Arousal in Drosophila

12 Pages·2003·0.2 MB·English

BehaviouralProcessesxxx(2003)xxx–xxx
Arousal in Drosophila
∗
Bruno van Swinderen , Rozi Andretic
TheNeurosciencesInstitute,10640JohnJayHopkinsDr.,SanDiego,CA92121,USA
Received2December2002;receivedinrevisedform16April2003;accepted24April2003
Abstract
Arousalcanbedescribedasanendogenouslygeneratedorexogenouslyinducedchangeinbehavioralresponsiveness.Changes
inlevelsofarousal,suchasoccurduringsleeporattention,mostlikelyaccomplishadaptivefunctionscommontomostanimals.
RecentevidencedemonstratingchangingarousalstatesinDrosophilamelanogastercomplementsotherbehavioralresearchin
thismodelorganism.Hereinwereviewthemethodologyrelatedtothestudyofcircadianrhythms,sleepandanesthesiawhere
arousal,orlackofit,playsanessentialrole.Weendthisreviewbydiscussinganewmethodthatallowsforthefirsttimeto
correlatechangesinbrainelectrophysiologytochangesinbehavioralarousalinthefruitfly.
©2003ElsevierB.V.Allrightsreserved.
Keywords:Anesthesia;Arousal;Circadian;Drosophila;Electrophysiology;Sleep
1. Introduction researcherintotheclassicfallacyoftheundistributed
middle:
Arousal is relative. Wakefulness is a state of in- Oilisfoundindeserts.
creasedarousalcomparedtosleep,justasattentionis TheMojaveisadesert.
a state of increased arousal compared to inattention. TheMojavehasoil.
In either case, a baseline is defined behaviorally and In other words, it is not the neuroanatomical phe-
changes from that baseline can be measured by vari- nomenon (the desert) that identifies arousal states; it
oustests.Behavioralmeasuresarethereforecentralto is always the behavior, which must be probed and
studying arousal as they represent the only real indi- calibrated to arousal stimuli in every new organism
cator of a change in state. For humans, one may rely investigated. This is especially the case for animals
on verbal reports, but for all other animals a behav- evolutionarily distant from humans that might have
ioral reaction to a test stimulus must be measured to verydifferentneuralarchitecturessupportingarousal.
ascertain arousal states. The verbal reports from hu- Theconceptofarousalasageneralstateofnervous
manshaveallowedneuroscientiststodiscovercertain system activity has been used in relation to anything
neuralcorrelatesofarousal,suchasthedifferentbrain fromphenomenaatthecellularleveltocomplexcog-
oscillationsseenbyEEG(Zeman,2001).However,us- nitiveperformanceandconsciousness(Zeman,2001).
ingsuchhumancorrelatestoidentifyarousalinother However, most often it is related to daily changes
animalswithoutfirstdefiningitbehaviorallytrapsthe in states of vigilance such as wakefulness, resting
sleepandrapid-eye-movement(REM)sleep(Steriade
et al., 1993). In addition, severe loss of behavioral
∗ Correspondingauthor. responsiveness is present as a consequence of action
E-mailaddress:[email protected](B.vanSwinderen). of anesthetics and in the extreme case of coma. The
0376-6357/$–seefrontmatter©2003ElsevierB.V.Allrightsreserved.
doi:10.1016/S0376-6357(03)00131-1
2 B.vanSwinderen,R.Andretic/BehaviouralProcessesxxx(2003)xxx–xxx
connectionbetweenarousalandsleep,withitsrhyth- arousal states. Internally generated periodic changes
mic daily changes, indicates an endogenous compo- in behavior and physiology that underlie sleep and
nent of arousal regulation. However, wake arousal wakefulnesshavebeenthefocusofstudyofcircadian
levels in the form of attention and awareness are in- and sleep scientists for many years (Magoun, 1958;
fluencedtoalargeextentbyenvironmentalfactorsas Steriadeetal.,1993;BorbelyandAchermann,1999).
well. Inthegenetics-friendlyfruitfly,sleepwasneglectedas
Experimentalresearchonarousalismostoftenper- anobjectofstudyforvariousreasons.First,theEEG
formedinanimalssuchascatsorrats,whichhavecon- cultureofarousalstudies,whereslowwaveshavebe-
tributedgreatlytoourknowledgeoftheneuroanatomy comethedefactosignatureofsleep,stymiedinvesti-
and pharmacology of arousal (Robbins, 1997). Lack- gationsofthephenomenoninDrosophilawheresuch
ingverbalreports,behaviorssuchastailflicksoreye electrophysiology was considered difficult. Second,
blinks are used as reporters of arousal levels in these automatedbehavioralmonitoringtechnologyhasbeen
animals.Asimilarneuroanatomybetweenhumansand limited to detecting gross fly movements rather than
highervertebratessupportssimilarneuralcorrelatesof themorecomplexresponsesthatmightcorrelatewith
arousal states, such as slow wave oscillations during changes in arousal level. Lastly, sleep in Drosophila
sleep(Pace-SchottandHobson,2002).Insects,onthe has been traditionally viewed as just another mani-
other hand, do not have a cortex, thalamus, or other festationofcircadianrhythms,therebyneglectingthe
such mammalian neuroanatomy. Yet, to those of us homeostatic regulation of sleep. Sleep and circadian
whoobserveflybehavioronadailybasisitisobvious rhythmsareofcourserelated(fliessleepatnight),but
that their arousal level changes—they still learn and they are not the same thing (sleep is homeostatically
sleep, for example (Tully, 1996; Shaw et al., 2000). regulated)(Hendricksetal.,2000;Shawetal.,2002).
