects in Drosophila

13 Pages·2015·3.64 MB·English

How deeply does your mutant sleep?
OPEN
Probing arousal to better understand
SUBJECTAREAS:
sleep defects in Drosophila
GENETICSRESEARCH
SLEEP
R.Faville1*,B.Kottler1*,G.J.Goodhill1,2,P.J.Shaw3&B.vanSwinderen1
Received
27August2014
1QueenslandBrainInstitute,TheUniversityofQueensland,Brisbane,Australia,2SchoolofMathematicsandPhysics,TheUniversity
Accepted ofQueensland,Brisbane,Australia,3WashingtonUniversitySchoolofMedicine,St.Louis,Missouri,USA.
15January2015
Published Thefruitfly,Drosophilamelanogaster,hasbecomeacriticalmodelsystemforinvestigatingsleepfunctions.
13February2015 Moststudiesusedurationofinactivitytomeasuresleep.However,adefiningcriterionforsleepisdecreased
behavioralresponsivenesstostimuli.HereweintroducetheDrosophilaARousalTrackingsystem(DART),
anintegratedplatformforefficientlytrackingandprobingarousallevelsinanimals.Thisvideo-based
platformdeliverspositionalandlocomotiondata,behavioralresponsivenesstostimuli,sleepintensity
Correspondenceand measures,andhomeostaticregulationeffects–allinonecombinedsystem.Weshowhowinsightinto
requestsformaterials dynamicallychangingarousalthresholdsiscrucialforanysleepstudyinflies.Wefirstfindthatarousal
shouldbeaddressedto probinguncoversdifferentsleepintensityprofilesamongrelatedgeneticbackgroundstrainspreviously
assumedtohaveequivalentsleeppatterns.Wethenshowhowsleepdurationandsleepintensitycanbe
B.v.S.
uncoupled,withdistinctmanipulationsofdopaminefunctionproducingoppositeeffectsonsleepduration
(b.vanswinderen@uq.
butsimilarsleepintensitydefects.Weconcludebyprovidingamulti-dimensionalassessmentofcombined
edu.au) arousalandlocomotionmetricsinthemutantandbackgroundstrains.Ourapproachopensthedoorfor
deeperinsightsintomechanismsofsleepregulationandprovidesanewmethodforinvestigatingtheroleof
differentgeneticmanipulationsincontrollingsleepandarousal.
*Theseauthors
contributedequallyto
thiswork. A betterunderstandingofthefunctionsofsleeprequiresbettermethodstostudysleepinmodelorganisms.
ThediscoveryoveradecadeagothatDrosophilafliessleep1,2hashadaprofoundimpactonthesleepfield3.
ThisisbecausethepowerfulgenetictoolsthathavemadeDrosophilaasuccessfulmodeltostudyneuronal
functioncouldnowbeappliedtounderstandingthefunctionsofsleep.Thereforekeyhypothesesandtheorieson
thefunctionsofsleep,suchasaroleinsynaptichomeostasis4–6couldbeinvestigatedinageneticallymalleable
modelorganism.Inaddition,agrowingnumberofresearchgroupsareturningtothisrelativelysimplebehavioral
readoutinDrosophilatocharacterizegenefunctionsandneuralcircuits7,separatefromanydirectinvestigationof
sleeporbrainfunctions.Sleepislikelytorecruitawidediversityofgeneticandneuronalpathwaysthatmaybe
more extensive than those controlling circadian rhythms, so sleep measures are highly likely to provide a
behavioralphenotypeforabroadvarietyofgeneticinvestigations.
OnereasonwhysleepstudiesinDrosophilahavebeensosuccessfulisbecauseexactlythesameparadigmsthat
have been used to monitor diurnal activity levels and circadian rhythms have been adapted to study sleep8.
Circadian rhythms in Drosophila have traditionally been measured using infrared beam interruption devices
(Trikinetics,Waltham,MA),whereasinglebeambisectinga65 mmtubeissufficienttodetecttheactivityofflies
housedindividuallyintubes(fliestendtowalkbackandforththelengthofthetube,thusbreakingtheinfrared
beamonceineverycrossing).Earlysleepstudiesrevealedthatfiveminuteswithoutbeam-crossingswasassoc-
iatedwithprolongedinactivityandincreasedarousalthresholds,suggestingasleep-likestate1.Subsequentstudies
confirmedthat5 minormoreofinactivitywasindeedareliableindicatorofsleepinDrosophila,atleastinwild-
typeflies8,9.AlmosteveryDrosophilasleepstudyhasthususedthis5 mincutoffasacriterionforquantifyingsleep
metrics,andmoststilluseinfraredactivitymonitors,althoughtherehasbeenarecentmovetovideotracking10–12.
However,athoroughgeneticinvestigationofsleeprequiresre-evaluationofarousalthresholdsindifferent
strains;behavioralresponsivenesslevelsindifferentmutantswillnotnecessarilyfollowthepatternshownbya
wild-type strain. In addition, sleep metrics are not restricted only to duration data, but should also consider
ongoingchangesinbehavioralresponsivenessandsleepintensity,inordertofullycapturesleepfunctionsin
differentstrains.Forexample,along-sleepingflymaybesleepinglightly,orashortsleepingflymightbesleeping
deeply;activitymonitorsandsimplewebcaminterfacescannotdistinguishbetweenthesepossibilities.Indeed,
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ongoingtrackingofanimalsandactiveprobingofarousallevelswith a variety of behavioral readouts together characterize sleep and
mechanical stimuli has revealed that flies may sleep in distinct arousalindifferentstrains.DARTthereforealsoprovidesapowerful
intensity stages, and that their arousal levels change throughout methodtomapdifferentgeneticvariantsinphenotypicspace.
