Behavioral profile and Fos activation of serotonergic and non-serotonergic raphe neurons after central injections of serotonin in the pigeon (Columba livia)

Behavioral profile and Fos activation of serotonergic and non-serotonergic raphe neurons after central injections of serotonin in the pigeon (Columba livia)
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  Behavioural Brain Research 220 (2011) 173–184 Contents lists available at ScienceDirect BehaviouralBrainResearch  journal homepage: Research report Behavioral profile and Fos activation of serotonergic and non-serotonergic rapheneurons after central injections of serotonin in the pigeon ( Columba livia ) Tiago Souza dos Santos a , Cristiane Meneghelli c , Alexandre A. Hoeller d , Marta Aparecida Paschoalini a ,Lut Arckens e , Cilene Lino-de-Oliveira a , José Marino-Neto a , b , ∗ a Dept. of Physiological Sciences, CCB, Federal University of Santa Catarina, 88040-900 Florianópolis, SC, Brazil b Institute of Biomedical Engineering, EEL-CTC, Federal University of Santa Catarina, 88040-900 Florianópolis, SC, Brazil c Metropolitan School of Blumenau, Blumenau, SC, Brazil d Dept. of Pharmacology, CCB, Federal University of Santa Catarina, Florianópolis, SC, Brazil e Lab. of Neuroplasticity and Neuroproteomics, Department of Animal Physiology and Neurobiology, K.U, Leuven, Belgium a r t i c l e i n f o  Article history: Received 11 November 2010Received in revised form 28 January 2011Accepted 1 February 2011 Keywords: FeedingDrinkingSleepRapheEvolutionAvian a b s t r a c t Central injections of serotonin (5-HT) in food-deprived/refed pigeons evoke a sequence of hypophagic, hyperdipsic and sleep-like responses that resemble the postprandial behavioral sequence.Fasting–refeeding procedures affect sleep and drinking behaviors “  per se”  . Here, we describe the behav-ioral profile and long-term food/water intake following intracerebroventricular (ICV) injections of 5-HT(50, 150, 300nmol/2  l) in free-feeding/drinking pigeons. The patterns of Fos activity (Fos+) in sero-tonergic (immunoreactive to tryptophan hydroxylase, TPH+) neurons after these treatments were alsoexamined. 5-HT ICV injections evoked vehement drinking within 15min, followed by an intense sleep.These effects did not extend beyond the first hour after treatment. 5-HT failed to affect feeding behav-ior consistently. The density of double-stained (Fos+/TPH+) cells was examined in 6 brainstem areasof pigeons treated with 5-HT (5-HTW) or vehicle. Another group received 5-HT and remained withoutaccess to water during 2h after treatment (5-HTØ). In the pontine raphe, Fos+ density correlated pos-itively to sleep, and increased in both the 5-HTW and 5-HTØ animals. In the n. linearis caudalis, Fos+and Fos+/TPH+ labeling was negatively correlated to sleep and was reduced in 5-HTØ animals. In the A8region, Fos+/TPH+ labeling was reduced in 5-HTW and 5-HTØ animals, was positively correlated to foodintake and negatively correlated to sleep. These data indicate that hyperdipsic and hypnogenic effectsof ICV 5-HT in pigeons may result from the inhibition of a tonic activity of serotonergic neurons, whichis possibly relevant to the control of postprandial behaviors, and that these relationships are sharedfunctional traits of the serotonergic circuits in amniotes. