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Behavioural Brain Research 220 (2011) 173–184

Contents lists available at ScienceDirect

Behavioural Brain Research

journa l homepage: www.e lsev ier .com/ locate /bbr

esearch report

ehavioral profile and Fos activation of serotonergic and non-serotonergic rapheeurons after central injections of serotonin in the pigeon (Columba livia)

iago Souza dos Santosa, Cristiane Meneghelli c, Alexandre A. Hoellerd, Marta Aparecida Paschoalinia,ut Arckense, Cilene Lino-de-Oliveiraa, José Marino-Netoa,b,∗

Dept. of Physiological Sciences, CCB, Federal University of Santa Catarina, 88040-900 Florianópolis, SC, BrazilInstitute of Biomedical Engineering, EEL-CTC, Federal University of Santa Catarina, 88040-900 Florianópolis, SC, BrazilMetropolitan School of Blumenau, Blumenau, SC, BrazilDept. of Pharmacology, CCB, Federal University of Santa Catarina, Florianópolis, SC, BrazilLab. of Neuroplasticity and Neuroproteomics, Department of Animal Physiology and Neurobiology, K.U, Leuven, Belgium

r t i c l e i n f o

rticle history:eceived 11 November 2010eceived in revised form 28 January 2011ccepted 1 February 2011

eywords:eedingrinkingleepaphevolutionvian

a b s t r a c t

Central injections of serotonin (5-HT) in food-deprived/refed pigeons evoke a sequence ofhypophagic, 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, 300 nmol/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 15 min, 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 2 h 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, which

e conroton

is possibly relevant to thfunctional traits of the se

. Introduction

Neurons that produce serotonin (5-hydroxytryptamine, 5-HT)re relevant to the fundamental homeostatic mechanisms in mam-als, 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, caudalart; A8 LoC, nucleus locus coeruleus, rostral part; BC, brachium conjunctivum; BCD,rachium conjunctivum descendens; CS, nucleus centralis superior; DBC, decussatiorachiorum conjunctivorum; flm, fasciculus longitudinalis medialis; GCt, substantiarisea centralis; LC, nucleus linearis caudalis; nIV, nucleus nervi trochlearis; PrV,ucleus sensorius principalis nervi trigemini; R, nucleus raphe pontis; TIO, tractus

sthmo-opticus; Zp-flm, zona peri fasciculus longitudinalis medialis.∗ Corresponding author at: Department of Physiological Sciences, CCB, Federalniversity of Santa Catarina, 88040-900 Florianópolis SC, Brazil.el.: +55 48 3721 8760.

E-mail addresses: marino@ccb.ufsc.br, marino@ieb.ufsc.br (J. Marino-Neto).

166-4328/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.bbr.2011.02.002

trol of postprandial behaviors, and that these relationships are sharedergic circuits in amniotes.

© 2011 Elsevier B.V. All rights reserved.

served attribute of vertebrate brains [8–10], which may epitomizeits importance in the control of primary brain functions. Genescoding for proteins of the 5-HT pathways are highly conserved inprimates and rodents without signals of positive selection, suggest-ing that functional constraints may act as major driving forces oftheir 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-nated throughout vertebrates or represent taxa-specific traits of themammalian brain remain unclear. The control of feeding and drink-ing and their consequences to post-ingestive phenomena may berelevant to the evolution of 5-HT circuit functions. The existence of

feeding-induced sequences of dramatic physiological changes dur-ing postprandial states is observed in all vertebrate species [13,14].In mammals, feeding induces a well-known postprandial succes-sion of drinking, maintenance and resting behaviors [15], and thesystemic injections of 5-HT or of serotonin reuptake inhibitors were

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hown to produce hypophagia and a behavioral sequence similaro 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-uitry in birds is strongly involved in the control of feeding, drinkingnd sleeping. In pigeons (Columba livia), fasting-induced feeding isollowed by increased drinking and preening, and by an increasedlow wave (SWS) and REM sleep [19–21]. IntracerebroventricularICV) 5-HT injections in food-deprived pigeons evoke an intenseipsogenic response that is followed by increased sleep and the

nhibition of fasting-induced feeding [22,23]. Although these datauggest that 5-HT-related postprandial behavior controls includeunctional traits shared by mammals and birds, the effects of 5-T in avian species may diverge. ICV injections of 5-HT elicitypophagia in food-deprived and in free-feeding Leghorn chick-ns (selected for increased oviposition and showing a low foodntake) and turkeys; hypophagia is also observed in free-feedingroilers (selected for increased weight gain and voracious feed-

ng) but not in food-deprived broilers. In broilers or turkeys, ICV-HT injections minimally affect water intake, and this procedureeduces drinking in food-deprived Leghorns [24–26]. ICV injectionsf 5-HT provoke intense sleep in neonatal broilers [27] and adulthode Island fowls [28], but not in turkeys [26] or Leghorns [25].hese data indicate that 5-HT mechanisms may play significantut species-dependent roles in sleep and ingestive behaviors, andhat the selection for growth/food intake in birds influences brainesponsiveness to monoamines [29]. Domestic pigeons are devoidf artificial selection for feeding or reproductive phenotypes, arecustomary laboratory species for behavioral pharmacology and

omparative anatomy studies (e.g., [30]), and thus may bettereveal potential plesiomorphic attributes of the avian brain.

Drugs acting selectively at different 5-HT receptors fail to com-letely reproduce the effects induced by 5-HT in pigeons. Drinkingnd feeding, but not sleep, are evoked by injections of metergolinea nonspecific 5-HT antagonist) or of a 5-HT1B/D agonist (GR-46611,hown to act presynaptically to reduce 5-HT efflux in mammals) inypothalamic [31] and amygdaloid [32] nuclei. ICV injections of DOIa 5-HT2 agonist [22]) evoke hypophagia (but no drinking), whileystemic cyproheptadine (a 5-HT2 antagonist) increases drink-ng and, to a lesser extent, feeding in 24 h food-deprived (24FD)igeons [33]. ICV injections of 8-OH-DPAT (a 5-HT1A agonist) oricroinjections of this drug in the midline mesopontine raphe

ncreases drinking, feeding and sleep (only after ICV injections) inree-feeding/drinking pigeons. Systemic injections of 8-OH-DPATeduce the activity of 5-HT neurons [34] and evoke hypophagia andleep, but not drinking, in free-feeding pigeons [35–37]. In 24FDigeons, systemic injections of zimelidine (a serotonin reuptake

nhibitor; [33]) reduce feeding and drinking, evoke no change inleep duration, increase SWS and decrease REM sleep [38].