thedayandnight,evenwithinasinglesleepbout13.Amajorchal-
lengeforthefieldhasbeentoprovideoneintegratedplatformthat
Results
examinesacomprehensivesetofsleepmetrics,fromvideotracking
Experimental setup and fly tracking. Adult virgin female
tocontrollingaprobestimulusandtestingsleep-deprivationeffects,
Drosophilamelanogasterflieswereloadedindividuallyinto65 mm
tofinallyprovidingprocessedlocomotion,arousal,andsleepintens-
glasstubes(Trikinetics)thatwerepluggedatoneendwithstandard
itydataattheend.Ideally,thisshouldbepossibleusingequipment
yeast-basedflyfood(seeMaterialsandMethods).Loadedtubeswere
andcomputationalpowerthatisreadilyavailabletoanyDrosophila
lab. Here, we describe such a platform, the Drosophila ARousal alignedonacustom-madeplasticplatform(17tubesperplatform),
Trackingsystem(DART). andmultipleplatforms (up to6atatime)placedonastagetobe
WeillustratethepowerofDARTbyexaminingkeymeasuresin filmed(Fig.1a),ina25uincubator(TritechResearch).Twoshaft-less
thestudyofDrosophilasleep,focusingonasetofgeneticvariants. motors(PrecisionMicrodrives)weregluedtotheundersideofeach
Weexaminetwocommonlyusedgeneticbackgroundstrains,aswell platform, and vibrations made by these motors were controlled
as two mutants predicted to have opposing sleep phenotypes. by the DART software interface, via a digital-to-analog convertor
Controllingforgeneticbackgroundeffectsisacrucialaspectofany (Measurement Computing). The DART interface was also used to
Drosophilabehavioralstudy.Yetoutcrossingtoacommongenetic record fly activity using a USB-webcam (Logitech) (Fig. 1a; and
backgroundcanbeadouble-edgedsword,asthismayintroducea Materials and Methods). Fly locations were determined by an
distinct set of behavioral effects. This is especially true for sleep image subtraction algorithm (Fig. 1b; Fig. S1; see Materials and
studies:forexample,exceptfortheCantonSstrain,fewbackground Methods), and quality control for tracking was performed via a
strainshavebeenwellcharacterizedforarousalthresholds1,9,13.We graphicaluser interface (Fig. 1b; Fig.S1). Whileourcurrent study
uncoverdifferentarousalthresholdsandsleepprofilesintheback- is adapted around an established preparation to study sleep in
groundstrains,andwerevealunexpectedsleepeffectsinthemutants Drosophila (single flies in narrow glass chambers), DART will
that are invisible without actively probing for arousal. Finally, we work with any chamber dimensions, different animal sizes, or
proposeamulti-dimensionalscalingapproachtobetterassesshow multipleanimalsperchamber.
Figure1|TheDrosophilaARousalTracking(DART)system.(a).Left:DARTsoftwarerecordsmoviesviaawebcamwhilesimultaneouslycontrolling
motorstimuliviaadigitaltoanalogconverter(DAC).Themultiplemotors(representedbyreddashedcircles)areconnectedtotheDACviaa
simplecircuitboard.Middle:Sixplatformsarepositionedina332arrangementof17fliesperplatform,withtwomotorsperplatform.Right:
Eachplatformhastwomotorsgluedunderneath.Theplatformhasfourpedestals(clearcircles)thatslotintoalargerbase.(b).Movementtracesfor17
fliesoverfourdays(yellowbackground)andnights(grey)outputtedfromDART.X-axisistimeofdayandy-axisishorizontaldisplacementinthe
tubeforeachfly.Thedifferentcolors(blue,red,greenpurple)areassignedsothattheindividualtracesareeasiertodistinguish.Graphicaluserinterface
supervisionallowsselectionofunusableflies:blacktraces(flies8and11)havebeenmarkedforremoval,asfliesappearedtohavedied.Artwork:
BenjaminKottlerandBrunovanSwinderen.
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Sleep tracking. To see whether our video-based strategy captured thresholds, 1 mm (the approximate length of a fly), 3 mm, and
basic sleep metrics as traditionally quantified by infrared beam 20 mm (equivalent to a beam-crossing experiment), and we kept
interruption devices8, we defined a virtual beam across the center thesleepcriterionat5 minimmobility(althoughthisvariablecan
ofeachtubeandcounted‘‘beam’’crossingsperminute(50%position alsobeadjusted).Notsurprisingly,wefoundthatforbothstrainsa
inFig.2a,seealsoMaterialsandMethods).Wetestedoursystemby 20 mm movement threshold strongly resembled the mid-position
examiningtwoclassicalgeneticbackgroundstrains,w1118andw2202 beamcrossingprofile,andthe1 mmthresholdrevealedsignificantly
(w1118 is a white-eyed Canton S strain, and w2202 is an isogenized lesssleep(Fig.2b,Fig.S2b,d,f,h),likelyduetohigh-resolutionjitter.
variant of this strain14, also known as isoCJ115). Most Drosophila We therefore chose 3 mm as a suitable threshold for subsequent
sleep studies define sleep as five minutes or more without any sleeptrackingexperiments.
beam crossing. Implementing this well-established sleep threshold Most animals prefer to sleep in defined locations16, and there is
in DART, we found that our video tracking system accurately some evidence that Drosophila flies prefer to sleep near a food
replicatedthesleepprofilesandassociatedmetricsfrompublished source2,12.SinceDARTaccuratelytracksflypositionsalongthetube,
datausingthetraditionaldevices1,8,althoughw1118appearedtosleep wenextinvestigatedwhetherthesewereunevenlydistributedacross
lessthanw2202(Fig.2a,Fig.S2). multipledaysandnightsinthetworelatedwhitebackgroundstrains.
Separating each fly tube into equal halves (50%) assumes that Wefoundthatflydistributioninthetubeswashighlyuneven,and
activity will be best reflected by the flies’ walking along the entire thatfliesindeedremainedclosertothefoodatspecifiedtimes,espe-
lengthofthetubeandback,whichtheytypicallydointhisconfined ciallyinthemiddleofthedayandthebeginningofthenight(Fig.2c,
context.However,flyactivitymaynotbeequallydistributedallalong Fig.S2i,j).However,thetwowhitestrainsbehaveddifferentlywhen
the tube length12, especially since food distribution is asymmetric tracked this way: while w1118 position preferences across 24 hours
(Fig. 2, top schemas). Variability in positioning the tubes in the weremoreevenlydistributedaroundthetubemidpoint,w2202spent
infrareddevicesmaythereforeleadtosomevariationinsleepand most of its time in the half closer to the food. Accordingly, w2202
locomotionphenotypes.Totestthis,wechangedthepositionofthe appears to sleep more than w1118 when analyzed by single-beam
virtualbeam,bringingitcloser(25%)orfurther(75%)fromthefood interruptionmethods(Fig.2a,Fig.S2).Clearly,othermeasuresare
(Fig.2a,topschema).Wefoundthatvariationinthepositionofthis required to ascertain whether w2202 indeed sleeps more than w1118,
virtualdetectorofflyactivitysignificantlyalteredsleepprofilesand becausew2202couldberemainingclosertothefoodforotherreasons.
associatedsleepmetricsinthetwowhite-eyed,wild-typebackground Asageneralrule,itappearsthatthe25%breakbettermatchesreal-
strains(Fig.2a;Fig.S2a,c,e,g).Positioningthebeamfurtheraway timemovementdatathanthe50%or75%break,forthesestrains.
fromthefoodoverestimatedsleepduration.