Thus, the mechanisms supporting arousal levels are Locomotion has been a phenotype used by circa-
notnecessarilytiedtotheneuralarchitecturesunique dian biologists for many years, and since recently by
tohighervertebrates. sleep biologists, as an indicator of arousal state in
In the following review, we will discuss different Drosophila (Konopka and Benzer, 1971; Hendricks
measures of arousal in Drosophila melanogaster. We et al., 2000; Shaw et al., 2000). Because locomo-
start with a discussion of circadian and sleep litera- tion is easily quantifiable it proved to be an excel-
ture from the perspective of endogenous control of lent phenotype to use in mutagenesis screens where
arousal states and continue by reviewing the spe- one searches for a single mutant fly among many
cial case of general anesthesia in Drosophila. These that differs in locomotion parameters. The first cir-
measures progress, as they did for humans, from the cadian locomotion mutants, isolated by Benzer and
purely behavioral to electrophysiological correlates Konopka in a screen looking for flies with unusual
which may serve as markers for arousal, so we end timing of eclosion, showed that a single gene could
by discussing a new methodological approach that affect a behavioral phenotype (Konopka and Benzer,
allows for correlating changes in neuronal activity to 1971). This discovery eventually induced Drosophila
changesinovertbehavior. circadian research to become molecular in character
and ultimately it resulted in our extensive knowledge
of the molecular basis of circadian behavior (Allada
2. Sleep et al., 2001). The field of sleep may follow a sim-
ilar path since the same high-throughput apparatus,
2.1. Endogenouscontrolofarousalstates the Activity Monitoring System (AMS), is used for
quantification.
Arousal,viewedintermsofthegeneralcapacityof The AMS, developed by TriKinetics (Waltham,
the nervous system to perceive and respond to envi- MA), is an automated method for measuring loco-
ronmentalstimuli,changesfromperiodsofhighvigi- motor activity of individual flies that has become a
lance or responsiveness (attention) to periods of very standard embraced both by circadian and sleep re-
lowresponsiveness(sleep).Thediurnalcyclesofsleep searchers. It consists of a monitor that can hold 32
and wake are the most salient form of changes in individual glass tubes 65mm in length with food on
B.vanSwinderen,R.Andretic/BehaviouralProcessesxxx(2003)xxx–xxx 3
one end and a cap on the other. Tubes fit into a slot Perhaps because the locomotor phenotype proved
equipped with an infrared emitter and detector, and sosuccessfultowardsunderstandingmolecularmech-
a single fly is placed in each tube. Activity is regis- anisms of circadian regulation, studies of other be-
tered each time a fly crosses the midline of the tube, haviors that might be under circadian regulation are
breaking the infrared beam. The data collection time sparse. In humans and laboratory animals, perfor-
windowdependsonthebehaviorunderinvestigation; mance efficiency on different cognitive and motor
a 30-min time window is regularly used in circadian tasks changes as a function of time of day (Carrier
studies whereas 5min has proven to be more appro- andMonk,2000).Bothsleep-relatedhomeostaticpro-
priate for sleep studies (Shaw et al., 2000). Impor- cessesandcircadianprocessesinfluenceperformance,
tantly, use of this device in combination with others however, there are few published reports investigat-
hasprovidedtwodistinctiveelementsofsleepregula- ing this in Drosophila (Allemand and David, 1984;
tion in flies; first, periods of immobility greater than Allemand and Bouletreau-Merle, 1989; McCabe and
5min result in decreased responsiveness to mechani- Birley, 1998; Ishida et al., 2001). Considering the
calstimulation(heightenedarousalthresholds)(Shaw results from several recent reports showing that ap-
et al., 2000; Nitz et al., 2002); second, sleep depriva- proximately 1% of all fly genes are regulated in a
tionintheformofcontinuousmechanicalstimulation circadianfashion(Cerianietal.,2002),itseemslikely
results in less locomotion (sleep rebound) compared that behavioral performance other than locomotion
to unperturbed levels (Hendricks et al., 2000; Shaw will be under circadian influence. In this respect it is
etal.,2000). interestingtonotethatgenesinvolvedinthesynthesis
Sleep studies in Drosophila focus on one extreme andmetabolismofseveralmonoaminesthathavebeen
ofthearousalcontinuum,theabsenceoflocomotion, correlatedwithchangesinarousalstatesinmammals
within a specified time window. Although measure- are rhythmically expressed in Drosophila, and we
mentofperiodsoflocomotion,ortheirabsence,inthe can speculate that they are involved in regulation of
AMS has proven ideal for gathering data from large arousalinthisspeciesaswell(Claridge-Changetal.,
numbers of flies, it lacks the sensitivity to capture 2001; McDonald and Rosbash, 2001; Ceriani et al.,
other nuances of behavior that reflect arousal levels. 2002;Linetal.,2002;Uedaetal.,2002).Drosophila
Flies also groom, taste and eat when in the AMS, so can be awakened by different stimulus modalities:
it seems unlikely that there exists a one-to-one rela- light, mechanical or auditory stimulation, indicating
tionbetweensleepandlocomotioneveninarelatively thatasinhighervertebratesmultiplesensorysystems
simple organism such as Drosophila. Indeed, insects might feed into a central arousal mechanism and can
can be inactive while awake. Predatory invertebrates cause a transition from sleep to waking (Nitz et al.,
such as scorpions and spiders remain vigilant while 2002).