These data indicate that a reduced activity of 5-HT circuits, pos-ibly mediated by 5-HT1A receptor activation, may contribute to thenitiation of feeding/drinking behaviors and facilitate sleep in theigeon, and that the expression of these behaviors may be undertonic inhibitory control by serotonergic circuits. However, the

-HT-induced behavioral effects have been thoroughly examinednly acutely in 24FD pigeons treated just before refeeding [22].ood deprivation in pigeons is associated with reduced drinking21] and strongly affects sleep-related phenomena [39]. As indi-ated above, refeeding after fasting is associated with an increase inigns of drinking and sleep. These effects influence the responses ofood-deprived/refed animals to 5-HT-related drugs and introduceotentially confounding effects evoked by refeeding after fast-

ng. Therefore, we present a detailed temporal behavioral profile,ncluding both ingestive and postprandial behaviors, and long-termngestive responses (e.g., food and water intake 1, 2, 3 and 24 h afterreatment) to ICV 5-HT injections in free-feeding/drinking pigeons.o further probe the brainstem mechanisms involved in the early 5-

Research 220 (2011) 173–184

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 infree-feeding/free-drinking pigeons

2.1.1. Animals, surgery and ICV injectionsAdult male domestic pigeons (C. livia, 390–480 g bw, N = 8), maintained in

individual cages (22–24 ◦C on a 12:12 light-dark cycle, lights on at 7:00 h) withfree access to food and water, were anesthetized with ketamine hydrochloride(50 mg/kg, i.p.)/xylazine (10 mg/kg, i.p.) and stereotaxically implanted with a stain-less steel guide cannula (26 G) aimed at the right lateral ventricle, according tocoordinates derived from the brain atlas of the pigeon [40]. The cannula wasanchored to the skull with jeweler’s screws and dental cement, and was maintainedpatent by an inner removable stylet. ICV injections were made through an innercannula (30 G) extending 1 mm 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 120 s, and a further 120 s 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 proceduresExperiments were performed between 10:00 and 16:00 h during the illumi-

nated part of the light/dark cycle, when the ingestive behavior is stable and low(see Fig. 1). At least 7 days 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 300 nmol, freshly dissolved in Veh),according to a Latin-squared design. Immediately after the injections, the animalswere returned to their home cages. During the first hour after drug injection, digitalvideo recordings (Sony Handycam MiniDV DCR-HC15) were taken from the homecage, and the latency to the first event, the total duration and frequency of drinking,feeding, preening, locomotor, exploratory, alert immobility and sleep-like behav-iors were scored using locally developed software (EthoWatcher® , freely availableat www.ethowatcher.ufsc.br). The definition and use of these behavioral units havebeen previously described [35–37,41], and are shown in a movie clip available onthe Internet [41] (doi:10.1016/j.regpep.2007.12.003). Food pellets were delivered inplastic 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 24 h aftertreatments.

2.1.3. Histological analysisAt the end of the experiments, the pigeons were deeply anesthetized (ketamine

hydrochloride: 50 mg/kg, and xylazine: 10 mg/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 brainswere removed and cut in the transverse plane (100 �m) on a vibratome (Vibratome1500 Sectioning System). Sections were stained with cresyl violet and examined ona light microscope to verify the cannula location.

2.1.4. Statistical analysis: ingestive and behavioral dataFood and water intake data were analyzed by a two-way repeated measures

ANOVA test (between-subject factor: treatment, within-subject factor: 1, 2 and 3 hafter the injection) using the Statistica 8.0 program (Stasoft, Tulsa, Oklahoma). One-way ANOVA tests (between-subject factor: treatment) were used to compare thebehavioral data and the intake of water or food 24 h after treatment. The post hocDuncan’s test was performed for the treatment effect where appropriate. Values ofP < 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 proceduresNine adult pigeons (390–430 g bw, submitted to the environmental conditions,

surgery for ICV cannulation and recovery procedures identical to the described for

Experiment 1 were randomly assigned to 1 of 3 groups: Group 1 (5-HTW, N = 3)received an ICV injection of 5-HT (150 nmol) and were returned to their cage withfree access to food and water for 2 h, 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 2 h, 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-l ) andl s of frs aminb the l

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abeled, Fos- and TPH-immunoreactive cells (Fos+/TPH+,⇐). Scale bar = 100 �m. (Bocation of the quantification fields in specific areas. (D) and (E) Schematic drawingquares), Fos+ (filled circles) and Fos+/TPH+ labeling (stars) in the different nuclei exrain are indicated in the right upper corner of each drawing. For abbreviations, see

ollowing 2 h. The 5-HTØ group was the control group for the intense drinkingehavior that occurred during the first 10 min after 5-HT treatment. Behaviors wereecorded during the first hour after treatment, and water/food intake were quan-ified as described in Experiment 1. Two hours after injections, the animals wereeeply anesthetized (ketamine hydrochloride: 50 mg/kg and xylazine: 10 mg/kg,

.p.) and perfused transcardially with heparin (intraventricular bolus of 1500 IU) andsucrose solution (9.25% in 0.02 M phosphate buffer (PB), pH 7.2, at 37 ◦C), followedy 4% paraformaldehyde in PB. The brains were removed, blocked and post-fixedor 4 h in the same fixative, transferred to a 0.01 M phosphate-buffered saline solu-ion (PBS, pH 7.2), cut on a vibratome at 40 �m in the frontal plane and stored in aryoprotectant at −20 ◦C, until required for the reactions.