Sincefilmingallowscontinuousandaccuratetrackingofflymove- Arousal probing. Although ongoing locomotion changes and
ments,wenextdeterminedsleepprofilesbasedonabsolutelocation position preferences can be a good predictor of sleep behavior in
for these strains (Fig. 2b). We contrasted 3 different movement Drosophila, an under-studied method of measuring sleep is to
Figure2|Distinctsleepprofilesintworelatedwhitebackgroundstrains,w1118andw2202.(a).Classicalsleepprofilesderivedfromvirtualbeam-crossing
experiments.Fliesweretrackedcontinuously,butavirtual‘‘beam’’waspositionedinthreedifferentpositionsalongthe65mmtubeshousingindividual
flies(25%,50%,and75%)tosimulateactivityreadoutsfrominfrared-basedtrackingsystems.Sleepwasdefinedas5minormoreofinactivity
(nobeamcrossing1).Theapproximatesizeoftheflyisindicatedbyadarkovalinthetube.(b).Sleepprofilesderivedfromabsolutelocationdata,forboth
whitestrains.Sleepwasdefinedas5minormorewithoutmovement,whichwasestablishedfor3differentmovementthresholds(1mm,3mm,and
20mm).The20mmmovementthresholdisessentiallyequivalentindistancetoabeam-crossingexperiment,asina.(c).Heatmapofposition
preferencesinthetubesforbothwhitestrainsovermultipledaysandnights.Theindividualflytubesweredividedinto8bins,andpositionpreference
(%perunittimeoverallbins)wasplottedasaheatmap,withwarmercolorsindicatingahigherprobabilityoffliesbeinginthatposition.Redline
indicatesaveragepositionforthestrain.Thethickerwhitedashedlineindicatesthemid-pointinthetube.SeeFig.S2foradditionalinformationonsleep
metricsinthesestrains.N545forw1118andN546forw2202.ConfidenceintervalsareSEM.
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actively probe an animal’s responsiveness to stimuli17. Sleeping better defined in w2202 than w1118 (Fig. 3h, i, yellow and grey dots).
animals should have increased arousal thresholds, compared to Thisraisesthequestionofwhethersleepintensityisdifferentbetween
awake animals that are simply not moving. Previous work has thestrains,especiallyatnight.
shownthatmechanicalstimuliofvaryingintensitiescanbeusedto
probesleepintensityinDrosophila9,13.However, incontrastto the Sleep intensity. Sleep in Drosophila is not uniform; wild-type
widelyapplied5 mininactivitycriterion,behavioralresponsiveness (Canton S) flies sleep deeper during the night than during the
is not probed in most Drosophila sleep studies and no standard day13, even though extended bouts of inactivity are prevalent
methodology exists to quantify behavioral responsiveness in flies. duringthedayandnight1.Evenwithinasleepbout,sleepintensity
Weattachedsmallmotorstotheundersideoftheplasticplatforms levelshavebeenshowntovarydependingonhowlongtheflieshave
holding the fly tubes13, so that we could probe the flies’ respon- beenimmobile13.Inactivityboutdurationandthenumberofsleep
siveness to mechanical stimuli (Fig. 1a); the motors are controlled boutsperunittimerevealsomeaspectsofsleepconsolidation8,9,but
such duration-based metrics remain insufficient to address the
by the same DART interface, and can be programmed to deliver
question of sleep intensity. To measure sleep intensity, respon-
patterns of stimuli over several days. Although we use motor
sivenesslevels insleeping fliesmustbeacutelyprobedthroughout
vibrations in this study, any other device can be used to probe
a sleep bout, and correlated with how long flies have been asleep.
differentarousalmodalities(e.g.,light,sound,odors).
SincetheDARTplatformtracksflybehaviorcontinuously(andall
Wefirstprobedarousalthresholdsinthetwowhitebackground
moviesaresaved),itisthereforestraightforwardtoclassifyfliesinto
strains,bydeliveringstimulustrainsofincreasingvibrationintensity,
differenttemporalbinsdependingonhowlongtheywerecompletely
everyhouroverseveraldaysandnights(Fig.S3a,seeMaterialsand
immobilepriortoamechanicalstimulus(Fig.4a).Inourdatasetfor
Methods).Wefoundthatarousalthresholdsinquiescentfliesfrom
bothwhitestrains,mostflieswereimmobileforlessthan5 minprior
bothstrainsfollowedatypical diurnalprofile (Fig. S3b),asshown
to thehourly stimulus, but some were immobile for up to 40 min
previouslyforwild-typeflies13.Atnight,fliesfrombothstrainswere
(Fig.4b,c,leftpanels).Binningthedatabypriorimmobilityallowed
morelikelytosleepthrougheventhestrongestvibrations,resulting
us to ask the question: is there a relationship between how long a
inaceilingeffect.Basedontheseresults,wedesignedasimplepro-
strain has been asleep and its average behavioral responsiveness?
tocol to probe behavioral responsiveness every hour over several
And,isonestrainsleepingmoredeeplyonaveragethantheother?
successive days and nights (Fig. S3c, see Materials and Methods).
Wefoundthattheproportionoffliesrespondingtothestimulus
These hourly probes (a train of five 200 ms pulses set at 1.2 g)
decreasedgraduallyinthefirst15 minofimmobilityinbothwhite
decreasesleepslightly,becauseasubsetofsleepingflieswillbeawo-
strains,dayandnight(Fig.4b,c;middlepanels),whichisindicative
kenhourly(datanotshown).Moreimportantly,thehourlyprobes
ofincreasingsleepdepth.However,daytimesleepbecamelighterin
did not alter the distinct sleep profiles shown by the two strains,
bothstrainsasimmobilityboutslengthened,whilenighttimesleep
basedontraditional5 minsleepcriteria,withw2202apparentlystill
continued to become deeper with time immobile. In general, the
sleepingmorethanw1118(Fig.3a).Also,wefoundinapreviousstudy
averageamplitudeoftheresponse(asinFig.3d)mirroredthepopu-
thatthehourlystimulidonotcausesleepdeprivationeffects13.
lationresponsivenessproportion(Fig.4b,c,rightpanels;Fig.S4).Itis
We next investigated the flies’ responsiveness to the stimuli, to
clearfromtheseanalysesthatw2202issleepingmoredeeplythanw1118
better understand how sleep quality may differ in these strains.
duringthenight,i.e.,itislessresponsive(seeTablesS1–4forstat-
Simplyexaminingtheflies’locomotion(i.e.,speed)revealedimport-
istics).Duringtheday,bothstrainssleepequallylightly.Thus,sleep
antdifferencesbetweenthestrains:w2202isclearlysloweronaverage
intensitydifferencesaremuchmoreclearlypartitionedbetweenday
than w1118 (Fig. 3b). In addition, w2202 displayed clearer day/night
andnightinw2202,comparedtow1118,andsleepisalsodeeperatnight
differences compared to w1118, with strong responses during the
inw2202.