immobile(Tobler,1988).Conversely,bodypartsmay
move while an animal is asleep; honeybees show an- 2.2. Central arousal mechanism versus changes in
tennal movements correlated to stages of sleep while thesensitivityofsensoryinputs
otherwiseimmobile(Kaiser,1988).Sleepdetectedby
the AMS was confirmed in additional studies using Most complex organisms in which circadian phe-
ultrasound (Shaw et al., 2000). Ultrasound monitor- nomena have been studied demonstrate a circadian
inghasanadvantageoverAMSinthatitcanregister architecture consisting of an intrinsically rhythmic
movements of the head, legs and wings, thus allow- master circadian clock or pacemaker, usually located
ingfordistinctionstobemadebetweentimeswhena inthebrain,andperipheraloscillatorswhich,depend-
flyisstandingstillbecauseitissleeping,versustimes ing on the species, can show varying degrees of au-
when it is not moving because it is eating or groom- tonomous rhythmicity (Helfrich-Forster et al., 1998;
ing.However,ultrasoundmonitoringislimitedinthat Giebultowicz, 2001; Ripperger and Schibler, 2001;
itdoesnoteasilyadapttomonitoringofmultipleflies, Tosini et al., 2001). Drosophila is no exception, and
it is time-consuming and requires substantial calibra- interactionsbetweencentralandperipheraloscillators
tion. Direct visual observation provides similar data havebeenstudiedusingtherhythmicexpressionofcir-
withfargreatersimplicity. cadiangenesasamarkertoelucidatethenatureofthe
4 B.vanSwinderen,R.Andretic/BehaviouralProcessesxxx(2003)xxx–xxx
interaction between central and peripheral clocks
(Hege et al., 1997; Plautz et al., 1997; Giebultowicz
etal.,2000).
There is an intriguing observation about the dif-
ference in circadian phases between general arousal
and the level of physiology of sensory systems. The
circadianpacemakerthatgeneratesrhythmicbehavior
in Drosophila is located in a small group of ventral
lateral neurons (LNv) in the brain and these contain
cyclically expressed molecules necessary for control-
lingcircadianaspectsoflocomotion(Helfrich-Forster,
1998;KanekoandHall,2000).Thissuggeststhatthe
LNvs, through their communication with other brain
regions,influencethearousalstateasevidentinquan-
titativechangesinlocomotionduring24h.Peripheral
clocksarepresentinbothneuronalandnon-neuronal
tissues and in vitro can reveal self-sustained rhythms
in the expression of clock genes (Plautz et al., 1997;
Giebultowiczetal.,2000).Thefunctionalrelevanceof
peripheralclocksremainsunclearsincethereareonly
fewattemptstolinktherhythmatthelevelofgeneex-
pression to changes in sensory physiology (Krishnan
et al., 1999, 2001). Furthermore, how the rhythm at
the level of sensory physiology is related to overall
change in circadian arousal seems to be complicated
by the fact that the responsiveness of most sensory
systemsstudiedisinverselyrelatedtotheoveralllevel
ofarousal.