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Neurons that produce serotonin (5-hydroxytryptamine, 5-HT)arerelevanttothefundamentalhomeostaticmechanismsinmam-mals, such as sleep [1,2], feeding and drinking behaviors (e.g., [3–7]). The existence of 5-HT neurons in the brainstem is a con-  Abbreviations:  Anl, nucleus annularis; A6 LoC, nucleus locus coeruleus, caudalpart;A8LoC,nucleuslocuscoeruleus,rostralpart;BC,brachiumconjunctivum;BCD,brachiumconjunctivumdescendens;CS,nucleuscentralissuperior;DBC,decussatiobrachiorumconjunctivorum;flm,fasciculuslongitudinalismedialis;GCt,substantiagrisea centralis; LC, nucleus linearis caudalis; nIV, nucleus nervi trochlearis; PrV,nucleus sensorius principalis nervi trigemini; R, nucleus raphe pontis; TIO, tractusisthmo-opticus; Zp-flm, zona peri fasciculus longitudinalis medialis. ∗ Corresponding author at: Department of Physiological Sciences, CCB, FederalUniversity of Santa Catarina, 88040-900 Florianópolis SC, Brazil.Tel.: +55 48 3721 8760. E-mail addresses:, (J. Marino-Neto). served attribute of vertebrate brains [8–10], which may epitomize its importance in the control of primary brain functions. Genescoding for proteins of the 5-HT pathways are highly conserved inprimatesandrodentswithoutsignalsofpositiveselection,suggest-ing that functional constraints may act as major driving forces of their evolution [11,12]. Therefore, any functional advantage pro- vided by the existence of the 5-HT system in the brain couldbe relevant to shaping the evolution of 5-HT circuits. However,the extent to which behavioral functions of 5-HT are dissemi-natedthroughoutvertebratesorrepresenttaxa-specifictraitsofthemammalianbrainremainunclear.Thecontroloffeedinganddrink-ing and their consequences to post-ingestive phenomena may berelevanttotheevolutionof5-HTcircuitfunctions.Theexistenceof feeding-inducedsequencesofdramaticphysiologicalchangesdur-ingpostprandialstatesisobservedinallvertebratespecies[13,14].In mammals, feeding induces a well-known postprandial succes-sion of drinking, maintenance and resting behaviors [15], and the systemicinjectionsof5-HTorofserotoninreuptakeinhibitorswere 0166-4328/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2011.02.002  174  T.S. dos Santos et al. / Behavioural Brain Research  220 (2011) 173–184 shown to produce hypophagia and a behavioral sequence similarto that induced by a food pre-load in food-deprived rats [4,15–18].Similar to the observations in mammals, the central 5-HT cir-cuitryinbirdsisstronglyinvolvedinthecontroloffeeding,drinkingand sleeping. In pigeons ( Columba livia ), fasting-induced feeding isfollowed by increased drinking and preening, and by an increasedslow wave (SWS) and REM sleep [19–21]. Intracerebroventricular (ICV) 5-HT injections in food-deprived pigeons evoke an intensedipsogenic response that is followed by increased sleep and theinhibition of fasting-induced feeding [22,23]. Although these data suggest that 5-HT-related postprandial behavior controls includefunctional traits shared by mammals and birds, the effects of 5-HT in avian species may diverge. ICV injections of 5-HT elicithypophagia in food-deprived and in free-feeding Leghorn chick-ens (selected for increased oviposition and showing a low foodintake) and turkeys; hypophagia is also observed in free-feedingbroilers (selected for increased weight gain and voracious feed-ing) but not in food-deprived broilers. In broilers or turkeys, ICV5-HT injections minimally affect water intake, and this procedurereducesdrinkinginfood-deprivedLeghorns[24–26].ICVinjections of 5-HT provoke intense sleep in neonatal broilers [27] and adult Rhode Island fowls [28], but not in turkeys [26] or Leghorns [25]. These data indicate that 5-HT mechanisms may play significantbut species-dependent roles in sleep and ingestive behaviors, andthat the selection for growth/food intake in birds influences brainresponsiveness to monoamines [29]. Domestic pigeons are devoid of artificial selection for feeding or reproductive phenotypes, area customary laboratory species for behavioral