dayandminimalresponsivenessatnight.Incontrast,responsesin
w1118 seemed equally strong during the night and the day. Closer Sleepdisruption.Sleepisunderhomeostaticcontrol,andsomeof
examination confirmed this observation (Fig. 3c), and also con- the best evidence for sleep need in any animal is determined by
firmedthatbaselinespeedwasconsistentlygreaterinw1118,dayor measuringasleepreboundfollowingsleepdeprivation19,20.Asleep
night,withincreasingaveragespeedasthenightprogressed(Fig.3c). reboundshouldthenbefollowedbyareturntonormalbehavioral
Thus,bothvideotrackingandarousalprobingrevealsstrikingdif- performance, indicating that the sleep deprivation methods did
ferencesbetweenthewhitestrainsthatwerenotevidentusingtra- not physically damage the animal. Achieving the right level of
ditionalbeam-crossingmethods. gentle handling to interfere with sleep yet not cause damage or
unduestressisdifficult,andpreviousDrosophilasleepstudieshave
Theshapeoftheresponse.While population measures (as in Fig. used automated, motorized devices that jolt or rattle the flies
S3b) effectively describe behavioral responsiveness in Drosophila, awake9,19. Since the DART platform is already equipped with
anotherapproachtostudyingarousallevelsinfliesistoaveragethe computer-controlled motors to test behavioral responsiveness, we
response kinematics in a strain, as a fitted curve18. Averaging the programmed these to deliver random stimuli trains during
stimuli response across several days reveals a typical locomotion 12 hours of night (Fig. 5a, purple box; Fig. S5a, and see Materials
responsecurvecharacterizedbyseveralkeyparameters:theaverage and Methods). We found that this gentle, unpredictable stimulus
pre-stimulusspeed(VPre-Stimuli),theresponseamplitude(VAmplitude), pattern produced a sleep rebound the following day and the
which represents the difference between the pre-and post-stimuli followingnight(Fig.5b,c;magentaplots).Interestingly,traditional
speeds, and the response decay time constant (tInactivation) (Fig. 3d, sleep duration metrics were not significantly altered during the
see Materials and Methods). This response averaging approach random stimulation (Fig. S5b, c). This suggests that it was sleep
confirmed major differences between the two background strains: quality that was compromised, rather than simply sleep duration.
w1118 flies are equally responsive during the day and the night, Suchsleepdisruptioneffects(withoutaffectingsleepduration)can
whilew2202issignificantlymoreresponsiveduringthedaythanthe also lead to behavioral deficits21. Nevertheless, behavior was still
night (Fig. 3e–g). Plotting post-stimulus speed against pre-stimulus clearly affected during the stimulation protocol: average walking
speed for all the data provides a snapshot of the diurnal respon- speed was higher and fly location in the tubes was more variable
siveness of a strain (Fig. 3h, i): both strains were responsive to the comparedtobaseline(Fig.S5c,d).
hourlystimulus(asevidentbythepositivegradientforboth:1.676 Afteradayandnightofrebound,sleepreturnedtobaselinelevels
0.09and2.1460.11forw1118andw2202,respectively).However,the bytheseconddayfollowingtherandomstimulation(Fig.5b,c;green
separation between daytime and nighttime responses was much plots).Closerexaminationofbehavioralresponsivenessbeforeand
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Figure3|Hourlyarousalprobingrevealssignificantdifferencesbetweenwhitestrains.(a).Averagesleepprofilesoverseveraldays(yellow)andnights
(grey)forw1118(blue)andw2202(red).Minutesofsleepperhourweredeterminedbyabsolutelocationdata,basedona3mmmovementthresholdasin
Fig.2b.Allflieswerestimulatedhourly(byvibrations,indicatedbytheblacklinesabovethegraph).N547forw1118andN550forw2202.(b).Average
speedofw1118(blue)andw2202(red)forthesamesetofflies,overthesametimeperiodasina.Theresponsestothevibrations(blacklinesabovethegraph)
areevidentasspikesinthespeed.Dataareaveragedforallflies,notonlyimmobileones.(c).Averagespeeddatafordayandnightforthesamesetofflies.
Theverticaldashedlinesindicatehourlyvibrationstestingforbehavioralresponsiveness.(d).Characterizationofbehavioralresponsivenesscurve.
Averagespeed(purple)(6SEM,lightblue)isshownforawild-typestrain,withfourmetricsderivedfromabestfit(yellowtrace)oftheaverageresponse.
Fittedparameterswere:theaveragespeed1minbeforethestimulus(orthebaselinespeed(V ),theaveragespeed1minafterthestimulus
Pre-Stimuli
(Post-StimuliSpeed),theamplitudeoftheresponse(V ),andthetimeconstantoftheresponse(t ).Greendashedlineindicatestimingof
Amplitude Inactivation
vibrationstimulus(T50).Fliesanalyzedforresponsivenessprofileswerenotnecessarilyimmobile.(e).Averageresponse(6SEM)forw1118,for
day(yellow)andnight(grey).(f).Averageresponse(6SEM)forw2202,forday(yellow)andnight(grey).(g).Bestfittingresponseparameters(asoutlined
ind)forw1118andw2202duringtheday(yellow)andnight(grey).N52256responsesforw1118andN52400responsesforw2202,dividedequallyamong
47and50flies,respectively,fordayandnight.(h).ScatterplotofPre-StimulispeedversusPost-Stimulispeed,forindividualday(yellowdots)and
night(greydots)eventsinw1118.Alinearregressionofthedata(6SEE)isshown,withassociatedslopeandcorrelationindicated.(i).Scatterplotof
Pre-StimulispeedversusPost-Stimulispeed,forindividualday(yellowdots)andnight(greydots)eventsinw2202.Aregressionofthedata(6SEE)is
shown,withassociatedslopeandcorrelationindicated.
after sleep disruption also revealed a homeostatic effect for this mutationinthedopaminetransportergene(dDAT),leadingtoan
measure of arousal: the average amplitude of the response to the increase in dopamine at the synapse, which has been shown to
vibrations was diminished thefirst dayfollowing sleep disruption, decrease sleep22. dumb2 is a hypomorphic allele of the dDA1
but returned to baseline the second day (Fig. 5d, e). Interestingly, receptor (also called DopR), which impairs arousal systems in
nighttime responsiveness was completely abolished following flies23, and has been shown to increase sleep14. Both mutations
sleep disruption, and remained significantly lower for two days were outcrossed to the w2202 genetic background, to ensure that
(Fig. 5d, e). This suggests that these homeostatic-sleep processes anyarousal-relatedphenotypeswerespecifictoeachgeneticlesion.