It appears as if a fly is hardest to awake (has high-
estarousalthresholds)attimeswhenhersensorysys-
temsaremostsensitive.Visualsensitivityindiurnally
active insects such as honey bees and Drosophila is
highestduringthenightwhentheyarenormallyrest-
ing (Kaiser, 1983; Chen et al., 1992). Similarly, in
Drosophila the responsiveness to different odorants
measured as a change in the electrical firing of sen-
soryneuronsintheantennaishighestduringthenight
(Krishnan et al., 1999; Fig. 1C.). Unfortunately, it is
Fig. 1. Circadian rhythms in wakefulness, mating and olfactory
unclear if high sensitivity during the night observed
sensitivity. (A) Average amount of time (in min) for each hour
in these experiments accurately reflects the status of
of the day that flies are awake. Data from CantonS female flies
sensory systems in sleeping animals since, as a part (n = 27) in 12h light:12h dark (and SEM). (B) Mating score
of the experimental procedure, flies are being kept in calculatedfromcopulationfrequencyinDDinCantonSfliesshown
anunnaturalenvironment(immobilizedinarecording as mean proportion mating out of possible 10 (and SEM). By
permissionfromTauberetal.(2003)[Curr.Biol.13(2003)140].
set-up, impaled with electrodes) and often are sleep
(C) Olfactory sensitivity measured as mean electroanntenogram
and food deprived for extended lengths of time. It is response upon administration of ethyl acetate (dilution 1:104) in
undeniable that arousal is likely to change as a con- CantonS females (filled squares) in free-running condition (DD).
sequence of experimental manipulation, and this will Each point represents mean response of 24 flies. No rhythm is
makereachingaconclusiveanswerevenharder,espe- observed in per01 (open circles) or tim01 (filled circles) flies. By
permissionfromKrishnanetal.(1999)[Nature400(1999)375].
B.vanSwinderen,R.Andretic/BehaviouralProcessesxxx(2003)xxx–xxx 5
ciallywhenthesubjectofstudyiscircadianregulation Tauberetal.,2003).However,circadianphasesofgen-
of arousal level. With this caveat in mind, it seems eralarousal,matingandolfactionarenotinclosecor-
thatitistheneuronalactivityinthecentralbrain,and relation indicating that there is no simple interaction
not peripheral nervous system, that determines gen- between them (Fig. 1). Increased olfactory respon-
eral behavioral arousal. So what is then the purpose siveness in the middle of the night is dependent on
ofcircadianchangesinsensoryperception? therhythmicexpressionofcircadiangenes(Krishnan
A preliminary explanation was proposed based on et al., 1999). Interestingly, this rhythm is abolished
asimilarobservationofthedisparitybetweenarousal if circadian genes are expressed only in the central
state and visual sensitivity in honeybees (Kaiser, clock LNv neurons (otherwise sufficient to drive cir-
1983; Kaiser and Steiner-Kaiser, 1983). In this study cadian locomotion) indicating that the olfactory re-
the electrical responsiveness measured intracellularly sponses are driven by changes in gene expression in
from a visual interneuron was found to be at its low- peripheral oscillators (Krishnan et al., 1999). In the
est while bees were sleeping, whereas at the same CantonS strain of D. melanogaster a copulation peak
time the visual sensitivity of the compound eye was occurs close to the day–night transition when olfac-
highest. The study proposes that it is advantageous torysensitivityisstilllowandcopulationremainsrel-
for a diurnal animal to have increased visual sensi- atively high throughout the night coinciding with the
tivity during the night. Dark adaptation of the visual peak of olfactory response (Fig. 1B; Krishnan et al.,
systemisslowcomparedtochangesinfiringofvisual 1999;Tauberetal.,2003).Increasedfemalereceptiv-
interneurons; therefore a more sensitive eye at night ityformatingduringthenight,wherefemalesaccept
ismorelikelytostimulatethesleepingbrainincases courting males more readily, could be, in part, ex-
of sudden visual change. Increased olfatory sensitiv- plainedbyincreasedolfactorysensitivity(Fig.1C).In
ity during the night in Drosophila could be similarly thiscase,itispossiblethatthehomeostaticregulation
advantageous. Although the change in sensory sen- ofsleepandarousal(Fig.1A)andthecircadianregu-
sitivity might be important in relation to arousal, it lationofolfactorysensitivity(Fig.1C)contributeina
is determined to a large extent by adaptation mech- timeofdaydependentmannertothecomplexbehav-
anisms operating in the periphery. For example, the ioral output. A more conclusive answer will become
sensitivity of sensory organs such as eyes or the an- availableonlywhenexperimentaltoolsformeasuring
tennae, on a circadian time-scale, could be inversely circadian and sleep-related phenomena advance to a
related to concentration and/or intensity of the stim- levelwheretheyinterfereminimallywithhomeostatic
uli in the environment and this could explain at least processofsleepandarousal.