.2.2. Immunohistochemistry proceduresUnless otherwise stated, all washing and incubation steps during the follow-

ng procedures were performed in a humid chamber under gentle shaking, and theashing steps consisted of three changes (5 min each) of 0.01 M PBS. Free-floating

ections were washed and blocked for 25 min in a solution containing 0.05% normaloat serum, 1% bovine serum albumin, 0.1% Triton X-100, 0.1% gelatin and 0.01%f sodium azide in PBS at room temperature (RT). They were incubated in the pri-ary antibody (anti-Fos primary antibody (1:3000), in 1% bovine serum albumin,

.1% gelatin and 0.01% of sodium azide in PBS, 48 h, at 4 ◦C). A polyclonal anti-Fosntibody, raised in rabbit and directed against a synthetic fragment correspondingo the 21 residues of the C-terminus (KGSSSNEPSSDSLSSPTLLAL; [42]) of the pro-ein product of the chicken’s c-fos gene [43], was used in these experiments. Fosxpression, as revealed by this antibody, was shown to be sensitive to a dipsogenictimulus (i.p. injections of hypertonic saline) in chickens, zebra finches and starlings

(C) Schematic drawings of frontal sections of the pigeon’s brainstem showing theontal sections of the pigeon’s brainstem showing the distribution of TPH+ (empty

ed. Approximate rostrocaudal levels of Karten and Hodos’s atlas [40] of the pigeon’sist provided.

[43]. They were washed and blocked (40 min) in 0.3% H2O2 in 50% methanol, washedagain and incubated (2 h, RT) in a goat anti-rabbit biotinylated secondary antibody(Vector Laboratories, 1:1000), followed by a 1.5 h incubation with the avidin–biotincomplex (Vector Laboratories, 1:1500). After washing, Fos labeling was visualizedusing 0.05% DAB (3. 3′- diaminobenzidine, Sigma) and 0.015% H2O2, and enhancedwith 0.05% nickel ammonium sulphate and 0.05% cobalt chloride in 0.01 M 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 0.01 M PBS). This polyclonal antibodywas produced in sheep from the recombinant rabbit TPH [44,45], and has been usedto 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 (2 h, RT) with a rabbitanti-sheep biotinylated secondary antibody (Vector Laboratories, 1:1000), followedby incubation with the avidin–biotin complex (1.5 h, Vector Laboratories, diluted1:1500). TPH labeling was visualized using 0.05% DAB and 0.015% H2O2 in 0.01 MPBS, which resulted in a reddish-brown staining. The sections were mounted on

chrome–alum–gelatin-coated glass slides, air-dried for 48 h, 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

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he TPH/Fos immunohistochemical reaction with pigeon sections. They were exam-ned through an optical microscope (Olympus, BH-2), and digital photomicrographsPixeLINK camera, Ontario, Canada) were taken from representative sections (seeefinitions of counting areas below). Contrast and brightness levels of the pho-omicrographs were adjusted in PhotoImpact SE software. The brain regions weredentified and named according to the stereotaxic atlas of the pigeon by Kartennd Hodos [40] and the review of prosencephalic nomenclature by the Avian Brainomenclature Forum [50].

.2.3. Cell counting and data analysisAll sections were first surveyed to identify areas expressing Fos, TPH and

ouble-labeled cells. According to this preliminary analysis, representative sectionsor each animal of each experimental group were selected for counting throughredetermined anteroposterior levels of the brainstem (Fig. 1). To ensure thatections at the same rostrocaudal level were compared across groups, sectionsere assigned for analysis by their position relative to landmarks specified for

ach nucleus. Fos-labeled (Fos+), TPH-labeled (TPH+) or double-labeled (Fos+/TPH+)ells were quantified in six brainstem nuclei (Fig. 1A), by a single blind-to-ondition person (TSS) on 3–6 entire field photomicrographs with ImageJ softwarewww.rsbweb.nih.gov/ij/) 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 tocover 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 overthe ventralmost cells of the LC, and the other 4 lined up dorsally (Fig. 1C). The CSpresented robust Fos immunoreactivity, but rarely showed TPH+ or double-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 thedorsolateral region of the Anl and extends dorsally, passing through and encirclingthe fiber bundles of the flm at the level of the nIV (Fig. 1C and E). The QFs wereplaced 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 mm2) of Fos+, TPH+ and Fos+/TPH+ells were analyzed separately for each nucleus by a Kruskal–Wallis one-way ANOVAtreatment as factor), followed by the Mann–Whitney U post hoc test, when appro-riate. Correlations between behavioral/ingestive indexes and Fos+ or TPH+/Fos+gures were performed using the non-parametric Spearman’s rank correlation coef-cient (Spearman’s �), by pooling the data of the 5-HTW, 5-HTØ and Veh-treatednimals. In all of these tests, values of P < 0.05 were accepted as being statisticallyignificant.

. Results

.1. Experiment 1: the ingestive and behavioral effects of ICVnjections of 5-HT in free-feeding/free-drinking pigeons

Injections of 5-HT consistently evoked a remarkable behav-oral sequence consisting of intense drinking behavior, followedy maintenance behavior and then by increased sleep-like pos-ures. The dipsogenic response was associated with an increaseduration (nearly 6-fold higher than controls) and frequency≈3-fold higher than controls) of drinking, that led to thentake of water amounts corresponding to 3.0–4.2% of the

igeon’s body weight (or 9.1–16.4 ml/pigeon with the two high-st doses), which is equivalent to an 8-fold increase from the-h water intake in Veh-treated animals. The two-way ANOVAest indicated significant effects of the different 5-HT dosesF3,84 = 267.67, P < 0.000001) and of the hourly periods after injec-

Research 220 (2011) 173–184

tions (F2,84 = 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±0.6 ml/100 gbw, Fig. 2); these effects were particularly intense after the150 nmol (3.42±0.1 ml/100 g bw) and 300 nmol 5-HT doses(3.11±0.24 ml/100 g bw) (Fig. 2). At all 5-HT doses, significantincreases in drinking duration (F3,28 = 70.41, P < 000.1) and fre-quency (F3,28 = 7.65, P = 0.006), and a reduced latency to the firstdrinking episode (F3,28 = 62.77, P < 0.0001) were observed (Fig. 3 andTable 1).