pharmacology andcomparative anatomy studies (e.g., [30]), and thus may better reveal potential plesiomorphic attributes of the avian brain.Drugs acting selectively at different 5-HT receptors fail to com-pletely reproduce the effects induced by 5-HT in pigeons. Drinkingand feeding, but not sleep, are evoked by injections of metergoline(anonspecific5-HTantagonist)orofa5-HT 1B/D  agonist(GR-46611,showntoactpresynapticallytoreduce5-HTeffluxinmammals)inhypothalamic[31]andamygdaloid[32]nuclei.ICVinjectionsofDOI (a 5-HT 2  agonist [22]) evoke hypophagia (but no drinking), while systemic cyproheptadine (a 5-HT 2  antagonist) increases drink-ing and, to a lesser extent, feeding in 24h food-deprived (24FD)pigeons [33]. ICV injections of 8-OH-DPAT (a 5-HT 1A  agonist) ormicroinjections of this drug in the midline mesopontine rapheincreases drinking, feeding and sleep (only after ICV injections) infree-feeding/drinking pigeons. Systemic injections of 8-OH-DPATreducetheactivityof5-HTneurons[34]andevokehypophagiaand sleep, but not drinking, in free-feeding pigeons [35–37]. In 24FD pigeons, systemic injections of zimelidine (a serotonin reuptakeinhibitor; [33]) reduce feeding and drinking, evoke no change in sleep duration, increase SWS and decrease REM sleep [38].Thesedataindicatethatareducedactivityof5-HTcircuits,pos-siblymediatedby5-HT 1A receptoractivation,maycontributetotheinitiation of feeding/drinking behaviors and facilitate sleep in thepigeon, and that the expression of these behaviors may be undera tonic inhibitory control by serotonergic circuits. However, the5-HT-induced behavioral effects have been thoroughly examinedonly acutely in 24FD pigeons treated just before refeeding [22].Food deprivation in pigeons is associated with reduced drinking[21] and strongly affects sleep-related phenomena [39]. As indi- catedabove,refeedingafterfastingisassociatedwithanincreaseinsignsofdrinkingandsleep.Theseeffectsinfluencetheresponsesof food-deprived/refed animals to 5-HT-related drugs and introducepotentially confounding effects evoked by refeeding after fast-ing. Therefore, we present a detailed temporal behavioral profile,includingbothingestiveandpostprandialbehaviors,andlong-termingestiveresponses(e.g.,foodandwaterintake1,2,3and24haftertreatment)toICV5-HTinjectionsinfree-feeding/drinkingpigeons.Tofurtherprobethebrainstemmechanismsinvolvedintheearly5-HT-induced dipsogenic response, we examined the patterns of Fosactivity in serotonergic and non-serotonergic brainstem neuronsafter this treatment using a series of double-labeling experimentsdesigned to reveal the expression of both Fos and tryptophanhydroxylase (TPH, the first-step and rate-limiting enzyme in thebiosynthesis of 5-HT). 2. Methods and materials  2.1. Experiment 1: the ingestive and behavioral effects of ICV injections of 5-HT in free-feeding/free-drinking pigeons 2.1.1. Animals, surgery and ICV injections Adult male domestic pigeons ( C. livia , 390–480g bw,  N  =8), maintained inindividual cages (22–24 ◦ C on a 12:12 light-dark cycle, lights on at 7:00h) withfree access to food and water, were anesthetized with ketamine hydrochloride(50mg/kg, i.p.)/xylazine (10mg/kg, i.p.) and stereotaxically implanted with a stain-less steel guide cannula (26G) aimed at the right lateral ventricle, according tocoordinates derived from the brain atlas of the pigeon [40]. The cannula was anchoredtotheskullwithjeweler’sscrewsanddentalcement,andwasmaintainedpatent by an inner removable stylet. ICV injections were made through an innercannula (30G) extending 1mm from the tip of the guide cannula and connected bypolyethylene tubing to a Hamilton microsyringe (5  l). The volume injected (2  l)was administered over 120s, and a further 120s was allowed for the solution todiffuse from the cannula. All of the experimental procedures were conducted inadherence to the recommendations of the “Principles of Animal Care” (NIH, 1985)and were approved by the local Committee for Ethics in Animal Research (CEUA –UFSC).  