Arousal and differential Fos expression in histaminergic neurons of the ascending arousal system

12 Pages·2005·0.85 MB·English

EuropeanJournal of Neuroscience, Vol.21, pp.1931–1942,2005 ªFederationof EuropeanNeuroscienceSocieties
Arousal and differential Fos expression in histaminergic
neurons of the ascending arousal system during
a feeding-related motivated behaviour
J. L. Valde´s,1 P. Far´ıas,2 A. Ocampo-Garce´s,3 N. Corte´s,3 M. Sero´n-Ferre´2 and F. Torrealba2
1ProgramadeDoctorado enCiencias Biome´dicas, ICBM,Universidad deChile, Santiago,Chile
2Facultadde CienciasBiolo´gicas,Pontificia Universidad Cato´lica deChile, Alameda340,Santiago, Chile
3ICBM,Universidad deChile, Santiago, Chile
Keywords:locuscoeruleus, orexinneurons,polysomnography, radiotelemetry,rat, tuberomammillarynucleus
Abstract
Arousal dependson the concertedactivity ofthe ascending arousalsystem(AAS) but specificstimuli mayprimarily activatesome
nucleiofthissystem.Motivatedbehavioursarecharacterizedbybehaviouralarousal,althoughitisnotknownwhichAASnucleiare
activeduringamotivatedbehaviour.Toaddressthisissue,ratswererenderedmotivatedforfoodbyfastingthemfor1dayandthen
wereenticedwithfoodthattheycouldnotobtainforvaryingperiodsoftime.Westudiedthelevelofarousalbypolysomnographyor
radiotelemetry, and Fos-ir in the AAS, during food enticing. We found a strong arousal and an early increase in Fos-ir in the
histaminergicneuronsfromthetuberomammillarynucleus,after30 minofenticing,followedbyincreasedFos-irinthewholeAASif
foodenticingwasprolongedto1or2hours.Incontrast,foodpresentationtonon-motivatedratsdidnotincreasearousalorFos-irin
the tuberomammillary nucleus. As opposed to the active arousal of the motivated rats, passive arousal induced by sensory
stimulationwasassociatedwithincreasedFos-irinthelocuscoeruleusandtheorexinneurons,butnotwithincreasedFos-irinthe
tuberomammillarynucleusorintheothernucleioftheAAS.Weconcludethatthearousalduringfeeding-relatedmotivatedbehaviour
isassociatedprimarilywiththeactivationofthetuberomammillarynucleus,whiletheotherarousal-relatednucleibecomeactivelater
on.
Introduction
The search for food, water, sexual partner or shelter are examples of 2004)actingonraphenucleineurons.Severalauthors(Stricker,1990;
motivated behaviours which are essential for the survival of the Robbins&Everitt,1995;Pfaffetal.,2002;Gareyetal.,2003)have
individualandthespecies.Allmotivatedbehavioursarecharacterized argued that motivated behaviours have an arousal component that is
by a behavioural arousal, particularly during the appetitive phase. In particularlynotoriousduringtheappetitivephase.Anopenquestionis
mammals, arousal results primarily from the concerted activity of howthedifferentnucleioftheAAScontributetotheincreasedarousal
waking-active neurons (Steriade & McCarley, 1990; PaceSchott & thatunderliestheseekingorappetitivephaseofamotivatedbehaviour
Hobson,2002;Jones,2003)collectivelynamedtheascendingarousal suchas feeding.
system(AAS)(Saperetal.,2001).TheAASneuronsresideinseveral Ithasbeenshownthathistamineaswellasorexinneuronsfromthe
subcorticalregionsthatincludethelocuscoeruleus,theraphenuclei, lateralhypothalamic–perifornicalarea,butnototherneuronsfromthe
the cholinergic basal forebrain and pontomesencephalic nuclei, the AAS, become active in close temporal relation to the behavioural
lateral hypothalamic areaandthe tuberomammillary nucleus. activationthatgoeswiththeanticipationofameal,whentheanimals
WhilethedifferentcomponentsoftheAAScontributetowakeful- have been entrained to a restricted feeding schedule (Inzunza et al.,
ness, each of them may account for particular instances of arousal- 2000; Angeles-Castellanos et al., 2004). This behavioural activation
related behavioural performance (Robbins & Everitt, 1995; Robbins, may be considered an expression of a particular food-searching
2000), so that disruption of a particular AAS component alters in a behaviour,whichissomehowtriggeredbysignalsfromahypothetical
specificwaytheperformanceinbehaviouraltests.Inadditiontothese food-entrainable circadian oscillator (Stephan, 2002). We decided to
subtle differences in the effects of AAS neurotransmitters on useasimplerandmoredirectfood-searchingparadigmnotinvolving
behaviour, there are indications that AAS nuclei may respond to circadian signals, to test the hypothesis that histamine and⁄or orexin
specific stimulation. Arousal can be elicited by sensory stimulation neuronsarethefirstcomponentsoftheAASthatbecomeactiveatthe
actingonthelocuscoeruleus(Aston-Jonesetal.,2000)orbychanges onsetofafood-searchingbehaviour.Thestimulususedtotriggerthe
in physiological parameters, such as an increase in CO (Richerson, behaviourwasthepresentationoffood.Weevaluatedthearousallevel
2
during this behaviour in motivated (fasted) vs. non-motivated (fed)
rats,andquantifiedFos-irasamarkerofneuronalactivation(Cirelli&
Tononi,2000)inAASnuclei.Totestwhethertheactivationpatternof
Correspondence:DrFernandoTorrealba,2FacultaddeCienciasBiologicas,asabove. theAASwasrelatedtothedevelopmentofamotivatedbehaviour,as
E-mail:[email protected] opposedtopassivearousal,wemappedFosexpressioninratsforced
Received13October2004,revised4January2005,accepted17January2005 to stayawake byexperimenters tapping ontheir cages.
doi:10.1111/j.1460-9568.2005.04013.x
1932 J.L. Valde´s etal.
Materials and methods Experimental protocols
Subjects Ratsweresubjectedtoafeedingprotocolofonedayadlibitum,oneday
offast(motivatedrats,n ¼9)oradlibitum(non-motivatedrats;n ¼ 5)
Subjects were 59 male Sprague-Dawley rats weighing 270–350g.