These were short-lived effects; most of the dipsogenic effectsoccurred within the first 15 min. When the behavioral effects ofa 150 nmol dose were analyzed at 15-min intervals during thefirst hour after injection, both drinking duration (dose factor:F1,56 = 29.28, P < 0.0001; time period factor: F3,56 = 38.07, P < 0.0001)and frequency (dose factor: F1,56 = 31.74, P < 0.0001; time periodfactor: F3,56 = 21.66, P < 0.0001) were higher than those for the con-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 (F2,28 = 86.34,P < 0.0001), third (F2,28 = 74.24, P < 0.0001) and twenty fourth hour(F2,28 = 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 (F3,28 = 37.06, P < 0. 0001), latency to the firstepisode (F3,28 = 6.46, P = 0. 001) and frequency (F3,28 = 13.18,P < 0.0001) of sleep-like postures. The two highest doses signifi-cantly increased the duration and frequency, and decreased thelatency of this behavior (Fig. 3; Table 1). When examined at 15 minintervals, the 150 nmol dose of 5-HT significantly affected theduration [treatment factor: F1,56 = 61.97, P < 0.0001; time periodfactor: F3,56 = 16.11, P < 0.0001; also with a significant interac-tion between factors: F3,56 = 15.64 P < 0.0001] and frequency ofsleep [treatment factor: F1,56 = 14.85, P = 0.0003; time period fac-tor: F3,56 = 5.72, P = 0.001; interaction: F3,56 = 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 peakedat the 30–45 min interval, and waned in the last 15 min of therecording period to levels higher than those for the controls, butlower than the preceding interval (Fig. 4). The duration and fre-quency of exploratory behaviors were significantly decreased at alldoses, although preening behavior was not affected by 5-HT injec-tions. However, both behaviors were mainly concentrated in the15 min interval preceding the sleep peak (data not shown).

5-HT injections failed to affect hourly food intake in any ofthe recording periods, but significantly changed the accumu-lated intake (treatment factor: F3,84 = 4.34 P = 0.006; time periodfactor: F3,84 = 25.25 P < 0.0001). The 50 nmol (P = 0.041) and the150 nmol (P = 0.045) doses reduced only the intake accumulated3 h after treatment, but this hypophagic effect was absent after24 h (Fig. 2). The effects of the 5-HT injections on feeding behav-ior were inconsistent: the 150 nmol dose decreased the duration(F3,28 = 3.47 P = 0.03) and frequency of feeding (F3,28 = 3.01, P = 0.04),but the 300 nmol dose affected only the latency to start offeeding (F3,28 = 3.18, P = 0.043; Fig. 3, Table 1). In the 15-min seg-ment analysis, significant effects on feeding duration (F1,56 = 12.12,P = 0.001) and frequency (F1,56 = 7.22, P < 0.009) were observed,which decreased in the two last 15 min intervals (Fig. 4). The impactof these changes on the water/food intake ratio was intense in the

first hour after the treatment, was reduced, but still significantin the accumulated 3-h intake, and was identical to those of theVeh-treated animals after 24 h (Fig. 2).

T.S. dos Santos et al. / Behavioural Brain Research 220 (2011) 173–184 177

F lateda ns. Alla

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ig. 2. Effects of ICV 5-HT injections (0, 50, 150 or 300 nmol) on hourly and accumund on the water/food intake ratio (1, 3 and 24 h) in free-feeding/free-drinking pigeonimals.

.2. Experiment 2: the effects of ICV injections of 5-HT on Fos

ctivity in pontine and mesencephalic serotonergic neurons

In this experiment, the behavior of Veh-treated and of 5-HT-reated birds with access to water and food (5-HTW animals)ere indistinguishable from those of the animals in experiment

food and water intake (first, second, third and twenty fourth hour after treatment)data are expressed as mean± S.E.M. values. (*) P < 0.05 compared to vehicle-treated

1. 5-HTW animals drank vigorously (3.42±0.3 ml/100 g bw) and

showed intense signs of sleep (sleep duration: 747.66±39.79 s).5-HTØ animals (treated with 5-HT but without access to water)displayed intense exploratory activity (duration: 304.46±81.37 s)in the first 15 min after injections, showed intense sleep behav-ior (duration: 1156.66±169.48 s; frequency: 17.33±2.96), and

178 T.S. dos Santos et al. / Behavioural Brain Research 220 (2011) 173–184

F e firstb s. All da

eotnewmett

d

ig. 3. Effects of 5-HT ICV injections (0, 50, 150 or 300 nmol) on the latency to thehaviors in the first hour following treatment in free-feeding/free-drinking pigeonnimals.

xhibited no feeding behavior. Fig. 5 depicts the localizationf the counted fields and of the general pattern of distribu-ion of TPH+, Fos+ and double-labeled cells in 5-HT-containinguclei of the pigeon’s rostral brainstem. In Fig. 5, samples ofach area double-stained for Fos and TPH are shown togetherith the counting results for each experimental condition. Theean densities of TPH+ somata in the different conditions for

ach nucleus were similar, suggesting that the sampling ofhese cells was not biased in any of the experimental situa-ions.