in part the increased visual and olfactory sensitivity
at night in Drosophila. However, it is tempting to
speculate that this increased peripheral sensitivity is 3. Studyingarousalwithgeneralanesthetics
also evolutionarily advantageous in how it relates to
centralarousalmechanismsinasleepinganimal. Volatile general anesthetics (VGAs) produce loss
Most tentative links between sensory physiology of consciousness in humans and a behaviorally sim-
and general arousal levels come from studies of ol- ilar loss of arousal in all animals (Franks and Lieb,
factionandmatingbehavior.Malesperformelaborate 1982, 1990). A long history of animal research using
courtship rituals before copulating, and odorant cues thesediverseclinicalagentshasrepeatedlyconfirmed
play an essential role in triggering and maintaining oneveryinterestingpropertyoftheiraction:thecon-
successful courtship (Greenspan and Ferveur, 2000). centration(measuredasvolumepercentatmospheres)
Matingalsoshowsacircadiancomponentwhichisde- requiredtoproducealossofarousalinmostanimals,
pendentuponfunctionalcircadiangeneexpressionin determined by lack of response to irritating stimuli,
femalesbutnotmales;althoughmalesareequallysex- is in the same range as that required to produce the
uallyarousedasevidentbythefrequencyofcopulation human unconscious state required during surgery
attemptsatallcircadiantimes,thenumberofsuccess- (Andrews and Bringas, 1993; Eger et al., 1994).
ful copulations has a clear species-specific circadian This concentration, termed “clinically-relevant,” or
component(Ishidaetal.,2001;SakaiandIshida,2001; “Minimal Alveolar Content” (MAC), varies with the
6 B.vanSwinderen,R.Andretic/BehaviouralProcessesxxx(2003)xxx–xxx
potencyofeachagent,yettheorderofVGApotency assaysinvolvecountingtheproportionofanimals“not
is remarkably consistent among all animals studied moving”(KrishnanandNash,1990)orquantifyingthe
(Campbell and Nash, 1994; Franks and Lieb, 1994; amountofmovementautomaticallyinapopulationof
Crowder et al., 1996). Such consistencies across di- flies(Baintonetal.,2000;Rodanetal.,2002).These
verse phyla, as well as a significant body of research types of experiments may not fully satisfy the funda-
showing that VGA targets are likely to be specific mentalcriterionforlossofarousal:thereisnochange
proteins rather than lipid membranes (Franks and inresponseperse;movementisnotalwaysinresponse
Lieb,1978,1981;Mossetal.,1991;Dickinsonetal., to something, and responses to anesthetic odor may
1993; Hall et al., 1994; Eger and Koblin, 1995), sug- be transient. Other movement assays involve the re-
gest a common mechanism whereby VGAs produce sponse to gravity, a naturally provided stimulus. The
loss of arousal in all animals, regardless of the spe- inebriometer,developedbyWeber(1988)andusedby
cific neuroanatomy. Consequently, natural changes Howard Nash and colleagues in the study of VGAs
in arousal (e.g. sleep, attention) might be supported (Weber, 1988; Krishnan and Nash, 1990; Leibovitch
by that common mechanism otherwise attenuated by et al., 1995) and by Ulrike Heberlein and colleagues
anesthetics. withethanol(Mooreetal.,1998),isadeviceamenable
EventhoughallDrosophilaworkersroutinelyanes- toprocessinglargenumbersofflies.Anesthetizedani-
thetizetheirsubjectswithCO orether,comparatively malstumblethroughaseriesofbafflesandthepropor-
2
littleworkhasbeendevotedtothestudyofanesthesia tionelutedatthebottomprovidesananesthesiascore.
in Drosophila. As in other animal studies, the loss of Severalmutantswerefoundtoresistelutionwhilesub-
arousalproducedbyVGAscanbequantifiedbyplot- jecttocertainVGAs(KrishnanandNash,1990).An-
ting a dose–response curve relating the effect of in- other measure of coordinated movement in response
creasingVGAconcentrationsonaspecificbehavioral to gravity is the “distribution test,” also used by the
endpoint. Logistic regression of the data provides an Nashlaboratory(Guanetal.,2000).Inthisassay,loss
anesthesiastatistic,theEC ,whichistheconcentra- of arousal is quantified as the proportion of flies un-
50
tion at which behavioral performance is half-optimal abletoclimbuptheslantedwallsofaplastictubeaf-
(Waud,1972;DeLeanetal.,1978).Behavioralperfor- terbeingstimulatedbytapping.Certainmutantsfound
mance can be any quantifiable response measured in to be resistant to VGAs in the inebriometer, notably
an individual or a population. The fundamental crite- har38,werefoundtoactuallybehypersensitivebythe
rionforgeneralanesthesiaisnon-responsivenesstoan distributiontest(Guanetal.,2000).Thisdiscrepancy
irritatingstimulus(FranksandLieb,1990;Leibovitch highlightscertainproblemswithinebriometer-typeas-
etal.,1995).Itis,likesleep,definedbyarousalthresh- says: resistance to elution does not necessarily imply
olds;alongwithbeingvisiblyinert(whichcanbemo- an unimpaired state of arousal at the level of more
tivational and hence a state of arousal), subjects fail complexbehaviors.