2.1.2. Experimental procedures Experiments were performed between 10:00 and 16:00h during the illumi-nated part of the light/dark cycle, when the ingestive behavior is stable and low(see Fig. 1). At least 7days after surgery, each animal was tested with vehicle (Veh, ascorbic acid 5% in distilled water), or 5-HT (5-hydroxytryptamine hydrochloride,Sigma Chemical Co., St. Louis, MO; 50, 150 or 300nmol, freshly dissolved in Veh),according to a Latin-squared design. Immediately after the injections, the animalswerereturnedtotheirhomecages.Duringthefirsthourafterdruginjection,digitalvideo recordings (Sony Handycam MiniDV DCR-HC15) were taken from the homecage,andthelatencytothefirstevent,thetotaldurationandfrequencyofdrinking,feeding, preening, locomotor, exploratory, alert immobility and sleep-like behav-iors were scored using locally developed software (EthoWatcher ® , freely previously described [35–37,41], and are shown in a movie clip available on theInternet[41](doi:10.1016/j.regpep.2007.12.003).Foodpelletsweredeliveredin plastic cups, and water was provided in plastic bottles. At the end of the recordingperiod, food pellets that spilled on the floor were recovered and weighed with thefood remaining in the feeder. Food and water were weighed 1, 2, 3 and 24h aftertreatments.  2.1.3. Histological analysis Attheendoftheexperiments,thepigeonsweredeeplyanesthetized(ketaminehydrochloride: 50mg/kg, and xylazine: 10mg/kg, i.p.), and Evans blue dye (1% indistilled water, 2  l) was injected through the guide cannula. The pigeons wereperfused transcardially with saline followed by a 10% formalin solution. The brainswereremovedandcutinthetransverseplane(100  m)onavibratome(Vibratome1500SectioningSystem).Sectionswerestainedwithcresylvioletandexaminedona light microscope to verify the cannula location.  2.1.4. Statistical analysis: ingestive and behavioral data Food and water intake data were analyzed by a two-way repeated measuresANOVA test (between-subject factor: treatment, within-subject factor: 1, 2 and 3haftertheinjection)usingtheStatistica8.0program(Stasoft,Tulsa,Oklahoma).One-way ANOVA tests (between-subject factor: treatment) were used to compare thebehavioral data and the intake of water or food 24h after treatment. The  post hoc  Duncan’s test was performed for the treatment effect where appropriate. Values of  P  <0.05 were accepted as being statistically significant.  2.2. Experiment 2: the effects of ICV injections of 5-HT on Fos activity in brainstemserotonergic neurons 2.2.1. Experimental procedures Nine adult pigeons (390–430g bw, submitted to the environmental conditions,surgery for ICV cannulation and recovery procedures identical to the described forExperiment 1 were randomly assigned to 1 of 3 groups: Group 1 (5-HTW,  N  =3)received an ICV injection of 5-HT (150nmol) and were returned to their cage with  free access to food and water   for 2h, Group 2 (5-HTØ,  N  =3) received the same treat-ment but had  no access to water (with free access to food ) in the following 2h, andGroup 3 (Veh,  N  =3) received an ICV injection of vehicle (5% ascorbic acid in dis-tilled water (2  l) and were maintained  with free access to food and water in the  T.S. dos Santos et al. / Behavioural Brain Research  220 (2011) 173–184 175 Fig. 1.  (A) Photomicrograph illustrating Fos expression in a representative counting field (in the nucleus annularis) indicating TPH-immunoreactive (TPH+, ← ) and double-labeled, Fos- and TPH-immunoreactive cells (Fos+/TPH+, ⇐ ). Scale bar=100  m. (B) and (C) Schematic drawings of frontal sections of the pigeon’s brainstem showing thelocation of the quantification fields in specific areas. (D) and (E) Schematic drawings of frontal sections of the pigeon’s brainstem showing the distribution of TPH+ (emptysquares),Fos+(filledcircles)andFos+/TPH+labeling(stars)inthedifferentnucleiexamined.ApproximaterostrocaudallevelsofKartenandHodos’satlas[40]ofthepigeon’s brain are indicated in the right upper corner of each drawing. For abbreviations, see the list provided.  following 2h . The 5-HTØ group was the control group for the intense drinkingbehaviorthatoccurredduringthefirst10minafter5-HTtreatment.Behaviorswererecorded during the first hour after treatment, and water/food intake were quan-tified as described in Experiment 1. Two hours after injections, the animals weredeeply anesthetized (ketamine hydrochloride: 50mg/kg and xylazine: 10mg/kg,i.p.)andperfusedtranscardiallywithheparin(intraventricularbolusof1500IU)andasucrosesolution(9.25%in0.02Mphosphatebuffer(PB),pH7.2,at37 ◦ C),followedby 4% paraformaldehyde in PB. The brains were removed, blocked and post-fixedfor 4h in the same fixative, transferred to a 0.01M phosphate-buffered saline solu-tion (PBS, pH 7.2), cut on a vibratome at 40  m in the frontal plane and stored in acryoprotectant at − 20 ◦ C, until required for the reactions.  2.2.2. Immunohistochemistry procedures Unless otherwise stated, all washing and incubation steps during the follow-ing procedures were performed in a humid chamber under gentle shaking, and thewashing steps consisted of three changes (5min each) of 0.01M PBS. Free-floatingsectionswerewashedandblockedfor25mininasolutioncontaining0.05%normalgoat serum, 1% bovine serum albumin, 0.1% Triton X-100, 0.1% gelatin and 0.01%of sodium azide in PBS at room temperature (RT). They were incubated in the pri-mary antibody (anti-Fos primary antibody (1:3000), in 1% bovine serum albumin,0.1% gelatin and 0.01% of sodium azide in PBS, 48h, at 4 ◦ C). A polyclonal anti-Fosantibody, raised in rabbit and directed against a synthetic fragment correspondingto the 21 residues of the C-terminus (KGSSSNEPSSDSLSSPTLLAL; [42]) of the pro- tein product of the chicken’s c-fos gene [43], was used in these experiments. Fos expression, as revealed by this antibody, was shown to be sensitive to a dipsogenicstimulus(i.p.injectionsofhypertonicsaline)inchickens,zebrafinchesandstarlings[43].Theywerewashedandblocked(40min)in0.3%H 2 O 2  in50%methanol,washedagain and incubated (2h, RT) in a goat anti-rabbit biotinylated secondary antibody(VectorLaboratories,1:1000),followedbya1.5hincubationwiththeavidin–biotincomplex (Vector Laboratories, 1:1500). After washing, Fos labeling was visualizedusing 0.05% DAB (3. 3 ′ - diaminobenzidine, Sigma) and 0.015% H 2 O 2 , and enhancedwith 0.05% nickel ammonium sulphate and 0.05% cobalt chloride in 0.01M PBS,which resulted in a black/dark brown nuclear staining.For the double-labeling experiments, the sections already submitted to Fosimmunohistochemistry were washed, incubated in the blocking solution and ina solution containing an anti-tryptophan hydroxylase (TPH) primary antibody(Chemicon International, AB 1541, diluted 1:2000 in 1% bovine serum albumin,0.1% gelatin and 0.01% of sodium azide in 0.01M PBS). This polyclonal antibodywasproducedinsheepfromtherecombinantrabbitTPH[44,45],andhasbeenused to describe the distribution of TPH-containing neurons and their co-localizationwith 5-HT in the brainstem of the pigeon [46], and to reveal TPH in rodent [47–49] and