(responsiveness) are not equivalently distributed between day and Using virtual beam-crossing metrics, we confirmed the published
nightsleep. sleep phenotypes for these mutants (Fig. 6b). Also consistent with
previouslypublishedwork,combiningbothmutationsinthesame
Sleepmutants.TotesttheDARTplatformonexistingDrosophila strain ‘‘rescued’’ the sleep defects of either mutant (Fig. 6b),
sleep mutants, we contrasted two mutations in the dopamine presumably because the effects of increased dopamine at the
pathway that have opposing effects on sleep, fumin and dumb2 synapse (in fumin) are balanced by defective postsynaptic DopR1
(Fig. 6a). The fumin (insomniac in Japanese) strain contains a function(indumb2)14.
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Figure4|Sleepintensitydiffersbetweenthewhitestrains.(a).Responseswerebinnedaccordingtothedurationofpriorimmobility.Vibrationstimuli
weredeliveredonceanhouroverseveraldaysandnights(greenarrow).Priorimmobilityepochswereautomaticallyclassifiedinto5minbins,as
determinedbythefirstretroactivedetectionofanymovement,basedona3mmthreshold.Fourexamplesareshown,whereflieshadbeenimmobilefor
differentlengthsoftimepriortothestimulus(red,blue,andpurplelines),andoneflythatwasmovingimmediatelybeforethestimulus(goldline).Only
immobileflieswereincludedinthesubsequentsleepintensityanalyses,whichsummedlocomotionresponses1minafterthestimulus(Actiontime
zone).They-axisrepresentshorizontalflymovementinthetubes.Notethattimebeforeandafterthestimulusisnotonthesamescaleintheschema.
(b).Sleepintensityinw1118.Leftpanel:frequencycountforevery5minimmobilitybin,forday(yellow)andnight(black).Middlepanel:theproportion
offliesresponding(6SEM)foreachimmobilitybin,fordayandnight.Rightpanel:theaverageamplitudeoftheresponseforeachimmobilitybin
(averagespeed6SEM),fordayandnight.(c).Thesamesleepintensitycalculationswereperformedforw2202N547forw1118andN550forw2202.See
TablesS1–4forcorrespondingstatistics.
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0 0 2 4 6 8 101214 0 2 4 6 T8im10e1 (2m14in) 0 2 4 6 8 101214 PProes-t-SPSoDsDt -DS1D PDPo2rset--PSSoDDs t-DS1 D D2
Figure5|Sleepreboundfollowingarandomstimulationprotocol.(a).Randompatternsofvibrationstimuli(setat1.2g)weredeliveredfor12hoursof
night.(b).Averagehourlysleepduration(6SEM)inw2202before(blue),during(purplebox),andafter(magentaandgreen)therandomstimulation.
Yellowbackgroundindicatesday,greyisnight.(c).Traditionalsleepdurationmetrics(6SEM)forthedatafromb,inthesamecolorscheme.*,p,0.05;
**,P,0.01,bypair-wise2-tailedt-test.(d).Averagespeed(6SEM)forthesamefliesasinb.(e).Averagedaytimeandnighttimebehavioral
responsiveness(6SEM)forbaselineandtwosuccessivedaysfollowingtherandomstimulation.Velocitydata(V )aresummarizedinthepanelon
amplitude
theright,forthesamethreedays.**,P,0.01,bypair-wise2-tailedt-test.Yellowplotsrepresentdaytimeresponseswhereasgreyplotsarenighttime
responses;bluesurroundingindicatesthedaybeforetherandomstimulation,magentasurroundingisonedayaftertherandomstimulation,andgreen
surroundingistwodaysafterrandomstimulation.N517flies.
We next investigated whether these DA mutants also displayed hourly mechanical vibrations during the day, but also during the
differentlevelsofbehavioralresponsiveness.Simpleflytrackingcon- night(Fig.S6d–f).Whileincreasedresponsivenessinfuminiscon-
firmedthatfuminfliesmovesignificantlymorethandumb2flies,day sistentwithincreaseddopamine inthisstrain, thissameresultfor
ornight(Fig.S6a–c).Bothfuminanddumb2respondedrobustlyto dumb2 (which has compromised DA signaling) was surprising.
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Figure6|SleepandarousalmeasurescanbedissociatedwithDART.(a).Opposingeffectsoffuminanddumb2ondopamine(DA)functionandsleep.
ReleasedDAimpactsDA1receptorstoinitiatecAMPsignalingpathways(leftblackarrow)beforebeingrecycledbackintothecell(redarrow).fuminis
mutantfortheDAtransporter,leadingtoincreasedDAlevelsinthesynapse,andconsequentlyincreasescAMPsignalinginpost-synapticneurons(right
thickblackarrow).dumb2ismutantfortheDA1receptor,leadingtodecreasedcAMPsignaling(dashedblackarrow).(b).Classicalbeam-crossingsleep
profiles(minsleep/hour(6SEM))basedona5mininactivitycriterion,forthew2202backgroundstrain(blue),fumin(red),dumb2(green),andthe
doublemutantfumin;dumb2(black).N568forw2202;N567forfumin;N562fordumb2;N566forfumin;dumb2.Thew2202profileisshowningreyfor
comparisoninthethreemutantpanels.(c).Sleepintensityprofiles(%reactive6SEM)forthesamefourstrainsasinb.(d).Multidimensionalscaling
(MDS)wasusedtoprojectthedataintoatwo-dimensionalspaceforeasiervisualizationofthemultidimensionalrelationshipsbetweendifferentstrains.
MDSanalyseswereperformedforfumin;dumb2(blueborder)comparedtoitsgeneticbackgroundstrain,w2202(blackborder),fordaytime(yellow)and
nighttime(grey)metrics.Leftpanel:fourbeam-crossingmetrics(asforb)wereusedincombinationforMDScomparingbothstrains.Thedifferent
metricsusedareindicatedinthegreenbox(bottomofthepanel).Thedaytimeeffectsoverlapwhilethenighttimeeffectsaredistinct.Rightpanel:four
arousal-basedmetricswerecombinedwithfourbeam-crossingsleepmetrics(indicatedbythelargermagentabox)forMDSanalyses,resultingina
completeseparationbetweendayandnighteffectsinbothstrains.a.u.,arbitraryunits.