to respond to pinpricks or other such irritating ma- Single animal responses provide a more accurate
nipulations.StudiesofanesthesiainDrosophilamake but also more laborious assessment of the effect of
use of a variety of behavioral endpoints, some more VGAsonarousalinDrosophila.Mostsuchassaysstill
clinically-relevant than others. As with all fly work, quantify arousal as the proportion of flies respond-
the different endpoints chosen represent a trade-off ing to a stimulus by locomotion. In an adaptation of
betweenlarge-scaleambiguityandsmall-scaletoil. the tail-flick assay of rodents, a hot (noxious) punc-
Most arousal assays in Drosophila are population tatelightbeamisfocusedonfreelymovingflies,one
assays where percentages of non-performing flies are at a time, and the proportion moving away into the
tallied.Thegrossestbehavioralendpointiswholean- shadowsistallied(Tinklenbergetal.,1991;Campbell
imalmovement,orlocomotion,asisusedinthestudy and Nash, 1994). This assay confirmed the hypersen-
ofcircadianrhythmsandsleep(reviewedabove).The sitivity of har38 to VGAs. Similarly, Gamo and col-
number of times a small population of flies (around leagues used a fine brush as an irritant to test arousal
10) interrupts a photodiode beam per unit time pro- levels in the presence of VGAs (Gamo et al., 1981).
videsthebehavioralresponseprobedbytheanesthetic Other single animal assays involve variations on the
agent(KrishnanandNash,1990).Relatedpopulation tendencyforfliestojumpwhenstartledbychangesin
B.vanSwinderen,R.Andretic/BehaviouralProcessesxxx(2003)xxx–xxx 7
luminosity, odor, or by tapping (Campbell and Nash, of various mutants were not recapitulated in headless
1998). The proportion of animals attempting such an flies(which,ifproperlyhumidifiedandstimulatedcan
escape response is tallied with respect to increasing still move around), showing that the body is insuffi-
concentrationsofanesthetic. cienttoproducetheVGAhypersensitivityphenotypes
Wholeanimalmovementssuchaswalkingorjump- (Leibovitch et al., 1995). Another instance where be-
ing by definition require movement of the animal’s havioral phenotypes were not replicated in headless
legs.Yet,theconnectionbetweenstatesofarousaland flies is behavioral sensitization to cocaine (McClung
leg movement is not necessarily clear; reflexive leg and Hirsh, 1998; Andretic et al., 1999; Andretic R.,
action may sometimes be independent of the state of personalcommunication).Third,inaseriesofpapers,
arousalofafly.Kirschfeld’sanalysisofVGAeffects Nashandcolleaguesusedtheelectrophysiologyofthe
ondifferentneuralfunctionsinalargerflyisinforma- Drosophilaescapereflex(stimulus-inducedjumps)as
tivehere(KirschfeldandBaier-Rogowski,1988).Leg ananesthesiaendpoint(LinandNash,1996;Walcourt
movement in response to a light flash (the escape re- and Nash, 2000). In recordings from neurons central
flex)inatetheredflywasfoundtobeaffectedonlyat to the reflex, they describe a short-latency neural re-
supra-clinical concentrations of halothane (2vol.%). sponse (1ms) unaffected by clinical concentrations
In contrast, head movement in response to moving ofVGAsandalong-latencyresponse(4ms)sensitive
visual stimuli (an optomotor response) was found to to VGAs. The short-latency response neurons bypass
be compromised at 0.2–0.8vol.% halothane, within the brain whereas the long-latency response involves
the clinically-relevant range causing unconsciousness the brain. Brain-related physiology is therefore af-
in humans. Sudden leg movements and the resulting fected at clinically-relevant VGA concentrations,
“behaviors”maythereforenotbethebestindicatorsof while motor-related physiology remains unaffected.
arousallevel.Directedmovement,suchasthegeotac- Therefore, should there be a common mechanism
ticeffortsinthedistributiontestormovementstowards whereby VGAs produce their effect at the same con-
a specific goal, probably recruit more complex neu- centrations in all animals, it is likely that the brain is
ronalassembliesthanthethoracicganglionandthere- central to supporting such arousal in Drosophila. For
foreitisnotsurprisingthatthesebehavioralendpoints this reason, it is crucial to develop techniques moni-
displaymoreclinically-relevantEC s(Campbelland toring levels of arousal within the Drosophila brain,
50
Nash,1994;Guanetal.,2000). asexistswithsuchhumanneuralcorrelatesofarousal
The forgoing results suggest that more complex asfoundintheEEG.
behaviors are more susceptible to VGAs. This is in-
deed the case in other organisms too (Harris, 1991;
Morgan and Sedensky, 1995; Crowder et al., 1996). 4. Electrophysiologyofarousalintheflybrain
In humans, short-term memory is more susceptible
than response to pain (Harris, 1991; Collins et al., Most research on arousal, including the forgoing
1995; Eger et al., 1997). The reflexive movement of discussion on general anesthesia in Drosophila, indi-
legs in Drosophila, as well as the reflexive response cates that the control of arousal states resides in the
to pain in humans, need not involve the brain. Is brain.Althoughchangesinarousalaremonitoredbe-
the brain important for arousal in Drosophila? And, haviorally by applying stimuli and testing responses,
do the arousal states affected by clinically-relevant certain neural correlates of arousal have been identi-
doses of anesthetic reside within its brain? Several fied in vertebrates. These correlates of arousal states
lines of research show that the brain is critical. First, are measures of activity in the brain such as EEG,
the fact that complex behaviors are most sensitive to MEG, fMRI, and unit recordings (Zeman, 2001).