human brainstem [45]. The sections were incubated (2h, RT) with a rabbit anti-sheepbiotinylatedsecondaryantibody(VectorLaboratories,1:1000),followedby incubation with the avidin–biotin complex (1.5h, Vector Laboratories, diluted1:1500). TPH labeling was visualized using 0.05% DAB and 0.015% H 2 O 2  in 0.01MPBS, which resulted in a reddish-brown staining. The sections were mounted onchrome–alum–gelatin-coated glass slides, air-dried for 48h, and dehydrated in agraded series of alcohols and xylene before being coverslipped with DPX (FlukaBioChemika, Sigma–Aldrich, St Louis, MO, USA).Control experiments consisted of the omission of the primary or secondaryantibody from each reaction, which produced no evident staining. As positive con-trols, we added sections of rat brainstem containing the dorsal raphe nucleus to  176  T.S. dos Santos et al. / Behavioural Brain Research  220 (2011) 173–184 theTPH/Fosimmunohistochemicalreactionwithpigeonsections.Theywereexam-inedthroughanopticalmicroscope(Olympus,BH-2),anddigitalphotomicrographs(PixeLINK camera, Ontario, Canada) were taken from representative sections (seedefinitions of counting areas below). Contrast and brightness levels of the pho-tomicrographs were adjusted in PhotoImpact SE software. The brain regions wereidentified and named according to the stereotaxic atlas of the pigeon by Kartenand Hodos [40] and the review of prosencephalic nomenclature by the Avian Brain Nomenclature Forum [50].  2.2.3. Cell counting and data analysis All sections were first surveyed to identify areas expressing Fos, TPH anddouble-labeledcells.Accordingtothispreliminaryanalysis,representativesectionsfor each animal of each experimental group were selected for counting throughpredetermined anteroposterior levels of the brainstem (Fig. 1). To ensure that sections at the same rostrocaudal level were compared across groups, sectionswere assigned for analysis by their position relative to landmarks specified foreachnucleus.Fos-labeled(Fos+),TPH-labeled(TPH+)ordouble-labeled(Fos+/TPH+)cells were quantified in six brainstem nuclei (Fig. 1A), by a single blind-to- condition person (TSS) on 3–6 entire field photomicrographs with ImageJ software( of sections containing the following areas: Nucleus raphe pontis  ( R ): Four quantification fields (QF) in sections correspondingto the A 1.00 stereotaxic level of the pigeon brain atlas [40] were positioned to cover this nucleus. Two QFs were placed horizontally on the more ventral aspectof the nucleus. The other two QFs were placed vertically and immediately dorsalto the former (Fig. 1B and D).  A6  (  formerly the caudal LoC  ; [50]): Three QFs were positioned between the ventro- lateral border of the fasciculus longitudinalis medialis (flm), the BC and the floorof IV ventricle at the A 1.00 stereotaxic level (Fig. 1B and D). Nucleus linearis caudalis  ( LC  ): Five QFs covered the 2 midline cell rows of the LC(A 2.25 stereotaxic level). This region also comprises the central superior nucleus(CS), laterally adjacent to the LC (Fig. 1E). The most ventral field was placed over the ventralmost cells of the LC, and the other 4 lined up dorsally (Fig. 1C). The CS presentedrobustFosimmunoreactivity,butrarelyshowedTPH+ordouble-labeledcells; immunoreactivity was not counted in CS.  A8  (  formerly the rostral part of the LoC   [50]): This area is located laterally adjacentto the flm, ventromedially to the tractus isthmo-opticus (TIO) and ventrolaterallyto the substantia grisea centralis (GCt). Four QFs were aligned vertically to the TIOand horizontally aligned to the flm at the A 2.25 stereotaxic level (Fig. 1C and E). Nucleus annularis  (  Anl ): Six QFs were positioned immediately ventral to the flmalong its entire mediolateral extent and dorsally to the DBC fibers, horizontallyaligned adjacent to each other at A the 2.25 stereotaxic level (Fig. 1C and E).  