Nevertheless, although both mutants respond more strongly than Fig. S7), as expected due to increased synaptic dopamine in this
w2202 (their common genetic background), their baseline arousal mutant14. Surprisingly, dumb2 nighttime sleep is also lighter, even
profilesarepredictablydifferentfromeachother,withfumingen- thoughdopaminergicfunctionisimpairedinthismutant23(Fig.6c,
erallymoreactivethandumb2(Fig.S6g–i). Fig.S7).Theneteffectofeitherdopaminergicmanipulationisthere-
TobetterunderstandtheeffectoftheseopposingDAmanipula- forelightersleep,dayornight,regardlessofoppositeeffectsonsleep
tionsonsleep,weexaminedtheirsleepintensity.Separatingthesleep duration. Combining the mutations (in fumin; dumb2 double
intensityprofilesfordayandnightinthesestrainsyieldedastriking mutants) appears to ‘‘rescue’’ nighttime sleep intensity, but also
result: both mutants remained highly responsive during the day, increases daytime sleep intensity (Fig. 6c). Thus, combining the
regardless of how long they had been immobile (Fig. 6c, Fig. S7). two mutations unexpectedly results in deeper sleep, even though
Thissuggeststhatneitherfuminnordumb2mutantsareeversleeping eachmutationaloneproducedlightersleep.
deeply during the day. Indeed, their average responsiveness level
(,80%)throughoutthedaymaysuggestthatinsteadbothmutants Multidimensionalscalingofarousaldata.UsingDART,aflystrain
arealwaysawakeduringtheday,eveniftheyarequiescent.During can be characterized by many locomotion-related metrics, from
thenight,fuminsleepisalsolighterthaninthecontrolstrain(Fig.6c, counting beam-crossings to fitting responsiveness profiles, and in
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manyinstancessimpleactivity-basedmeasuresmaybesufficientto Surprisingly,theserelatedbackgroundstrainswerefoundtobequite
characterizemoleculargeneticeffectsinamutantstrain.However,a differentforawiderangeofsleepandlocomotionphenotypes,high-
globalassessmentofarousalmeasuresinastrainmayhelpprovidean lightingthefactthatevensimilar‘‘wild-type’’backgroundsmightbe
overallpictureofhowonestraincomparestoanother,andwhether significantlydifferent.Idiosyncrasiesineachstrain,suchasthepro-
‘‘rescue’’experiments,forexample,onlyrelatetosomemeasuresbut pensityforw2202toremainnearafoodsource,arelikelytocloudany
not others. To provide such a global assessment of locomotion genetic investigations without access to positional information.
andarousal-relatedmeasures,weincorporatedamultidimensional Similarly,ourfindingthatw1118respondsequallywelltomechanical
scaling(MDS,Fig.S8;seeMaterialsandMethods)optioninDART, vibrations during the night as during the day suggests decreased
whereselectedmeasurescouldbeassessedincombinationtoprovide arousal thresholds at night in thisclassic background strain, com-
amoreglobalpicture ofbehavioral effects. MDSisan exploratory paredtow2202.Outcrossingamutationtoaw1118backgroundmight
data analysis technique which projects the absolute differences consequently lower arousal thresholds at night, and thereby alter
in data points from high-dimensional (Fig. S8a–c) to low- sleep phenotypes (and potentially any other phenotypes regulated
dimensionalspace(Fig.S8d).Thisenablesvisualizationofthedata by sleep functions). In contrast, outcrossing to w2202 would more
structure within high-dimensional space, which in turn can reveal likelyproduceflieswithastrongerday/nightsleepdichotomy,pos-
underlyingpatterns,suchasthepresenceofclusters.ApplyingMDS siblyimbuingmutantswiththedistinctsleepfunctionsthatmightbe
on eight combined measures (sleep bouts per hour, total sleep associatedwithlightversusdeepsleep.
duration,sleepboutduration,wakingactivity,responseamplitude, Thatdifferentformsofsleepexistinmammalsandbirdsiswell
pre-stimulusspeed,post-stimulusspeed,decaytimeconstant–asin known16. It is becoming apparent that different forms of sleep are
Fig. 6d) to the two genetic background strains supported the view alsolikelyininsects13,25.Someflystrains,suchasCantonS13andw2202
thatday/nightactivitywasdifferentforw2202,butnotforw1118.(Fig. (this study) sleep significantly more lightly during the day, even
S9a).MDSanalysisonthemutantsfuminanddumb2,ontheother though prolonged daytime inactivity, especially around mid-day,
hand,supportsthesuggestionthateachmutantoccupiesadistinct might suggest otherwise. What is the function, if any, of lighter
phenotypic space, day and night (Fig. S9b). Such visualization daytime‘‘sleep’’,andhowisitdifferentthannighttimesleep?One
techniquesprovideavaluablestartingpointforassessingtheeffect possibility may be that different sleep functions, such as memory
ofsubsequentgeneticmanipulations. consolidation and synaptic homeostasis, might be segregated by
WenextusedMDStoexaminetheextentofphenotypicrescuein dayand night in insects. However, itis interesting to observe that
the double mutant. Previous work using beam-crossing methods nighttimesleepinfliesalsoincludesepochsofcomparativelylighter
predictthatthedoublemutant,fumin;dumb2,revertstowild-type sleep.Thisobservationcouldonlybemadebyactivelyprobingbeha-
behavior, because opposing dopaminergic effects are combined14. vioralresponsivenessinimmobileflies,andthenplottingthepopu-
Whileclassicalsleepmetrics onun-stimulated fliesindeed suggest lation responses as sleep intensity curves by retroactively binning
phenotypicrescue(Fig.6b),activelyprobingtheflieswithvibration flies into different immobility duration groups (Fig. 4), which
stimuli(Fig.S10)revealedonlyapartialrescue,assuggestedbyMDS DART does automatically. Finally, it remains possible that lighter
on the same data (Fig. 6d, left panel: nighttime measures do not sleep in fliesaccomplishes the same functions as deeper sleep, but
overlap). MDS of more arousal metrics, combining beam crossing maybetoalesserdegree;fliesmayneedtobemorealertduringthe
andresponsivenessmeasures,suggestsafurtherseparationbetween day for obvious ethological reasons. In any case, the DART sleep
fumin;dumb2andthew2202backgroundstrain(Fig.6d,rightpanel).A intensity measures now allow these important aspects of sleep to
global assessment of all these measures therefore provides greater beaddressedinwildtypeandmutantstrains.
insight into phenotypic differences between the two strains, high- Asproofofprinciple,wethereforeappliedDARTtotwoknown
lightingwhichmeasuresmightbeaffectedbyageneticmanipulation, sleepmutantswithpredictedeffectsonsleepneed.fumindecreases
andwhichremainunaffected.Inthisspecificgeneticmanipulation,it sleepdurationinflies,anddumb2increasessleep14,andtheseoppos-
appearsthatwhilesleepphenotypesareindeedrescued(Fig.6b,c),
ingphenotypesasaconsequenceofdefectsinwell-understoodmole-
average speed(dayornight)remains lowerin fumin;dumb2,com-
cules have been convincingly used to understand the role of
paredtothegeneticbackgroundcontrol(Fig.S10),andresponsive- dopamineinpromotingarousalinDrosophila14,26.Weplacedfumin
nessingeneral(dayornight)remainslower(Fig.S10).Thislackof anddumb2inaw2202background,topreservetheday/nightdicho-
correspondencebetweensleepdurationdataandarousalmetricsis
tomymoreevidentinthisbackground.Whilethesemutantsbehaved
illustratedbytheclearseparationbetweenthestrainsshownbyMDS
as expected for inactivity-based sleep profiles (Fig. 6b), their res-
ofalleightmeasurescombined(Fig.6d,rightpanel).