However, Fos+ and Fos+/TPH+ densities were affected by theifferent treatments. In the R nucleus, Fos+ density increased after

episode, the duration and the frequency of ingestive, sleep-like and exploratoryata are expressed as mean± S.E.M. values. (*) P < 0.05 compared to vehicle-treated

5-HT injections (H2,9 = 7.2, P = 0.03) in both the 5-HTW and 5-HTØanimals compared to the Veh group, with no changes in Fos+/TPH+densities (Fig. 5). The fraction of the total Fos+ somata that werealso TPH+ was reduced significantly (H2,9 = 5.79, P = 0.05) in boththe 5-HTW (P = 0.49) and 5-HTØ (P = 0.49) animals, although thehigh percentage (circa 70%) of TPH+ cells that were double labeleddid not change with the treatments (Table 2). Spearman’s correla-

tion tests indicated that Fos+ density (but not Fos+/TPH+) in 5-HTW,5-HTØ and Veh-treated animals was strongly and positively corre-lated to sleep duration (�: 0.85, P = 0.003), and that frequency (�:0.88, P = 0.001) was negatively correlated with the latency to thefirst sleep episode (�: −0.88, P = 0.009).

T.S. dos Santos et al. / Behavioural Brain Research 220 (2011) 173–184 179

Table 1Effects of 5-HT ICV injections (0, 50, 150 or 300 nmol) on the latency to the first episode, the duration and the frequency of ingestive, sleep-like, exploratory and maintenancebehaviors in the first hour following treatment in free-feeding/free-drinking pigeons.

5-HT dose (nmol) 0 50 150 300

FeedingDuration (s) 27.8 ± 4.6 30.5 ± 5.5 10.8 ± 3.9* 17.6 ± 5.3Frequency 3 ± 0.6 3.7 ± 0.8 1.3 ± 0.4* 2.5 ± 0.8Latency (s) 1461 ± 235 618.7 ± 134.9 1863.2 ± 569.7 2685.6 ± 235.7*

DrinkingDuration (s) 9.3 ± 3.4 26.6 ± 2.5* 70.5 ± 1.2* 61.7 ± 5.1*

Frequency 1.7 ± 0.6 3.8 ± 0.4* 5.3 ± 0.5* 5.3 ± 0.8*

Latency (s) 2665 ± 271 478 ± 44.5* 228.8 ± 49.7* 251.6 ± 47.03*

Sleep-likeDuration (s) 67.3 ± 25.8 162.1 ± 67.9 562.2 ± 23.6* 1097.6 ± 11.1*

Frequency 2.5 ± 0.9 4.3 ± 1.3 12.2 ± 1.3* 14.5 ± 2.4*

Latency (s) 2666.7 ± 274.1 1939.3 ± 401 1409.6 ± 421.4* 692.5 ± 130*

ExploratoryDuration (s) 1409.1 ± 157.8 938.2 ± 154* 602.1 ± 156.6* 246 ± 72.7Frequency 36.8 ± 3.2 23.7 ± 2.5* 19.2 ± 5.7* 12.1 ± 3.1*

Latency (s) 5.2 ± 3.9 7.5 ± 5.1 6.7 ± 3.4 23 ± 11.2

Alert ImmobilityDuration (s) 973.1 ± 279.9 1261.6 ± 206 1608.8 ± 319.8 1376.7 ± 59.1Frequency 11.8 ± 1.9 18.8 ± 2.6* 19.3 ± 3.7* 20.2 ± 1.5*

Latency (s) 739.2 ± 217.5 522.7 ± 156 581.8 ± 282.3 97.1 ± 67

PreeningDuration (s) 865.8 ± 108.8 1031.3 ± 154 695.3 ± 175.3 564.5 ± 211.7Frequency 16.5 ± 3.7 20.3 ± 3.4 14.2 ± 1.7 13.5 ± 2.9Latency (s) 377.2 ± 78.1 536.6 ± 129.9 176.7 ± 39.2 478.2 ± 168.9

LocomotionDuration (s) 247.2 ± 35.1 149.5 ± 32.4 187 ± 60.7 73.2 ± 25.7*

Frequency 21.37 ± 3 15.3 ± 2.4 13.8 ± 3.7 6.1 ± 2.3*

.3 ± 4

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Latency (s) 65.8 ± 20.7 79

ll data are expressed as mean± S.E.M. values.* P < 0.05 as compared to vehicle-treated animals.

In the LC nucleus, total Fos (H2,9 = 5.06, P = 0.05; Mann-Whitneyost hoc test: P = 0.049) and Fos+/TPH+ densities (H2,9 = 5.60,= 0.04; Mann–Whitney post hoc test: P = 0.048) were significantly

educed in 5-HTØ animals. Furthermore, the percent of the totalPH+ somata that were also Fos+ was significantly lower in 5-HTØnimals (H2,9 = 6.25, P = 0.043) than in 5-HTW (P = 0.049) and Veh-reated pigeons (P = 0.047) (Table 2). Correlation tests indicatedhat Fos+/TPH+ density in 5-HTW, 5-HTØ and Veh-treated animalsas negatively correlated to sleep duration (�: −0.74, P = 0.02) and

requency (�: −0.78, P = 0.01), but positively correlated with theatency to the first sleep episode (�: 0.71, P = 0.03). Fos+/TPH+ den-

ity was also negatively correlated to the total water intake (�:0.92, P = 0.01).

In the A8 area, 5-HT injections affected only Fos+/TPH+ cell den-ity (H2,9 = 7.2, P = 0.03), which was significantly reduced in bothhe 5-HTW and 5-HTØ animals, when compared to the Veh group

able 2ffects of 5-HT (150 nmol) or vehicle (VEH) ICV injections on Fos expression in rostral rapreatment.

Nucleus R LC A 6

% Fos+/TPH+ from the total Fos+ cellsVEH 44.3 ± 2.3 60 ± 3 48.6 ± 1.35-HTW 29 ± 2* 58.6 ± 5.2 48.3 ± 2.95-HTØ 32 ± 3.7* 46 ± 8.4 63.3 ± 3.3

% Fos+/TPH+ from the total TPH+ cellsVEH 71 ± 8 75.6 ± 4.8 86.6 ± 1.65-HTW 75 ± 2.3 57.6 ± 10.7 81.6 ± 5.25-HTØ 77.6 ± 3.8 21 ± 2.*,# 42.3 ± 3.3

ata is presented as percentage of double-labeled somata (Fos+/TPH+) from the total FoPH-immunoreactive (TPH+) somata. All data are expressed as mean± S.E.M. values.