Zone peri-fasciculus longitudinalis medialis  (  Zp-flm ): The Zp-flm area occupies thedorsolateralregionoftheAnlandextendsdorsally,passingthroughandencirclingthe fiber bundles of the flm at the level of the nIV (Fig. 1C and E). The QFs were placed laterally to the IV ventricle and medially to the flm, dorsomedial to the flmand lateral to the fourth ventricle, and the last 2 fields were placed laterally to theflm.The densities (number of labeled cells per mm 2 ) of Fos+, TPH+ and Fos+/TPH+cellswereanalyzedseparatelyforeachnucleusbyaKruskal–Wallisone-wayANOVA(treatment as factor), followed by the Mann–Whitney  U post hoc   test, when appro-priate. Correlations between behavioral/ingestive indexes and Fos+ or TPH+/Fos+figureswereperformedusingthenon-parametricSpearman’srankcorrelationcoef-ficient (Spearman’s   ), by pooling the data of the 5-HTW, 5-HTØ and Veh-treatedanimals. In all of these tests, values of   P  <0.05 were accepted as being statisticallysignificant. 3. Results  3.1. Experiment 1: the ingestive and behavioral effects of ICV injections of 5-HT in free-feeding/free-drinking pigeons Injections of 5-HT consistently evoked a remarkable behav-ioral sequence consisting of intense drinking behavior, followedby maintenance behavior and then by increased sleep-like pos-tures. The dipsogenic response was associated with an increasedduration (nearly 6-fold higher than controls) and frequency( ≈ 3-fold higher than controls) of drinking, that led to theintake of water amounts corresponding to 3.0–4.2% of thepigeon’s body weight (or 9.1–16.4ml/pigeon with the two high-est doses), which is equivalent to an 8-fold increase from the1-h water intake in Veh-treated animals. The two-way ANOVAtest indicated significant effects of the different 5-HT doses( F  3,84 =267.67,  P  <0.000001) and of the hourly periods after injec-tions ( F  2,84 =25.30,  P  <0.00001) on water intake, but with nosignificant interactions. All 5-HT doses significantly increasedwater intake compared to Veh-injected birds (0.23 ± 0.6ml/100gbw, Fig. 2); these effects were particularly intense after the 150nmol (3.42 ± 0.1ml/100g bw) and 300nmol 5-HT doses(3.11 ± 0.24ml/100g bw) (Fig. 2). At all 5-HT doses, significant increases in drinking duration ( F  3,28 =70.41,  P  <000.1) and fre-quency ( F  3,28 =7.65,  P  =0.006), and a reduced latency to the firstdrinkingepisode( F  3,28 =62.77, P  <0.0001)wereobserved(Fig.3and Table 1).These were short-lived effects; most of the dipsogenic effectsoccurred within the first 15min. When the behavioral effects of a 150nmol dose were analyzed at 15-min intervals during thefirst hour after injection, both drinking duration (dose factor: F  1,56 =29.28, P  <0.0001;timeperiodfactor: F  3,56 =38.07, P  <0.0001)and frequency (dose factor:  F  1,56 =31.74,  P  <0.0001; time periodfactor: F  3,56 =21.66, P  <0.0001)werehigherthanthoseforthecon-trols in the first ( P  <0.0001 for drinking duration and frequency),but not in the 3 subsequent 15 min-periods (Fig. 4). Furthermore, although accumulated water intake remained significantly higherthan that for Veh-treated animals in the second ( F  2,28 =86.34, P  <0.0001), third ( F  2,28 =74.24,  P  <0.0001) and twenty fourth hour( F  2,28 =5.26,  P  =0.005) after treatment, the absolute hourly intakevalues were statistically similar to those of the controls at the endof the second and third hour after the injections (Fig. 2).5-HT injections increased sleep-like behavior. A one-wayANOVA test indicated significant effects of these treatmentson the duration ( F  3,28 =37.06,  P  <0. 