andadayoffoodpresentation.Placingaclosedwire-meshboxfullof
They were kept in individual cages, under 12: 12-h cycles of
foodpelletsontopoftheratcage,for2h,inducedtheappetitivephase
light:darkness,ataconstanttemperatureof22 (cid:1)Candwithadlibitum
offeedingbehaviour.Thewireboxmeasured10cmoneachsideand
provisionof waterandratchow.All experiments were carried outin
thesizeofthemeshsquareswas0.4cm,sothattheratscouldsmell,
accordance to the NIH Guide for the Care and Use of Laboratory
touchandseethefoodbutcouldnotgetitoutoftheboxorgnawthe
Animals(NIH Publicationsno.80–23,revised 1996).Thesurgeryto
pellets through the mesh. To assess the temporal pattern of Fos
implantelectrodesforpolysomnographicorthetelemetricsensorswas
expression in the different nuclei of the AAS, three other groups of
performedunderasepticconditions.Theratswereanaesthetizedwith
ketamine(50 mg⁄kg;ImalgeneTM,RhodiaMerieux,Santiago,Chile) fastedratswerepresentedwiththewireboxwithfoodfor0 min(n ¼ 5
andxylazine(10 mg⁄kg;i.p.;RompunTM,Bayer,Santiago,Chile)i.p. rats),30min(n ¼6rats)orfor1 h(n¼ 5).Twoadditionalgroupsof
rats were used as controls for arousal that was independent of food-
At the end of the surgical procedure the rats received an antibiotic
seekingbehaviour.Onegroupconsistedoffivena¨ıveratsunderbaseline
(BaytrilTM 5 mg⁄kg, i.p.; Bayer) and an analgesic (KetofenTM,
conditions of adlibitum feeding; thisgroupwas usedtoassess basal
0.2mg⁄kg, s.c.)
Fos-irexpressionbetween10.00and12.00 h.Theothercontrolgroup
(fast+forcedwaking;n ¼5)correspondedtofastedratsthatwerekept
awakefor1hbygentlytappingtheratcageevery5 min.Tocontrolfor
Polysomnographicrecordings
the Fos-ir expression thatisrelated tothe act ofeating, westudieda
Twocranialepiduralelectrodes(stainlesssteelscrews)wereplacedas group of five rats that had food for 2h after a 24-h fast. Table 1
describedelsewhere(Ocampo-Garces&Vivaldi,2002).Twostainless summarizestheratsusedtostudyFos-irexpressionintheAAS.
steel neck muscle electrodes were sutured for EMG recording. A All the rats were killed between 10.00 and 12.00h. They were
piezoelectric device at the base of the animal’s cage detected the deeply anaesthetized with 7% chloral hydrate (350mg⁄kg) and
horizontallocomotoractivity.After10daysofpostoperativerecovery, perfused through the left ventricle with a saline flush (100mL)
animals(n ¼12)wereconnectedtoaflexible,counter-balancedcable followed by 500mL of 4% paraformaldehyde in 0.1m phosphate
attached to a slip ring and maintained under a stable 12:12-h light buffer(PB;pH7.4).Thebrainswereremoved,postfixedinthesame
dark cycle in recording cages (30 ·40·30cm) contained in fixativefor2 h,transferredto30%sucroseinphosphate-bufferedsaline
individual isolation chambers (65 ·60·60cm). Ambient tempera- (PBS)with0.02%sodiumazidefor2 daysandblockedinthecoronal
ture was maintained at 21–23(cid:1)C, with food and water ad libitum. planetobesubsequentlycutunderdryiceusingaslidingmicrotome.
Sessions started after at least 2 days of adaptation to recording
conditions. A computer-based data acquisition system sampled,
displayed and stored EEG, EMG and locomotor activity signals. Immunohistochemistry
Sampling was performed at 250Hz after analogue filtering and
WeassessedFos-irexpressioninsevennucleiandareasoftheAAS.
conditioningofsignals.Anon-linedisplayallowedforqualitycontrol.
Thetuberomammillarynucleus(TMN)wasidentifiedbyimmunocyto-
Data were stored in 15-s epochs (240epochs⁄h). Two independent
chemistry against the marker enzyme adenosine deaminase (ADA)
scorers assigned epochs to wakefulness, rapid eye movement (REM)
(Senbaetal.,1985).Theventraltegmentalarea(VTA),andthelocus
sleepornon-REM(NREM)sleepbyoff-linepolysomnographicvisual
coeruleus (LC) were identified by tyrosine hydroxylase (TH) immu-
analysis.Wakefulnesswasidentifiedbythepresenceofadesynchro-
noreactivity, and the orexin neurons of the lateral hypothalamic area
nizedEEGandphasicEMGactivity.NREMsleepconsistedofahigh-
amplitudeslow-waveEEGtogetherwithalowEMGtonerelativeto
waking. REM sleep was identified by the presence of regular theta Table 1. Summary of the control and experimental groups of rats used to
activitycoupledwithlowEMGtonerelativetoNREMsleep.Epochs
studyFos-irexpressionintheAAS
scored as wakefulness were further analysed to determine active
waking or quiet waking by means of visual inspection of locomotor Groups n Descriptionoftreatments
activity.Thepresenceofhorizontalmovementdefinedactivewaking.
Epochs with artifacts were excluded from analysis. Hours containing Experimental(motivated)
Fast+0minfoodpresentation 5 24hfast
>10% of damaged data were excluded from statistics. The scoring
Fast+30minfoodpresentation 6 24hfastfollowedby30min
procedureassignedeachepochtothestatethatoccupied>50%ofthat
presentationofboxedfood
epoch. The brains of these rats were not used for Fos immunohist- Fast+1hfoodpresentation 5 24hfastfollowedby1h
ochemistry. presentationofboxedfood
Fast+2hfoodpresentation 11 24hfastfollowedby2h
presentationofboxedfood
Control
Locomotoractivityand coretemperaturerecordings
Na¨ıve(baseline) 5 Adlibfeeding
Fourteenratswereimplantedwithradiotelemetricsensors(PDT-4000 Non-motivated 5 Adlibfeedingfollowedby2h
presentationofboxedfood
E-mitter sensor from MiniMitter, Sun River, OR, USA) in the
Fast+forcedwaking 5 24hfastfollowedbywaking
peritoneal cavity, after a small midline abdominal incision. The rats
uponceevery5minfor1h
hadaweektorecoverfromsurgerybeforethecontinuousrecordings Fast+2hfeeding 5 24hfastfollowedby2h
started. Core body temperature and locomotor activity data were feeding
acquiredusingtheVitalViewsoftware(MiniMitter).After2 hoffood Total 47
presentation, these 14 rats were anaesthetized and their brains
processedfor Fos-immunoreactive (-ir) immunohistochemistry. n,numberofrats.