* P < 0.05 as compared to vehicle-treated animals.# P < 0.05 as compared to 5-HTW animals. For nuclei abbreviation, see list.

4.6 46.5 ± 11.9 793.6 ± 285.7*

(Fig. 5). The percent of the total TPH+ somata that were also Fos+was significantly lower in both the 5-HTW and 5-HTØ animals(H2,9 = 7.26, P = 0.023), when compared to the Veh-treated pigeons.The fraction of the total Fos+ somata that were also TPH+ waslower in the 5-HTØ group than in Veh-treated pigeons (H2,9 = 6.25,P = 0.044; Table 2). Fos+/TPH+ cell density was significantly and pos-itively correlated to food intake (�: 0.74, P = 0.02), feeding duration(�: 0.83, P = 0.003), frequency (�: 0.76, P = 0.03) and to the latencyto sleep onset (�: 0.73, P = 0.01). On the other hand, Fos+/TPH+cell density was negatively correlated to sleep duration (�: −0.80,P = 0.004) and frequency (�: −0.83, P = 0.004).

Marginally significant effects of the 5-HT injections wereobserved in the A6 area. In the latter nucleus, Fos labeling(H2,9 = 5.42, P = 0.066) and Fos+/TPH+ cell densities (H2,9 = 5.60,P = 0.061) suggested a slight reduction in total Fos labeling(P = 0.050) and in Fos+/TPH+ cell density (P = 0.049) in 5-HTØ

he serotonergic neurons in pigeons allowed (5-HTW) or not (5-HTØ) to drink after

A 8 Anl Zpflm

67.8 ± 1.4 41 ± 3.6 71.6 ± 8.859.6 ± 2.7 43 ± 3 47.3 ± 8.1

* 50 ± 1* 42.3 ± 5.4 36.3 ± 4.9

71.6 ± 2.4 87.3 ± 1.7 50 ± 3.256.6 ± 3.3* 92.3 ± 2.3 35.3 ± 12.3

* 32 ± 3.7*,# 75.6 ± 4.8 18 ± 5.8

s immunoreactive cells in each region (in the three upper rows) or from the total

180 T.S. dos Santos et al. / Behavioural Brain Research 220 (2011) 173–184

F stive at ive 15v

acwMFcPrFna

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ig. 4. Effects of 5-HT ICV injections (150 nmol) on the temporal distribution of ingereatments in free-feeding/free-drinking pigeons. All data are presented in successehicle-treated animals.

nimals, when compared to Veh-treated pigeons (Fig. 5). The per-entage of Fos+/TPH+ cells to the total of Fos+ nuclei in the A6as significantly increased in 5-HTØ animals (H2,9 = 5.51, P = 0.05;ann–Whitney post hoc test: P = 0.043), but the percentage of

os+/TPH+ cells compared to the total TPH+ somata was signifi-antly decreased (H2,9 = 5.85, P = 0.05; Mann–Whitney post hoc test:= 0.046) in the same 5-HTØ animals (Table 2). No significant cor-

elations between sleep or drinking parameters and Fos+/TPH+ oros+ labeling were observed in A6. Injections of 5-HT failed to sig-ificantly affect Fos+/TPH+ or Fos+ labeling in the Anl and Zp-flmreas.

. Discussion

Similar to the results observed in 24FD pigeons [22], ICV injec-ions of 5-HT in free-feeding/free-drinking pigeons induced arompt, transient and vehement increase in drinking. This dipso-

nd sleep-like behavior (SLB) duration and frequency during the first hour following-min segments and are expressed as mean± S.E.M. values. (*) P < 0.05 compared to

genic response led to an intake of water in amounts equivalent to an8-fold increase from the 1-h water intake of Veh-treated animals.Drinking induced by similar doses of 5-HT was even more vigorousin 24FD-refed pigeons (27–41 ml/animal 1 h after a 155 nmol doseof 5-HT vs. 9–14 ml in Veh-treated birds; [22]), but this increasewas proportionally lower (only 3-fold compared to controls) thanthe increase observed in the free-feeding/free-drinking pigeons.Because refeeding after food deprivation is accompanied by anincrease in drinking [19–21], it is possible that 5-HT injectionsjust potentiate food deprivation-induced drinking in 24FD animals.Drinking returned to its usual low levels at the end of the firsthour. After 24 h, the differences between the water intake in Veh-and 5-HT-treated animals were of a magnitude (3–4 ml/100 g bw)

similar to levels observed 1 h after treatment, suggesting that thisexcess intake was rapidly corrected by excretion, with no furtherbehavioral adjustments in drinking.

Hyperdipsic effects were observed after ICV injections of the 5-HT1A/7 agonist 8-OH-DPAT, both in free-feeding/free-drinking and

T.S. dos Santos et al. / Behavioural Brain Research 220 (2011) 173–184 181

F -cont( to watb rticall imals.