0001), latency to the firstepisode ( F  3,28 =6.46,  P  =0. 001) and frequency ( F  3,28 =13.18, P  <0.0001) of sleep-like postures. The two highest doses signifi-cantly increased the duration and frequency, and decreased thelatencyofthisbehavior(Fig.3;Table1).Whenexaminedat15min intervals, the 150nmol dose of 5-HT significantly affected theduration [treatment factor:  F  1,56 =61.97,  P  <0.0001; time periodfactor:  F  3,56 =16.11,  P  <0.0001; also with a significant interac-tion between factors:  F  3,56 =15.64  P  <0.0001] and frequency of sleep [treatment factor:  F  1,56 =14.85,  P  =0.0003; time period fac-tor:  F  3,56 =5.72,  P  =0.001; interaction:  F  3,56 =3.87,  P  =0.013]. Sleepduration increased at all periods, and frequency of sleep episodeswas higher than those for Veh-treated animals during the last tworecording periods (Fig. 4). However, both sleep parameters peaked at the 30–45min interval, and waned in the last 15min of therecording period to levels higher than those for the controls, butlower than the preceding interval (Fig. 4). The duration and fre- quencyofexploratorybehaviorsweresignificantlydecreasedatalldoses, although preening behavior was not affected by 5-HT injec-tions. However, both behaviors were mainly concentrated in the15min interval preceding the sleep peak (data not shown).5-HT injections failed to affect hourly food intake in any of the recording periods, but significantly changed the accumu-lated intake (treatment factor:  F  3,84 =4.34  P  =0.006; time periodfactor:  F  3,84 =25.25  P  <0.0001). The 50nmol ( P  =0.041) and the150nmol ( P  =0.045) doses reduced only the intake accumulated3h after treatment, but this hypophagic effect was absent after24h (Fig. 2). The effects of the 5-HT injections on feeding behav- ior were inconsistent: the 150nmol dose decreased the duration( F  3,28 =3.47 P  =0.03)andfrequencyoffeeding( F  3,28 =3.01, P  =0.04),but the 300nmol dose affected only the latency to start of feeding ( F  3,28 =3.18,  P  =0.043; Fig. 3, Table 1). In the 15-min seg- mentanalysis,significanteffectsonfeedingduration( F  1,56 =12.12, P  =0.001) and frequency ( F  1,56 =7.22,  P  <0.009) were observed,whichdecreasedinthetwolast15minintervals(Fig.4).Theimpact of these changes on the water/food intake ratio was intense in thefirst hour after the treatment, was reduced, but still significantin the accumulated 3-h intake, and was identical to those of theVeh-treated animals after 24h (Fig. 2).  T.S. dos Santos et al. / Behavioural Brain Research  220 (2011) 173–184 177 Fig. 2.  Effects of ICV 5-HT injections (0, 50, 150 or 300nmol) on hourly and accumulated food and water intake (first, second, third and twenty fourth hour after treatment)andonthewater/foodintakeratio(1,3and24h)infree-feeding/free-drinkingpigeons.Alldataareexpressedasmean ± S.E.M.values.(*) P  <0.05comparedtovehicle-treatedanimals.  3.2. Experiment 2: the effects of ICV injections of 5-HT on Fosactivity in pontine and mesencephalic serotonergic neurons In this experiment, the behavior of Veh-treated and of 5-HT-treated birds with access to water and food (5-HTW animals)were indistinguishable from those of the animals in experiment1. 5-HTW animals drank vigorously (3.42 ± 0.3ml/100g bw) andshowed intense signs of sleep (sleep duration: 747.66 ± 39.79s).5-HTØ animals (treated with 5-HT but without access to water)displayed intense exploratory activity (duration: 304.46 ± 81.37s)in the first 15min after injections, showed intense sleep behav-ior (duration: 1156.66 ± 169.48s; frequency: 17.33 ± 2.96), and
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