ª2005FederationofEuropeanNeuroscienceSocieties,EuropeanJournalofNeuroscience,21,1931–1942
Arousalsystem andfeeding 1933
(LHA) were identified by using an antiorexinA antibody. We used VTA or the LC, or orexin-ir neurons in the LHA. The second
cytoarchitectoniccriteria(seebelow)toidentifythedorsalraphe(DR), immunostaining was done after an overnight rinse in PBS with
the latero dorsal tegmental nucleus (LDT) and the substantia innom- 0.02% sodium azide. The second immunostaining was revealed with
inata(SI). DAB with no nickel intensification, which yielded a brown
Weobtainedthreealternateseriesof50-lmsections.Oneserieswas cytoplasmic precipitate that contrasted with the dark violet nuclear
stained with Cresyl Violet and the other two series were used for DAB–nickellabellingoftheFos-ir.Theantiserausedwereanti-ADA
immunohistochemistry.Free-floatingsectionswereincubatedin0.3% (polyclonal,raisedinrabbit,diluted1 :10,000,fromChemicon,CA,
H O inPBSfor30min,rinsedinPBSandtransferredtotheblocking USA); anti-TH (rabbit polyclonal, 1 :10,000, Chemicon); antiorex-
2 2
solution (0.4% Triton X-100, 0.02% sodium azide and 3% normal in A (rabbit polyclonal, 1 :2000; from Phoenix Pharmaceutical Inc.,
goatseruminPBS)for1 h.Thesectionswerethentransferredtothe CA, USA).
primaryantibodyincubationsolutionandleftthereovernightatroom The specificity of the antibodies we used has been tested by
temperature. This incubation solution contained the Fos polyclonal preadsorption of the antisera with the respective antigens: Orexin-A
antibody (Ab-5, rabbit polyclonal, from Oncogene, San Diego, CA, (Chen et al., 1999), ADA (Gerashchenko et al., 2001), c-Fos
USA) diluted 1 : 20000 in the blocking solution. The sections were (Constandil et al., 1995) and TH (Strack et al., 1989). We also
rinsed in PBS for 1 h before being incubated in the secondary checked and confirmed the published distribution of the different
antibody solution [Biotin-SP-conjugated AffiniPure goat antirabbit antigens in braintissue.
IgG (H +L) from Jackson ImmunoResearch, PA, USA; diluted
1 : 1000in0.4%TritonX-100and1.5%normalgoatseruminPBS].
Quantificationof Fos-irexpression
After rinsing for 40min, the sections were incubated for 1 h in
Vectastain ABC Elite kit (Vector Laboratories, CA, USA) diluted Theactivationofthedifferentnucleioftheascendingarousalsystem
1 : 500in PBS,rinsedandincubated ina0.05%3–3¢diaminobenzi- was assessed by counting Fos-ir neurons bilaterally in three coronal
dine hydrochloride (DAB) solution containing 0.003% H O and sections from each nucleus, per rat. In the TMN, the LHA and the
2 2
0.05% nickel chloride to get a dark blue reaction product. Selected VTAwecountedneuronsdoublylabelledforFos-irandeitherADA,
sectionsfrombothseries,alreadyimmunostainedfornickel-enhanced orexinorTH,respectively.FortheLC,LDT,SIandDRweusedgrids
Fos-ir, were subjected to a second immunostaining to identify of appropriate size to count Fos-ir neurons. The areas used to count
histaminergic ADA-ir neurons in the TMN, or TH-ir neurons in the Fos-ir neuronsareshown inFig.1.
Fig.1. Photomicrographs ofrepresentativecoronalsectionsthroughtheAASnucleianalysedinthepresentstudy(openrectangles).WecountedFos-irneuronsin
threesectionsfromeachnucleus,andthepicturesshowtheintermediatesection.(a) ADA-ir neuronsfromtheventralTMN(level)4.2mmAP;Swanson,1998).
(b) Orexin-ir neuronsintheLHA–perifornicalarea,atalevel)2.85mm.(c) The VTAasoutlinedbyTH-ir,level)5.2mm.(d) The LCwasidentifiedbythe
presence of TH-ir neurons; the )9.8mm level is shown. The overlying rectangle of 100·450lm delimits the LC area where we counted Fos-ir neurons.
(e) The LDTandtheDRat)8.6mm;the250-lmsquareusedtocountFos-irneuronsintheLDTandthe200·600lmrectangleusedtocountneuronsintheDR
areshown.(f) The 500-lmsquareusedforthesamepurpose,butintheSI.Thephotomicrographshowsa)0.11-mmlevel,justventraltothetemporallimbofthe
anteriorcommissure.Abbreviations:aco,anteriorcommissure,olfactorylimb;act,temporallimboftheanteriorcommissure;AQ,cerebralaqueduct;CP,caudate–
putamen;f,fornix;fr,fasciculusretroflexus;ME,medianeminence;mlf,mediallongitudinalfascicle;MM,medialmammillarynucleus;mp,mammillarypeduncle;
NDB,nucleusofthediagonalband;och,opticchiasm;PCG,pontinecentralgrey;pm,principalmammillarytract;scp,superiorcerebellarpeduncle;SNc,substantia
nigra,parscompacta;SUV,superiorvestibularnucleus;V3,thirdventricle;V3m,thirdventricle,mammillaryrecess;V4,fourthventricle.Scalebar,0.5mm.
ª2005FederationofEuropeanNeuroscienceSocieties,EuropeanJournalofNeuroscience,21,1931–1942
1934 J.L. Valde´s etal.
For the TMN, the intermediate rostrocaudal section (Fig. 1a) nucleiandunderdifferenttreatmentswasanalysedusingtheKruskal–
corresponded to the level of the mammillary recess of the third Wallis one-way anova followed by Dunn’s multiple pairwise
ventricle(approximatelevel)4.2oftheatlasofSwanson,1998).The comparison method.
other two sections were 150lm rostral or caudal to the intermediate
level shown in Fig. 1a. We counted neurons from the ventral TMN
only.