VGAs implicates the brain; directed movement in- By such measures, different states of arousal can be
volves a coordination of sensory and motor systems inferred along with behavioral changes; these brain
that coalesce in the brain (Rein et al., 2002). Sec- signatures correlate with the behavioral markers of
ond, studies of decapitated flies and anesthesia have arousal, and therefore can serve as proxy markers
highlighted the importance of the head for arousal of arousal for the species. These markers include
phenotypes. The VGA hypersensitivity phenotypes alpha, delta, and gamma oscillations in humans and
8 B.vanSwinderen,R.Andretic/BehaviouralProcessesxxx(2003)xxx–xxx
other mammals. Slow waves (delta, 0.5–3Hz), for
example,areassociatedwithdeepsleep,alphawaves
(10–15Hz) with “idling” while awake, and gamma
oscillations (30–60Hz) with heightened states of at-
tention(Zeman,2001;Nieburetal.,2002).Multi-unit
activity and Local Field Potential activity within the
brain of animals show similar associations to arousal
statesandhavehencebeenusedtostudyarousalitself
as well as serving as reliable markers of such states
(Steriadeetal.,1993,2001).
Such neural correlates of arousal states were not
available for Drosophila research until recently. It
is remarkable how little is known about the electri-
cal activity within the Drosophila brain, in contrast
to how much is known about the rest of the organ-
ism. There are several reasons for scarcity of data
onDrosophilabrainactivity.Althoughcomparatively
small (∼120,000 neurons in the central brain), the
neuropil is a tangled mess of projections lacking
the nuclei landmarks common to mammalian brains.
The cell bodies are mostly very small (∼2(cid:1)m in
diameter) and therefore not amenable to unit record-
ings. Further, most electrophysiological preparations
require the immobilization of the recording device
(electrodes and supporting connectors) resulting in
the consequent immobilization of the whole fly, thus
making the necessary behavioral correlations prob-
Fig. 2. Relating brain activity to arousal. (A) Differential volt-
lematic.Thesedifficultieshavediscouragedanyelec- age recordings between two electrodes implanted in the brain of
trical investigation of the Drosophila brain beyond a tethered, yet otherwise freely-moving fly provide a measure of
studyingsensorystructures(eyes,antennae,andtheir brainactivity.Simultaneousrecordingsofthoracicvibrationsquan-
tify gross movements from the fly’s legs, wings, and abdomen.
respective lobes) in immobilized animals. It is un-
On-line analysis of the fly’s movement (or lack thereof) allows
likely that such sensory structures will be central to
arousalthresholdstobedeterminedbyvariousautomatedstimuli
thegenerationofdifferentarousalstates,asdiscussed such as light flashes or mechanical taps. An optional arena sur-
intheearliersleepsection.Opticalrecordingsofcer- rounding the fly provides visual stimuli that can also be used as
tain central brain structures, such as the mushroom arousalstimuli.(B)Sleepepochsdefinedbylackofmovementand
heightened arousal thresholds are characterized by lower power
bodies,havebeenattempted(Wangetal.,2001;Fiala
of all frequencies compared to neighboring waking epochs. An
etal.,2002).These,however,havenotestablishedthe
overnight average is shown for a sample fly. (C) Presentation of
correlations to behavior in order to confirm states of visual stimuli (a moving bar) results in greater brain activity in
arousal. How else to know if the animal has actually the 20–30Hz range when the stimulus is in front of the fly. The
smelled or seen something than by a behavioral re- magnitude of this evoked response is increased (thicker line) by
stimulusnovelty,classicalconditioning,andothersalienceeffects
port? This issue is separate from establishing neural
(vanSwinderenandGreenspan,2003).
correlates of sensory stimulations, an extensive arena
of research in other insects such as cockroaches and
locusts(Perez-Oriveetal.,2002). electrodes and tethered by their head and thorax, are
Recently, Nitz et al. (2002) have described a novel able to “walk,” “fly,” groom, eat, and react to visual
preparation that allows for the recording of Local and olfactory stimuli in this preparation (Fig. 2A).
FieldPotentialsfromtheDrosophilabraininbehaving A thorax electrode, as well as video monitoring,
animals(Nitzetal.,2002).Fliesimplantedwithbrain continuously records gross fly movements and other
B.vanSwinderen,R.Andretic/BehaviouralProcessesxxx(2003)xxx–xxx 9
behaviors such as proboscis extensions, flight, 20–30Hz responses in the Drosophila brain (power
twitches,orgrooming. and coherence) are modulated by image saliency.