ponsestoprobingstimuliwereverydifferentfromthoseexpected:
DARTrevealedbothmutantstobemoreresponsivethanwildtype
Discussion whilequiescent,dayornight.Thissuggeststhat,toachievewild-type
The discovery that flies sleep has revolutionized sleep research, responsiveness profiles, the level of dopamine signaling has to be
largely because Drosophila molecular genetic tools could be effi- maintainedwithinanoptimalrange.Increasedordecreaseddopa-
ciently deployed to address various hypotheses relevant to sleep mine signaling leads to equivalent sleep intensity defects, which is
function. We introduce here the Drosophila ARousal Tracking consistentwiththe‘‘invertedU’’hypothesisofdopaminefunction27,
(DART) system, which is a video-based platform that effectively andwithareviewofseveralotherdopaminemanipulations28:most
provideswell-establishedactivitymetricsalongwithcrucial beha- dopaminergic manipulations seem to increase behavioral respon-
vioralresponsivenessmeasurestomorethoroughlydescribesleep, siveness, regardless of whether dopamine function is enhanced or
arousal, and locomotion in a strain. DART provides a variety of impaired. This observation is also consistent with a recent study
arousal metrics for Drosophila, from activity levels and position showing increased visual responsiveness in flies that have experi-
preferences, to behavioral responsiveness, sleep intensity, and encedanaltereddopamineenvironmentduringdevelopment,ina
homeostatic effects. Data can be processed, analyzed, and visua- Drosophila model for schizophrenia29. It will be interesting to see
lized entirely within DART graphical user-interfaces, or extracted whether sleep duration and sleep intensity can be dissociated by
as text files for use in other programs. altering dopamine levels in different dopamine sub-clusters30.Our
We used DART to first compare behavior in two classic white arousal-basedapproachpredictsthattherewillbedistinctdopami-
strains,whichare oftenusedaswild-type backgrounds forgenetic nergic effects on sleep duration and responsiveness, because com-
studies.Indeed,w2202(alsocalledisoCJ1)isanisogenizedvariantof biningfuminanddumb2rescuedoneactivitymeasurebutnotothers.
w1118, which was derived from the Canton-S wild-type strain15. ThisresultisconsistentwithotherDrosophilastudiessuggestingthat
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sleepandgeneralarousalarecontrolledbynon-overlappingdopa- usingestablishedDrosophilasleep/activitymetrics.Guidelinesareavailableinthe
minergiccircuitsindifferentbrainstructures18.Finally,thelackof DARTinstructionmanual,providedathttp://web.qbi.uq.edu.au/dart/index.html.
Thelocationsofthefliesfromtheavi/mp4videosarecalculatedinasemi-auto-
any significant changes in sleep intensity during the day in both
maticfashionviaanimagesubtractionmethodthatusesanestimateoftheimage
dopaminemutants(Fig.6c)suggestsapotentialbiologicalbasisfor backgroundthatisspecifictoeachrecordedvideo.Thismethodispreferredtoimage
theregulationofarousalthresholdsasafunctionofpriorimmobility. subtractionusingastaticreferenceimage11becausetheimagepixelintensitycan
It is possible that dopaminergic signaling regulates behavioral fluctuatesignificantlyoverthedurationofamulti-dayexperiment.Thebackground
imageestimateisdeterminedbyfirstreadingastackof10–20imagesthatareselected
responsivenessspecificallyduringtheday,andthatanotherneuro-
randomlyfromthecandidatevideo.Foreachimagewithinthestack,flylocationsare
modulatorregulatesresponsivenessatnight.Interestingly,daytime determinedwithinmanuallydefinedtuberegionsbycalculatingthecross-correlation
arousalthresholddynamicsarerescuedwhenfuminanddumb2are oftherawimagecomplimentwithanempirically-defined2Dflytemplateimage.The
combined (Fig. 6d), although these do not exactly phenocopy the twotypesoftemplateimagedistributionsusedinDARTarethe2DBoltzmannand
Gaussiandistributions,whichtakethefollowingfunctionalform:
daytimesleepintensitydynamicsseeninthecommongeneticback-
groundstrain. Boltzmann:W~ 1 ð1Þ
The concept of phenotypic rescue is central to genetic analysis; 1zekðD{DhÞ
geneticmanipulationsdisruptaphenotype,andothermanipulations
(cid:2) (cid:3)2
aredonetorestorethephenotype,thusprovidinganunderstanding Gaussian:W~e{ D{sDmD ð2Þ
of the underlying mechanisms. Half a century ago, this working
concept was first applied to understanding the genetic underpin- whereD~pffiDffiffiffiXffiffiffi2ffiffizffiffiffiffiffiDffiffiffiYffiffiffi2ffiffi,k/D aretheslopefactorandhalf-inactivationdistance,and
h
nings ofdifferent behaviors, suchas circadian rhythms, courtship, m /s arethemean/standarddeviationdistances.
D D
and learning, and genes such as period, fruitless, and dunce were Afterdeterminingtheflyregionsoverallthebackgroundimageestimatestack,
identifiedandsincethenslottedintoagrowingbodyofknowledge imageregionsnotoccupiedbythefliesarestoredforeachimageframe.Fromthis,the
weightedaverageofthenon-occupiedimageregionsisusedtorepresenttheback-
about nervous system function31. Sleep is a relative newcomer to
groundimage.Thelocationofthefliesfortheremainingframesinthevideoarethen
Drosophila behavior genetics1,2, and already dozens of genes have calculatedfromthesubtractedimageresidualwiththemostlikely(brightest)region
beenidentifiedaskeytotheregulationofsleep,aswellasavariety withineachtuberepresentingthefly’slocation.Theprogramreadsthevideointosub-
ofdistinctcircuitsandmolecularpathways3,32.Sleepandarousalin imagestacksforeachapparatus(50frames/stack)andcalculatestheflylocations
fromtheimagesubtractionresiduals(seeFig.S1a–hforasummaryoftheprocesses
Drosophila can be characterized by several sub-phenotypes: total
describedabove).Allflylocationsweretrackedatarateof1Hz,whichisconsistent
sleepduration,daytimesleep,nighttimesleep,sleepboutnumber withothervideotrackingmethods12.Trackingfalsepositives,usuallysignifiedby
(dayornight),boutlength(dayornight),activityperwakingminute, largejumpsinflylocation,canbefixedmanuallyinpost-processingusingtheDART
aswellasthenovelmetricswehavepresentedhere.Clearly,many flytrackingGUI.Fliesthathaveseveretrackingissues,oraredeceased,canbecon-
firmedandremovedviavisualinspectionthroughtheDARTdatacombinationGUI.