Results
For the orexin-ir neurons in the LHA the rostral section had as
Effect of foodpresentationonarousal
landmarktheanteriorpoleofthemedianeminence;thecaudalsection
had the tip of the ventral horn of the lateral ventricle, and the Food presentation to fasted rats caused an intense arousal character-
intermediate section (Fig. 1b), 450 lm from the rostral and caudal izedbyadesynchronizedandfastEEG,increasedlocomotoractivity
sections,correspondedtolevel)2.85oftheatlasofSwanson(1998). andariseincorebodytemperature.EEGandlocomotoractivitysigns
For the VTA, the intermediate rostrocaudal section (Fig. 1c) was ofarousalincreasedfromtheonsetoffoodpresentationandlastedfor
identifiedbythecaudaltipofthemammillarybodyandcorresponded nearly 1h, while the temperature increase was delayed by about
to level )5.2mm of the atlas of Swanson (1998). At this level, the 20min andalsolasted for 1 h.
mammillary peduncle clearly separated the VTA from the TH-ir Thetemporalprofileofwakeandsleepstatesinthe09.00–12.00 h
substantianigra. Theother twosections were 300lm apart. interval wasstudiedfor3 consecutive days,includingonedayunder
IntheLC(Fig.1d)theTH-irneuronsweresotightlypackedthatit ad libitum feeding, one day under fast (starting at 10.00 h), and the
was not possible to reliably count doubly labelled cells. Here we daywhenfoodwaspresented(at10.00h).Valuesobtainedduringthe
counted Fos-ir neurons within a rectangle of 450lm by 100lm first 2days were averaged to obtain the baseline of the sleep-state
(0.045mm2) centred in the nucleus. The intermediate section profile. Figure 2 shows the proportion of time spent in the different
corresponded to the )9.8 mm section of the atlas of Swanson states of the sleep–wake cycle during baseline compared to the hour
(1998),thatis,justcaudaltotheparabrachialnucleus.Theothertwo precedingfoodpresentationandtothefirsthouroffoodpresentation.
sections were 300lm rostral and caudal to the intermediate level, During the hour before food presentation (09.00–10.00 interval,
respectively. Fig. 2a), fasted rats spent significantly more time in active waking
The LDT was identified in adjacent Nissl-stained sections as a (32.8 ±3.9% of the hour; P¼ 0.02, paired t-test) and less time in
magnocellularcellgroupformingaroundnucleusneartheaqueduct. NREM sleep (42.9± 3.7%;P¼ 0.007,paired t-test) than during the
The intermediate section, shown in Fig. 1e, corresponds to level baseline days (20.2 ±3.4 and 53.4 ±2.7%, respectively). During
)8.6mm of the atlas of Swanson (1998), where the LDTabuts the the 10.00–11.00h interval, food presentation to fasted rats increased
superiorcerebellarpeduncle.Weuseda250-lmsquare(0.0625 mm2) theproportionoftimespentinactivewakingto72.1%,mainlyatthe
to count Fos-ir neurons and to evaluate LDT activation. The first expense of the time spent in NREM sleep (16.5%) but also to time
sectionwas300lmrostralandthethirdsection600lmcaudaltothe spent in REM sleep (4%). These changes were significant (paired
intermediatelevel. t-test; P< 0.001 for active waking and NREM sleep; P ¼0.002 for
TherostralDRwasidentifiedinNissl-stainedsectionsatthesame REM sleep). The time spent in quiet waking did not change with
levels as the LDT. We used a vertical 200·600-lm rectangle respect to baseline days. During the 10.00–11.00 interval of the
(0.12mm2), centred on the midline, and just below the cerebral baseline days the rats spent 13.5% in quiet waking, 34.1% in active
aqueduct or below the floor of the fourth ventricle. The intermediate waking, 9.9% in REM sleep and 42.5% in NREM sleep. During the
section,600lmapartfromtheothertwosections,isshowninFig.1e, second hour of food presentation, the rats did not show significant
anditwasfrom the same )8.6 mmlevel usedfor the LDT. differencesin sleep–wake profilerelative tobaseline days.
Inthe SIwe useda500-lmsquare(0.25 mm2)as acounting grid Oncewewerecertainthatfoodpresentationinducedastrongarousal
(Fig. 1f). The intermediate section is depicted in Fig. 1f and was infastedrats,asdemonstratedbythelargeincreaseinactivewaking,
identified as the section where the temporal limb of the anterior we conducted studies to correlate locomotor activity and core
commissure was longest. This section corresponded to level temperature with the pattern of Fos-ir expression in the AAS nuclei
)0.11 mmoftheatlasofSwanson(1998).Atthislevel,thecounting in motivated (fasted) and non-motivated (fed) rats. We continuously
squarewasplacedjustbelowthecentralportionofthetemporallimb measured locomotor activity and core temperature for 3 days in two
oftheanteriorcommissure.Thelateraledgeofthesquareabuttedthe groups of rats.Nine rats spent one dayunder adlibitum feedingand
fundusofthestriatum.TherostralSIsectioncorrespondedtothemost onedayunderfasting,andthenwerepresentedwiththeboxedfoodon
caudal section where the anterior commissure displayed no temporal themorningofthethirdday.Fiveratsspent2 daysunderadlibitum
limb (level +0.10mm). Here the counting square was placed just feedingandthenwerepresentedwiththeboxedfood.Therecordings
below the anterior commissure. The caudal section corresponded to performedduringthe2 daysbeforefoodpresentationshowednormal
themostanteriorsectionwithafusedanteriorcommissure(atlaslevel circadianrhythmsoflocomotoractivityandofcoretemperatureinboth
)0.26mm),andherethecountingsquarewascentredalongavertical groups of rats (Fig. 3 shows data from the group of rats that fasted).