2[rt

ig. 5. Photomicrographs illustrating the induction of Fos expression in 4 serotonin150 nmol) in pigeons with free access to food and water (5-HTW), with no accessut treated with vehicle. Scale bar = 50 �m. The graph columns show the mean (ve

abeling for each condition and nucleus. (*) P < 0.05 compared to vehicle-treated an

4FD pigeons [22,35], and after 8-OH-DPAT injections into the LC37]. This effect is attenuated by p-MPPI (an antagonist at 5-HT1Aeceptors). Similar to mammals, 8-OH-DPAT acts pre-synapticallyo reduce the activity of 5-HT neurons and the extracellular 5-HT

aining brainstem nuclei (for abbreviations, see list) 2 h after ICV injections of 5-HTer (with free access to food, 5-HTØ animals) or with free access to food and water,bars) and the individual data (empty circles) density (number of cells per mm2) of(#) P < 0.05 compared to 5-HTØ animals.

levels in pigeons [34], suggesting that drinking induced by thisdrug may be secondary to a reduction in the activity of midlineserotonergic neurons in the pigeon. Consistent with this proposal,injections of metergoline (a 5-HT1/2/7 antagonist) or GR46611 (a 5-

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T1B/1D agonist) evoke a prompt and long-lasting increase in waterntake when injected into the arcopallium intermedium (part ofn avian brain region comparable to the mammalian amygdala)n free-feeding/free-drinking pigeons [32]. These data indicate thexistence of a tonic inhibitory influence on drinking behavior,ossibly exerted by serotonergic inputs directed to the pigeon’smygdala, and suggest that the 5-HT-induced drinking reportedere might be associated with a 5-HT1A receptor-mediated inhi-ition of serotonergic neurons. Such a inhibition may act uponrinking-related angiotensinergic mechanisms, because the pre-reatment ICV with [Sar1,Ile8]-ANGII, a nonspecific ANGII receptorntagonist, dose-dependently attenuates the 5-HT-induced drink-ng in the pigeon [23].

Drinking was followed by increased sleep and by decreasedocomotion/exploratory behaviors after the administration of thewo highest 5-HT doses. The incidence of sleep episodes peaked at0–45 min after injections before waning near the end of the firstecording hour. The occurrence of sleep appears to be independentf the previous intense drinking because the 5-HTØ animals sleptntensely after 5-HT, despite having no water to drink. Further-

ore, the lower dose of 5-HT evoked dipsogenic effects, but failedo affect sleep. In 24FD animals, however, the absence of water after-HT injections precludes the appearance of sleep in the first hourfter injection [22]. Besides affecting drinking mechanisms (as dis-ussed above), food-deprivation evokes profound changes in sleepnd sleep-related thermal and energy metabolism phenomena inhe pigeon and in mammals [39,51]. These results suggest thatasting/refeeding procedures may heavily affect the response of-HT-sensitive sleep- and ingestive-behavior-related mechanisms.

The hypnogenic effects of 5-HT may be mediated by 5-HT1Aeceptors. Systemic injections of 8-OH-DPAT in free-feeding/free-rinking pigeons increase the duration and incidence of sleep [36],nd these effects are blocked by pretreatment with WAY100635 (a-HT1A antagonist). When injected alone, WAY100635 decreaseshe frequency of sleep-like responses, and increases the latencyo the first sleep-like episode. As judged by visual and spectralnalyses of hippocampal EEG activity, the hypnogenic effects of-OH-DPAT include increases in frequency and duration of SWSpisodes electrographically similar to the diurnal sleep in untreatedigeons. In addition to an increase in drinking [35], ICV injectionsf 8-OH-DPAT (at doses higher than those necessary to increaseater intake) can also increase sleep (Dos Santos et al., unpublished

esults), suggesting that sleep may be under the tonic inhibitorynfluence of serotonergic neurons, at least during the light phase inhis diurnal species.

Feeding behavior was only marginally affected by 5-HTnjections. A subtle, short-lasting, but significant hypophagia

as detected only for the 3-h accumulated intake. This weakypophagic effect may be related to a floor-effect, possibly inducedy the low food intake (1.2±0.3 g/h) usually observed in the dayeriod (10–16 h) when the present experiments were performed.evertheless, 5-HT treatments failed to appreciably affect this

nibbling-like” (i.e., low but continuous food intake) eating pattern,ypical of the midday in the pigeon. Furthermore, feeding boutsere totally absent only during the sleep peak, which occurred in

he 30–45 min interval after the treatments, suggesting that thisypophagic effect might be a by-product of the 5-HT-induced sleep.

n contrast, ICV injections of 5-HT (in the dose range used here), ofOI or MK-212 (5-HT2 agonists, [22]) can produce severe hypopha-ia in refeed 24FD pigeons. These data suggest the existence ofifferent controls for the “nibbling” and “fasting-induced” feed-

ng behaviors, and that central 5-HT circuits may not control theormer, residual eating pattern in the pigeon.

In free-feeding/free-drinking pigeons, however, hyperphagicffects are observed after ICV injections of GR46611 (5-HT1B/1D ago-ist that reduce 5-HT efflux in rodents) and after metergoline or

Research 220 (2011) 173–184

GR46611 injections into three medial hypothalamic areas [31,35],and into the nucleus taeniae (TnA, comparable to the medial amyg-dala of mammals [32]). ICV-injected 8-OH-DPAT increases feedingonly at doses higher than those sufficient to produce drinking andsleep ([35], Dos Santos et al., unpublished results). When injectedinto the midline group of 5-HT-immunoreactive containing neu-rons in the rostral brainstem (mainly into the LC; [37]), 8-OH-DPATalso evokes a modest, but significant hyperphagia.

Therefore, it is apparent that similar to the speculations abovefor drinking and sleep, tonically active serotonergic inputs tofeeding-related hypothalamic and amygdaloid districts operate infree-feeding/free-drinking pigeons, and that these feeding mech-anisms are mediated by 5-HT1A and 5-HT1B/D receptors. Because5-HT injections do not evoke feeding in free-feeding/free-drinkingpigeons, these tonically active feeding-inhibitory circuits (at leastthose under the control of 5-HT1 autoreceptors whose activationreduces the presynaptic activity of 5-HT neurons) may not besusceptible to ICV 5-HT injections. It is worth noting that in free-feeding rats, the hyperphagic effects are observed after systemicor intra-raphe 8-OH-DPAT injections (through a somatodendritic5-HT1A autoreceptors-mediated decrease in 5-HT neurons activity,e.g., [52,53]) and by systemic, ICV or intra-amygdaloid injections ofmetergoline (a nonspecific 5-HT 1/2/7 receptor antagonist) [54,55].This evidence indicates that tonic inhibitory 5-HT activity mayrestrain feeding in free-feeding, nibbling birds and mammals [56].