Nitz et al. (2002) demonstrated by this preparation Hence, the 20–30Hz response reflects a level of
that arousal, as defined behaviorally, is accompanied arousal elicited by the salience of the object at hand
with changes in brain activity in Drosophila. As dis- (Fig. 2C). It is a narrower correlate of arousal levels
cussedearlier,Shawetal.(2000)showedthatperiods than the overall increase in frequency power associ-
of immobility lasting more than 5min are associated ated with a waking brain. Sleep, wake, and salience
with heightened arousal thresholds in the fly. Teth- may thus, each have their fly neural correlates, much
eredflieswithimplantedelectrodesshowthisaswell, like alpha, delta, and gamma oscillations represent
and hence also sleep. An automated arousal-testing suchstatesinhumans.Theseneuralcorrelateswillbe
paradigmconfirmedthatinthispreparationfliesdoin- a valuable tool in the investigation of mechanisms of
deedhaveincreasedarousalthresholdsfollowinglong arousalinDrosophilaandotheranimals.
(5min) periods of immobility. The arousing stimuli
tested included light flashes and mechanical tapping,
both of which satisfied the necessary criteria for 5. Conclusions
identifying states of decreased arousal or sleep. Con-
current brain recordings revealed that during such This review covers only a portion of the behav-
behaviorally defined sleep epochs flies displayed ioralstudiesinvestigatedinDrosophilawherearousal
an overall decrease in brain activity (measured as levels might play an integral part of the observed re-
1–100Hzpower).Assuch,thesignificantdecreasein sponse.Mostbehavioralassaysrequirethattheflybe
power (activity) found in the Drosophila brain dur- active or aroused either in response to some salient
ing sleep is a neural correlate of decreased arousal stimulus from its environment (learning and memory
(Fig. 2B). By the same token, increased power is a assays)orgeneratedendogenously(circadianlocomo-
neural correlate of increased arousal, independent of tion).Drosophilabehavioralresearchisextensive,and
fly movement. This last point is crucial as it indicates many behavioral paradigms such as those involving
thatarousalisabrainstatecorrelatedwithbehaviorbut operantandclassicalconditioningsuggestthatperfor-
independent of moment-to-moment movement. Like mance is partly under genetic control (Waddell and
theEEG,flybrainLocalFieldPotentialscantherefore Quinn, 2001). Yet fly behaviors have rarely been ex-
be used as an quasi-behavioral phenotype on their amined in relation to the homeostatic processes char-
own as well as serving as markers of arousal states. acteristicofarousal.Rather,sincemostbehaviorsare
UnlikethehumanEEG,theDrosophilabraindoesnot typicallyfickle,muchcareistakentoreduce“environ-
appear to generate slow waves during sleep, which is mental variance” by keeping test conditions identical
not surprising given the very different neuroanatomy. to certain empirical standards. Some of this environ-
Finally, the Drosophila brain activity described by mentalvarianceismostlikelyhiddenarousalvariance
Nitz et al. is sensitive to VGAs (CO and Isoflurane) that is just not properly understood. For starters, ex-
2
at clinically-relevant concentrations, further suggest- tended quiescence, or sleep, decreases behavioral re-
ing a connection to a common arousal mechanism sponsivenesstostimuli.Itislikelythatothercomplex
(vanSwinderen,manuscriptinpreparation). behavioral responses and not just sleep also depend
In a subsequent study, van Swinderen and onhomeostaticprocesses.
Greenspan (2003) use the fly brain signal pheno- ThelimitedstudyofarousalinDrosophilahaspro-
type to investigate neural correlates of salience in ducedthreeimportantconclusionsthatwewouldlike
Drosophila—a narrower manifestation of increased to emphasize. First, the preponderance of behavioral
arousal. Salience is discernable as a heightened be- evidence suggests that control over general arousal
havioral response to one object at the expense of and responsiveness is located in the brain. Decapi-
othersimultaneouspresentations(Nieburetal.,2002). tated flies are mostly immobile and perform reflex
Thus,arousalshiftsdependingonobjectsalience.By behaviors only if stimulated (Yellman et al., 1997;
conditioning flies to associate heat, odor, or novelty Andretic and Hirsh, 2000). Most other lesion stud-
to select images, the authors were able to show that ies (genetic and surgical), with perhaps one curious
10 B.vanSwinderen,R.Andretic/BehaviouralProcessesxxx(2003)xxx–xxx
exception(BookerandQuinn,1981),indicatethatthe alambosKyriacouforpermissiontoreproducefigures
brain controls behavioral performance, learning, and fromtheirpublishedwork:Krishnanetal.(1999)and
“motivation.” The brain is also crucial for producing Tauberetal.(2003).Thisworkwassupportedbythe
clinically-relevantgeneralanesthesiaphenotypes,and NeurosciencesResearchFoundation.
itdisplaysneuralcorrelatesofarousalindependentof
moment-to-momentlocomotion.Asecondconclusion
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