measurescanbegroupedasbehavioralsyndromes,andtheseshould
change(orbe‘‘rescued’’)together,whileothersareirrelevanttothe
Analysistools.Arousalthresholds.Arousalthresholdsweretestedwithsequentially
problem being investigated. Multidimensional scaling (MDS) of increasingvibrationintensities,from0to1.2g,in0.3g(200ms)increments,every
multiple phenotypes, as we have shown via the DART platform, 15s,onceanhourover24hours.Arousalthresholdswerecalculatedbyassigningthe
providesonewayofmorethoroughlyinvestigatingbehavioralrescue vibrationintensity(g)thatevokedalocomotionresponse(walkingatleasthalfthe
lengthoftheglasstube)inquiescentanimals(i.e.,fliesthathadnotshownany
ortestinghypotheses,evenforstudiesnotdirectlylinkedtosleepand
movementintheprecedingminute),anddeterminingthedistributionofgvaluesfor
arousal.WeenvisionthattheDARTplatformcouldthereforealsobe astrain13.
effectively used more generally for characterizing different genetic
effectsinbehavioral-phenotypicspace. ElementaryAnalysisCalculations.AbsoluteLocationKinematicCalculations.Dueto
thedimensionsoftheflytubes,wecanapproximateflylocationspatiallyby1
dimension(i.e.,thex-locationoftheflywithinthetube),which,fortheithmovie
Methods frame,isdenotedbyX.Therefore,thetotaldistancetravelledbyanindividualflyover
i
Flystocksandrearingconditions.Flieswereraisedinplasticbottles(Genesee Nmovieframesiscalculatedasfollows:
Scientific)at22uConstandardyeast-basedDrosophilamedia,ona12hourlight-
1th2enhoaugreddafrokr(3L–D5)drahyysthinmg.rVoiurpgsinoffe2m0a–l5e0swflieerseicnolplelacstteidcuvinadlseryeCaOst2baanseesdthaegsaira,falynd D~XNi~{11jXiz1{Xij ð3Þ
media.Forallexperiments,individualflieswereaspiratedinto65mmglasstubes Fromthis,theaveragespeedtravelledbyaflyoverthistimedurationcalculatedby:
(Trikinetics,Waltham,MA)containingflymediaononeendandsealedwithacotton
D
plugontheotherend.Twowhitestrainswerecompared:w1118andw2202.Theseare V< ð4Þ
standardwhite-eyed,wild-typebackgroundstrains,typicallyusedascontrolsfor TN{T1
transgenicexperiments.w2202isanisogenizedvariantofw1118,alsoknownasisoCJ115.
whereT denotesthetimestamp(giveninseconds)oftheithmovieframefromthe
fuminanddumb2werekindlyprovidedbyK.Kume.Thesewereoutcrossedatleastsix i
startoftheexperiment.Themeanpopulationdistance/speed,overthegiventime
generationstoaw2202geneticbackground14.
period,iscalculatedastheaverageoftheindividualkinematicmetricsoverall
memberswithinthepopulation.Itshouldbenotedthatallkinematicsforthisstudy
Behaviorialplatform.Flieswereindividuallyhousedin65mmglasstubes
werecalculatedusingthismethod,unlessotherwiseexplicitlystated.
(Trikinetics,Waltham,MA),17tubesonaplastictray(1438cm),2–6traysper
filmedexperiment.Flieswererecordedcontinuouslyinaviormp4formatat5frames MovementDetection.Consideraflythatatatime,T,hasalocationofX.Aflyis
persecondthroughoutmulti-dayexperiments,assetbytheDARTsoftware(see consideredtohavemovedif,foragiventimeperiod,D0T,thedistancerang0eisgreater
below),usingaUSB-webcam(Logitech)fittedwithawide-anglelens(Zeiss).The
thanthemovementdistancethreshold,D :
DARTinterfacealsocontrolledvibrationstimulideliveredtotheflies.Shaft-less Move
vibratingmotors(PrecisionMicrodrivesTM;model312–101)weregluedunderneath i:e: jXi{X0jwDMoveðforTi [½T0,T0zDT(cid:2)Þ ð5Þ
eachtrayof17flies(2motorspertray),andthesewerecontrolledbymodulating
voltageoutputwithDART,byinterfacingwiththeanalogueoutputchannelsofaUSB Conversely,thedurationofinactivityofanindividualflyiscalculatedasthetotal
dataacquisitiondevice(MeasurementComputing;1280LS).Theunitgisusedto time,fromaninitialtimeT0,thatthedistancerangeoftheflyislessthanDMove:
eexarptrhe,s9s.8vibmra/st2io).nTahmepPlrietucidseio(n1Mgiecqroudalrsivtehemgortaovristahtaiovneaallfionrecaeraretltahtieosnusrhfiapcebeotfwteheen DT~Tj{1{T0(cid:6)forthefirstjwhere(cid:5)(cid:5)Xj{X0(cid:5)(cid:5)wDMove(cid:7) ð6Þ
inputvoltageandvibration,supportingamaximumvoltageof3.5Vor2.4g13.The
vibrationmotorswereoperatedatastimulationfrequencyof50Hzforall
experiments.Arousalprobingexperimentsweretypically4–5dayslong,withthefirst
VirtualBeamKinematicCalculations.Torecreatethevirtualbeamkineticsfromthe
dayandnightdiscardedtoallowforthefliestoacclimatizetothetubes.
DARTvideotracking,anidealbeamisplacedwithinagiventubeatalocation,X ,
Beam
whichiscalculatedas:
Flytrackingandvideoimagesegmentation.TheDARTexperimentsuiteisa
graphicaluserinterface(GUI)basedprogramthatwasdevelopedusingMatlab XBeam~XMinzPBeam|ðXMax{XMinÞ ð7Þ
(Mathworks).TheDARTframeworkprovidesthemechanismsbywhichtoA)
custom-designstimuli-basedexperimentalprotocols,B)tracktheflylocationsfrom whereX /X istheminimum/maximumx-locationsoverallflieswithinasingle
Min Max
therecordedvideofiles,andC)combineandquantitativelyanalyzetheflies’activity apparatustray,andP istheproportionalextentofthetubetakenfromtheleft
Beam
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