The pattern of Fos expression in serotonergic neurons after 5-HT injections supports some of these speculations, and indicatesadditional roles for ponto-mesencephalic 5-HT-producing cells inthe control of drinking, feeding and sleep behaviors. In the LC, Fos+and Fos+/TPH+ labeling were reduced in 5-HT-treated animals notallowed to drink, and were negatively correlated with drinking andsleep-like behaviors. This decrease appears to be relatively selectivefor the serotonergic neurons in this nucleus, and to be linked tothe absence of drinking in this situation (because these animalsshowed increased sleep), suggesting that the 5-HT-induced “thirst”is associated with a reduced activity of 5-HT neurons in the LC, andthat this activity is restored by post-ingestional drinking-evokedsignals.

In the pontine raphe (R), Fos+ density increased after 5-HT injec-tions in both the 5-HTW and 5-HTØ animals, and was positivelycorrelated only to sleep behavior indexes. Furthermore, 5-HT injec-tions induced intense sleep in both groups but failed to affect Fosexpression in the serotonergic neurons of this nucleus. Therefore,the activation of non-serotonergic neurons in this nucleus may berelated to the intense 5-HT-induced sleep. It should be noted thatsleep peaked just (nearly) 1 h before the perfusion, and the activa-tion of these non-5-HT cells of the R nucleus may only precede thesleep surge itself. However, 5-HT injections reduced Fos+/TPH+ inboth the 5-HTW and 5-HTØ animals in a more rostral and lateral5-HT-containing nucleus, the A8. Therefore, this effect is appar-ently unrelated to the intense drinking in 5-HTW pigeons, or tothe intense “thirst” in the 5-HTØ animals. Furthermore, the num-ber of Fos+/TPH+ cells was negatively correlated with sleep indexes,suggesting that a reduction in the activity of 5-HT cells at this siteunderlies the expression of sleep in the pigeon.

It should be noted that the injections of both Veh and 8-OH-DPAT into the A8 tend to decrease sleep and to increase alertimmobility, when compared to untreated animals [37]. These dataindicate that serotonergic neurons in A8 may be related to sleep, buttheir activity is not associated to a 5-HT1A autoreceptor-mediatedsleep (as suggested by the ICV 5-HT and 8-OH-DPAT experiments).

The hypothesized 5-HT1A receptor-mediated reduction in 5-HTactivity associated with 5-HT-evoked sleep, if it exists, may occurin serotonergic populations not examined in this study. The den-sity of Fos+/TPH+ cells in A8 was also positively correlated to foodintake, but this may be secondary to the strong negative correlation

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ith sleep, because feeding was reduced only at the peak of sleepehavior. Furthermore, injections of 8-OH-DPAT in this region doot affect feeding in free-feeding/free-drinking pigeons [37].

Ingestive behavior-dependent changes in Fos activity of rostralaphe neurons are also observed in the rat [57]; food-intake antici-ation, searching, ingestion and satiety states are accompanied by aifferential activation of the raphe nuclei. Interestingly, extensivevidence in rodents and carnivores indicate that, during waking,-HT neurons of the dorsal raphe nucleus are steadily active, buthat they reduce their firing just before and during SWS, and nearlytop during REMS (e.g., [1,2,58]). Although we cannot directly com-are each avian raphe nuclei to a mammalian correspondent, theseata suggest that the tonic activity of certain groups of raphe neu-ons (both 5-HT and non-5-HT) may contribute to the maintenancef waking and to the sleep-waking changes that occur during theeri-prandial scenario in both mammals and birds.

It should be noted that these conclusions may be only partiallyalid for avian species in general. As indicated in the introduction,CV 5-HT injections in a dose range similar to the one used here thatroduce hypophagia fails to affect (or even decrease) drinking inood-deprived chickens and turkeys (poultry species of the Orderalliformes; [24–26]). In chickens, long-lasting sleep is observedfter intravenous [59,60], ICV and intrahypothalamic 5-HT injec-ions [27,28,61]. In the quail (another Galliformes poultry species),ystemic injections of the 5-HT precursor L-hydroxytryptophan,ut not of 5-HT, increase water intake [62–64]. Systemic injectionsf hydroxytryptophan or 5-HT in quails evoke intense sleep [62,64].ecreased food intake has been observed in free-feeding and food-eprived quails after a tryptophan-enriched diet [62,65], but theffects of these treatments on drinking or sleep were not reported.

Therefore, although the 5-HT circuitry appears to be importanto the hydromineral balance, energy homeostasis and sleep controln birds, intense inter-taxa variation in the serotonergic mecha-isms involved in these behaviors may exist. Further scrutiny mayelp to identify the widespread and shared functional attributesf the generally ancient 5-HT circuitry. The association of a partic-lar pattern of 5-HT circuit operation to the behavioral sequencef satiety (BSS, [15]) may indicate a conserved functional attributef the amniote brain. It should be noted, however, that the par-icular mechanisms by which this coordination is achieved mayiffer significantly in the pathways and synaptic aspects betweenhese classes. These functional specializations or apomorphic char-cters may indicate that, despite some general similarities, both theSS and the possibly underlying serotonergic mechanisms relatedo energy balance, hydromineral homeostasis and sleep may beulnerable to extensive change during their evolution.

cknowledgments

This study was supported by CNPq and FAPESC research grantso J. Marino-Neto (proc. 471888/03-6 and 300308/2007-8), andy a FAPESC grant (CON04539/2008-0) and an Alexander vonumboldt Research grant (Equipment subsidy 3-8151/07073) to. Lino-de-Oliveira. T.S. dos Santos and C. Meneghelli receivedSc. fellowships from Capes. We wish to thank the excellent and

evoted technical help and animal care provided by Mr. Carlos H.spíndola, Mrs. Joanésia M.J. Rothstein, Mr. Marco A. de Lorenzo, Mr.merson V. Fornalski, Mrs. Sandra R B de Oliveira, and Mr. Sandro. de Jesus throughout the experiments.

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