line that was tangential to the ventricular border of the caudate– After24hoffasting,theratsshowedafallincoretemperature(arrow
putamen;the upperedgeof the squareabutted the internal capsule. inFig.3)relativetothesameclocktimeunderadlibitumfeeding.In
the06.00–10.00hinterval,fastedratshadamean± SEMtemperature
of37.1 ±0.03(cid:1)C,whilethesameratsunderadlibfeedingcondition
Statistical analysis
hadacoretemperatureof37.4 ±0.04(cid:1)C(P¼0.005,Mann–Whitney
We usedthe SigmaStat 3.0 software (SPSSInc., Chicago, IL, USA). rank-sum test). A fall in core temperature after a 24-h fast has been
Accumulatedlocomotoractivityandthepatternofsleep–wakecycles describedpreviously(Nagashimaetal.,2003).
were analysed with paired Student’s t-tests. The temporal course of A representative actogram illustrates (Fig. 4a) the daily rhythm of
locomotor activity and of temperature were analysed with two-way locomotoractivityofasingleanimalduringtheadlibitumday,during
repeated-measures anova, followed by the Holm–Sidak multiple the day of fast and during the day of food presentation. This rat
comparison method. The number of Fos-ir neurons in the different showed a circadian rhythm of activity with the expected maximum
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Arousalsystem andfeeding 1935
Fig.2. Percentage oftimespentindifferentstatesofthesleep–wakecycle,basedonpolysomnographicrecordings(n¼12rats),(a) during thehourpreceding
foodpresentationand(b)duringthefirsthouroffoodpresentation.Asterisksindicateasignificantdifferencewithrespecttothebaselinecondition(P<0.05,paired
t-test).AW,activewaking;NREM,slow-wavesleep;QW,quietwaking;REM,rapideyemovementsleep.*P<0.05.
Fig.3. Circadian rhythmsofmeanlocomotoractivityandcoretemperature(includingtheSEMingrey)fromthegroupofnineratsthatwerepresentedwithfood
for2h.Inopenbackgroundisthedayunderadlibitumfeeding(dayd1ofFig.4a),andingreybackgroundisshownthedayoffasting(from10.00hofdayd2,asin
Fig.4a).Thearrowpointstothefallintemperaturecausedbytheovernightfast.Theemptyandfilledbarsbelowthegraphindicatethelights-onandlights-off
intervals,respectively.
during the dark hours (Fig. 4a) and a minimum during the day. The tion(Fig.4c)wasalmosttwiceasmuchasthebaselinedays(P<0.001,
presentationoffoodfor2 hinthemorningofexperimentalday3(bar pairedt-test).Themotoractivityreturnedtobaselinevaluesduringthe
inFig.4a)inducedalargeincreaseinlocomotoractivitycomparedto secondhouroffoodpresentation(P ¼0.39,pairedt-test).
the previous days when the rat was under ad libitum and then was During food presentation core temperature steadily increased from
fasting. 36.94 ±0.16(cid:1)Cattimezero(10.00h)to37.54 ±0.2(cid:1)Cafter50min
Theanalysisofthe10.00–12.00hintervalforthegroupofninerats of food presentation (Fig. 4d). The two-way repeated-measures
thatfasted(Fig.4b)confirmedthattheyweremuchmoreactiveduring anova showed a time-dependent change in temperature
foodpresentation.Theambulatoryactivityduringthepresentation of (P<0.001). The Holm–Sidak method showed that at the times
foodonday3(sinFig.4b)washigherthanthepreviousdays(d),and indicated by asterisks in Fig. 4d the temperatures were higher than
remainedsoatleastforanother25min.Atwo-wayrepeated-measures 5 min before food presentation (P <0.05). The increase in tempera-
anova test revealed that there was a time-dependent change in ture followed the increase in locomotor activity with a delay of
locomotoractivity(P< 0.001),thatthelocomotoractivitywashigher (cid:1)20min,andlastedabouthalfanhour,toslowlydeclinethereafter.
in the food presentation condition (P ¼0.011), and that there was a We detected no differences between baseline and food presentation
significant interaction (P<0.001) between time and treatment. The (P¼ 0.651),probablybecausethepresentationcurvebeganatalower
multiple-comparison procedure (Holm–Sidak method; P< 0.05) temperature dueto the priorfasting.
showed that, at the times indicated by the asterisks in Fig. 4b, the Incontrast,foodpresentationtonon-motivated(fed)ratsinduceda
experimentalratshadmorelocomotoractivitythanduringthebaseline weak locomotor activation (Fig. 5a; P¼ 0.014, two-way repeated-
days and than at the time 5min before food presentation. The measures anova, followed by a multiple pair-wise comparison,
cumulativeambulatoryactivityduringthefirsthouroffoodpresenta- Holm–Sidak method, P< 0.05), that did not increase the cumulative
ª2005FederationofEuropeanNeuroscienceSocieties,EuropeanJournalofNeuroscience,21,1931–1942
1936 J.L. Valde´s etal.
Fig.4. Effects offoodpresentationonlocomotoractivityandcoretemperatureinmotivatedrats.(a) Actogram showingthelocomotoractivityofasinglerat
recordedthrough3consecutivedays(d1–d3)under12:12hoflight:darkcycle(indicatedinthefirstrow);thebarind3indicatesthe10.00–12.00hinterval
duringwhichtheratwaspresentedwithawireboxfullofratchow.Theratwasunderadlibitumfoodduringd1,andfoodwasremovedat10.00hofd2.The
quantitativeanalysisoflocomotoractivityandofcoretemperaturewascentredinthetimeintervalspanningthe2hfrom10.00to12.00h.Thebaselinecondition
wasobtainedbyaveragingtheactivityofthe10.00–12.00-hintervalduringdaysd1andd2.(b) Mean locomotoractivityofninemotivatedratsduringthe10.00–
12.00h period of day d3. The asterisks indicate the values that were statistically different from the baseline (*P<0.05; two-way anova for repeated
measurements).(c) The cumulativeactivityduringthefirsthour(10.00–11.00h)andsecondhour(11.00–12.00h)ofenticingwithfood(openbars)wascompared
totheactivityatthesametimeduringdaysd1–d2(baselinecondition,darkbars).Theactivitywassignificantlyhigherduringthefirsthourofpresentationtofood
(*P<0.05,Student’st-test).(d) The averagecoretemperatureresponsetofoodpresentation(s),comparedtothetemperatureduringthebaselinecondition(d).
Theasterisksindicatesignificantdifferences(*P<0.05;two-wayanovaforrepeatedmeasurements)relativetotime5minbeforefoodpresentation.
locomotor activity (Fig. 5a, inset; P¼0.38 for the first hour and Fos-irabovethelevelsfoundinna¨ıveratsonlyintheLDTandtheLC
P¼0.75 for the second hour; paired t-test) or the temperature (P <0.001, Kruskal–Wallis one-way anova; P <0.05, Dunn’s
(Fig. 5b; P¼ 0.99, two-way repeated-measures anova) relative to method for multiple comparisons), but not in the TMN, the orexin
baseline days. neuronsintheLHA,theDR,theSIortheVTA.After30minoffood
presentation,fastedratsshowedasignificantincreaseinthenumberof
Fos-ir neurons in the TMN (P<0.001, Kruskal–Wallis one-way
Timecourse ofFos-irexpression in theAAS in fasted rats anova; P< 0.05, Dunn’s method for multiple comparisons), invol-
enticedbyfood
ving (cid:1)15% of the ADA-ir neurons (Fig. 6a and c). The other AAS
The AAS nuclei showed very few Fos-ir neurons in the na¨ıve rats nucleididnotshowthisearlyincreaseinthenumberofFos-irneurons
during the 10.00–12.00h time interval (Fig. 6a and b), as has been relative to the na¨ıve rats after 30min of presentation (Fig. 6). The
described (Ko et al., 2003), indicating a low level of neuronal situationchangedafter60minoffoodpresentation.TheTMNshowed
activation.Intheratskilledattimezerooffoodpresentation,wefound increased levels of Fos-ir while the orexin neurons in the LHA
ª2005FederationofEuropeanNeuroscienceSocieties,EuropeanJournalofNeuroscience,21,1931–1942
Arousalsystem andfeeding 1937
Fig.5. Locomotor activityandbodycoretemperatureduringfoodpresentationtonon-motivated(fed)rats.(a) Mean locomotoractivityoffivenon-motivatedrats
duringthe10.00–12.00hofdayd3.Theasterisksindicatethevaluesthatwerestatisticallydifferentfromthebaseline(*P<0.05;two-wayanovaforrepeated
measurements).Theinsetshowsthetotallocomotoractivityduringthefirstandsecondhouroffoodpresentation,comparedtothebaselineconditionforthesame
rats.(b) The averagecoretemperatureresponsetofoodpresentation(s),comparedtothetemperatureduringthebaselinecondition(d).Therewerenosignificant
differencesbetweenthetwocurves.
(Fig.6a)aswellastheLDT,SI,DRandLC(Fig.6b)hadsignificantly kept forcibly awake for 60min by tapping the rat cage every 5 min.
more Fos-ir neurons than the na¨ıve controls (P<0.001, Kruskal– Theyspentthehourwiththeireyesopen,butmotionless,inastateof
Wallis one-way anova; P< 0.05, Dunn’s method for multiple quiet waking most of the time. These rats had a pattern of AAS
comparisons). By 120min of food presentation every AAS nucleus activation (Fig. 7c and d) entirely different from that of the rats
studied had significantly (same test) more Fos-ir neurons than the activelytryingtogetthefoodfromthewireboxfor1 h.Wefoundthat
na¨ıve controls. The photomicrographs in Fig. 6c–h illustrate the keepingtheratsforciblyawakefor1 hinducedasignificantincrease
increaseinthenumberofFos-irneurons,inresponseto30or120min in the number of Fos-ir neurons in the LC and in orexin-ir neurons
of food presentation, in the TMN and the LC and in the orexin-ir from the LHA (P <0.001, Kruskal–Wallis one-way anova;
neurons ofthe LHA–perifornical area. P< 0.05, Dunn’s method for multiple comparisons). In marked
Theweakbehaviouralactivationofthenon-motivatedratsbyfood contrasttotheratskilledafter1 hofactivearousal(Fig.6aandb),the
presentationwasreflectedinadramaticallydifferentactivationpattern rats kept forcibly awake showed no increase in the number of Fos-ir
of the AAS than that observed in motivated rats. The marked neuronsintheTMN,theDR,theLDT,VTAortheSI.Thenumberof
activation of the TMN, the DR and the orexin-ir neurons, and the orexin neurons with Fos-ir was significantly lower in the forced-
milderactivationofLC,SIandofVTA,observedinthemotivatedrats wakinggroupthaninthe60-minfood-presentationgroup(P<0.05).
after120minoffoodpresentationcompletelydisappearedinthenon- To further analyse the activation pattern of AAS nuclei during the
motivated rats (Fig. 7a and b). Non-motivated rats showed a different phases of feeding behaviour, we studied Fos-ir expression
significant increase in Fos-ir in the LDT (asterisk in Fig. 7b) with after the consummatory phase. For this purpose we studied Fos-ir in
respect to the na¨ıve controls (P <0.001, Kruskal–Wallis one-way ratsthathadfoodavailablefor2haftera24-hfast(n¼ 5).Theyate
anova, followed by Dunn’s method for multiple comparisons, 5.2± 0.7gofchowduringthattime.Theseratsdisplayedactivation
P <0.05), but not different from the increase observed after 2 h of of every AAS nucleus studied exceptthe VTA (Fig. 8). Weusedthe
foodpresentation (Fig. 6b) tomotivated rats. Kruskal–Wallis one-way anova, and Dunn’s method for multiple
TobetterunderstandtherelationshipbetweenFos-irexpressionand comparisons. In the TMN (cid:1)8% of the ADA-ir neurons were also
increasedlocomotoractivityasamanifestationofarousal,wecarried Fos-ir. While this percentage was significantly higher that na¨ıve
out a correlation study between these variables during food presen- controlsvalues(P< 0.05),itwasnotsignificantlydifferentfromthe
tation.Theresults,showninTable2,indicatethattherewasapositive 23% observed after 2 h of food presentation (Fig. 6a). The orexin-ir
and significant correlation for the TMN, VTA, SI and LHA orexin neurons of the LHA also became active during feeding, with nearly
neurons. In contrast, we found no such correlation, except for the 8% of neurons doubly labelled (P <0.05 relative to na¨ıve rats), not
VTA, when the absolute locomotor activity was used instead of the significantly different from the 22% found after 2 h of food
change inlocomotor activity. presentation. The LC, DR, LDT and SI also became active during
The activation of the AAS in the motivated rats described above feeding(P<0.05relativetona¨ıvecontrols),butonlytheLCandthe
could have been related to the waking-up of the rats caused by the LDT had significantly more Fos-ir neurons following feeding than
sensorystimulationthatnecessarilywentwiththeplacingofthewire following 120min offoodpresentation (P<0.05).
boxoffood.Toassesshowspecificwasthepatternofactivationofthe
AAS relative to the active seeking of food in motivated rats, as
Discussion
opposedtoapassivearousalduetosensorystimulation,weanalyseda
group of five rats that had fasted for the previous 24h. These Thepresentstudyshowsthatfood-seekingbehaviourhasanimportant
experimentswerealsoconductedfrom10.00to12.00 h.Theratswere arousalcomponentthatisrelatedtoaspecifictemporalpatternofFos-ir
awakened by placing the empty wire box on top of their cages, and oftheAASnuclei,wheretheTMNisthefirstnucleusoftheAASto
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1938 J.L. Valde´s etal.
Fig.6. Activation ofthedifferentnucleioftheAAS,assessedbytheexpressionofFos-ir,duringthepresentationoffoodtomotivatedrats.Wecountedthenumber
ofFos-irneuronsinratsunderadlibitumfeeding(na¨ıvecontrols)thatwerekilled,astheexperimentalrats,inthe10.00–12.00hinterval.Theexperimentalratswere
killedafter0,30,60or120minoffoodpresentation.(a) Mean±SEM percentageofADA-irthatalsoexpressedFos-irintheTMN,oforexin-irneuronswithFos-
irintheLHA,andofTH-irneuronsintheVTAthatalsohadFos-ir.TheasterisksindicatethetimepointswhenthesenucleihadmoreFos-irneuronsthanthena¨ıve
rats(P<0.05;one-wayanova).(b) Number ofFos-irneurons⁄mm2intheLC,DR,LDTandSI.Labelsasin(a).(c–h)PhotomicrographsofFos-irneuronsin
(candf)theventraldivisionofthetuberomammillarynucleus(TMN),doublyimmunostainedforadenosinedeaminase(ADA)-ir,of(dandg)orexin-irneuronsin
theLHA–perifornicalarea(LHA),andof(eandh)thelocuscoeruleus(LC)immunostainedfortyrosinehydroxylase(TH).Theratswerekilledafter(c–e)30minor
(f–h)120minoffoodpresentation.Thebluearrowspointtodoublylabelledneurons.Scalebar,50lm.*P<0.05.
show Fos-ir during this behaviour. The other AAS nuclei become motivatedrats,orthanduringabaselineday.Theaveragetimespent
activenearly30minaftertheactivationofthehistamineneurons.The in active waking during the first hour offood presentation was 72%,
early activation of the histamine neurons seem specifically related to which is a large increase from the 34% found under baseline
the increased arousal during this feeding-related behaviour, because conditions.Arousalandcoretemperatureoften,butnotalways,covary
non-motivatedratshadlowerlevelsofarousalandnoTMNactivation in healthy animals, as exemplified by the circadian rhythms of both
during food presentation. Moreover, the arousal forced by strong variablesandbytheobservationthatarousal-inducingstimulantssuch
sensory stimulation failed to activate the tuberomammillary nucleus asd-amphetamine(Pigeauetal.,1995a)orcaffeine(Bonnet&Arand,
andonlyactivatedthelocuscoeruleus. 1992),butnotmodafinil(Pigeauetal.,1995b;Scammelletal.,2000),
The rats motivated to get food had significantly more waking increase body temperature. Other states of high arousal, such as
episodes,spentmoretimeinactivewaking,hadmuchmorelocomotor immobilisation stress (Shibata & Nagasaka, 1984), are known to
activity and had larger increases in core temperature than the non- increase body temperature. The increase in temperature during food
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Arousalsystem andfeeding 1939
Fig.7. Fos-ir expressionintheAASnucleiof(aandb)non-motivatedratspresentedwithfoodfor120min,andof(candd)ratskeptforciblyawakefor60min.
TheasterisksindicatetheAASnucleithathadsignificantlymoreFos-irneuronsthanthena¨ıvecontrols.TheyweretheLDTinthenon-motivatedratsandtheLCand
theorexinneuronsintheforciblyawakegroup(seetextfordetails).
presentationmaybeoneconsequenceoftheincreasedmotoractivity Table2. Pearson product–momentcorrelationcoefficients(CC)andP-values
because the motivated rats were significantly more active. It is also obtained after plotting the number of Fos-ir neurons (as in Fig. 6) in the
possible that the rise in core temperature reflected anticipatory differentAASnucleiagainsttheamountoflocomotoractivityafter1hoffood
physiological priming toobtain andconsumeameal. presentationtomotivated(n¼7)versusnon-motivated(n¼5)rats.
IthasbeenshownthatthedifferentnucleioftheAASincreasetheir
discharge of action potentials as soon as the animal is awakening TMN VTA SI LHA DR LC LDT
(Trulson&Jacobs,1979;Footeetal.,1980;Alametal.,2002;Jacobs
ChangeinLA(cid:1)
et al., 2002; VanniMercier et al., 2003). At time zero of food
P 0.006 0.008 0.022 0.03 0.105 0.129 0.883
presentationwefoundFos-irexpressionabovebackgroundintheLC CC 0.738 0.719 0.649 0.624 0.491 0.463 0.047
andtheLDToffastedrats.ThisincreasedFos-irintheLCandLDTof
TotalLA(cid:2)
fasted ratsmay reflecttherelativelyhigh frequencyofactive waking P 0.185 0.012 0.271 0.212 0.314 0.328 0.782
episodeswefoundduringthehourprecedingfoodpresentation(after CC 0.411 0.695 0.346 0.389 0.318 0.309 )0.089
fasting), compared to the previous baseline days when the rats were
feeding adlibitum. LA,locomotoractivity.(cid:1)This valueisthedifferencebetwenLAduringfood
presentationandbaseline.(cid:2)Corresponds toLAduringfoodpresentation.
Whenthemotivatedratswerekilledafter30minofenticing,only
the TMN showed Fos-ir while the other components of the AAS,
including the LC and the LDT, were silent. After 1 h of food Tononi, 2000), so smaller activation of the AAS nuclei relative to
presentationmostoftheAASnucleiwereengaged,andtheyremained TMN activation may go undetected by Fos-ir expression. Fos
active if the enticing with food was prolonged to 2 h. The relative expressionmaydependonaparticularlyhighrateofactionpotentials
action potential discharge rate of the AAS nuclei during food that increases the intracellular levels of second messengers long
presentation remains an open question. Fos expression may not be a enough to induce Fos expression (Cirelli & Tononi, 2000). The
goodtoolfordiscerningsmallchangesinneuronalactivity(Cirelli& increase in Fos expression in the TMN suggests that arousal might
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1940 J.L. Valde´s etal.
Fig.8. The effectoffeedingfor2honthenumberofFos-irneuronsintheAASnuclei.Whenthefastedratswereallowedtoeat,mostoftheAASnuclei,except
fortheVTA,hadsignificantlymoreFos-irneuronsthanthena¨ıvecontrols(*).MostnucleihadthesamenumberofFos-irneuronsafterfeedingasafterfood
presentationfor2h,exceptfortheLCwhichhadmoreFos-irneuronsafterfeeding,andtheVTAwhichhadmoreFos-irneuronsafterfoodpresentation(seetext).
trigger activity-dependent cascades of second messengers leading to activatedinsequenceduringfeeding,firstinducearousalbyactingon
earlier changes in genes expression in this nucleus. Consistent with corticalandthalamicneuronsandthencontributetosatiety,aneffect
our finding that the TMN gets active first, there are examples of ofhistamineonventromedialandparaventricularhypothalamicnuclei
differentialexpressionofFos-irintheAASnucleiofratskeptawake that isdriven bymastication (Sakataet al., 2003).
by different treatments. In rats kept awake >90% of the time with Our findings are consistent with the observation that histamine is
intermediatedosesofmodafinil,Fos-irwasincreasedintheTMNand releasedduringfeeding.Histaminewasreleasedinthehypothalamus
inorexinneuronsbutnotinotherAASnucleisuchastheLC,LDTor of fasted rats when they ate for 15min (Itoh et al., 1991).
DR (Scammell et al., 2000). In the same line, orexin neurons show Interestingly,histaminereleaseincreasednotonlyduringactualeating
largeincreases inFos-ir inassociation withhigh-arousal waking,but butalsoduringfoodpresentationfor15min.Theseauthorssuggested
not with spontaneous waking, suggesting that orexin neurotransmis- thatreleaseofhistamineisenhancedduringfeeding,butalsodidnot
sion is responsive to high-arousal and not to waking per se (Espana exclude the possibility that behavioural excitation induced by the
etal.,2003).ItispossiblethattheearlyTMNactivationmayreflecta presentation of food caused the histamine release. Histamine is also
faster kinetics of Fos expression in the TMN than in the other AAS released in the hypothalamus of rats forced to swim for 1 h (Endou
nuclei. However, in the forced waking group the LC and the orexin et al., 2001), suggesting that histamine may have been released
neurons, but not the TMN, had increased Fos-ir, indicating that the becauseofthestressor,alternatively,becausetheratsareperforminga
patternofFos-irintheAASnucleiisbetterrelatedtotheprocedureof motivatedbehaviourtosurvive.Intheseexperiments,thecontrolrats
inducingarousalratherthantodifferentkineticsofFos-irexpression. that were just handled, and were presumably awake, showed no
AstraightforwardexplanationfortherecruitmentoftheotherAAS histaminerelease(Endouetal.,2001),suggestingthatgentlesensory
nucleiwouldbethattheTMNcontributedtotheactivationoftheother stimulation is not a potent releaser of brain histamine. The latter
AAS components by means of its known excitatory effects on them. finding agrees with our results showing no Fos-ir expression in the
Histamine excites dorsal raphe neurons (Brown et al., 2002), TMN during gentle sensory stimulation.
cholinergic pontomesencephalic neurons (Khateb et al., 1990; Lin The increased arousal during food presentation to fasted rats
etal.,1996)andbasalforebrainneurons(Jones,2004;Rameshetal., reportedheremaybeconsideredoneconsequenceofTMNactivation,
2004). The central administration of histamine, acting on H1 which was, hypothetically, triggered by an internal decision of the
receptors, increases the activity of noradrenergic, probably LC, hungry rats to actively try to get the food, once food seemed to be
neurons projecting to the diencephalon (Fleckenstein et al., 1994). closeathand,asduringpresentation.Underthisview,theearlyTMN
The cortical activation during early food presentation and the activationmayreflectsomeendogenoussignalsthatpreparetheratsto
increasedsensoryresponsivenessandmotoractivationmayalsohave obtain food by increasing the arousal level. In this sense, our results
contributed to engaging the other AAS nuclei and so maintaining a support thesuggestionsthat histaminergicneuronsplayakeyrole in
levelof arousal for aprolongedtime. exploratory behaviour (Inoue et al., 1996) and in maintaining
Thereisacomplex,andnotwell-understood,relationshipbetween vigilance faced with behavioural challenges (Parmentier et al.,
brain histamine and feeding. The circadian rhythm of extracellular 2002). If exploratory behaviour is considered a motivated behaviour
brainhistaminehasamaximumduringtheactivephase,thatis,when (Hebb, 1955), it is not unreasonable to suggest that TMN neurons
animals eat more (Morimoto et al., 2001). However, central admin- increase arousal in both feeding-related andin exploratory motivated
istration of histamine decreases food intake via H1 brain receptors behaviours. Because arousal is normally accompanied by increased
located in the ventromedial and paraventricular hypothalamic nuclei locomotoractivity,thereisapossibilitythattheearlyTMNactivation
(Morimoto et al., 2001; Sakata et al., 2003). Mice with genetic was actually a consequence of changes in this variable and was not
ablation of H1 receptors (Masaki et al., 2001) or of histidine relatedtoincreasedmotivationforfood.ThenumberofFos-irneurons
decarboxylase (Fulop et al., 2003) are not hyperphagic, but show in the TMN, VTA, SI and LHA showed a positive and significant
reducedresponsestoleptin.ItispossiblethatdifferentTMNneurons, correlation with the change in locomotor activity, but not with the
ª2005FederationofEuropeanNeuroscienceSocieties,EuropeanJournalofNeuroscience,21,1931–1942