European Journal of , Vol. 21, pp. 1931–1942, 2005 ª Federation of European Neuroscience Societies

Arousal and differential Fos expression in histaminergic of the ascending system during a feeding-related motivated behaviour

J. L. Valde´s,1 P. Farı´as,2 A. Ocampo-Garce´s,3 N. Corte´s,3 M. Sero´n-Ferre´ 2 and F. Torrealba2 1Programa de Doctorado en Ciencias Biome´dicas, ICBM, Universidad de Chile, Santiago, Chile 2Facultad de Ciencias Biolo´gicas, Pontificia Universidad Cato´lica de Chile, Alameda 340, Santiago, Chile 3ICBM, Universidad de Chile, Santiago, Chile

Keywords: locus coeruleus, orexin neurons, polysomnography, radiotelemetry, rat, tuberomammillary nucleus

Abstract Arousal depends on the concerted activity of the ascending arousal system (AAS) but specific stimuli may primarily activate some nuclei of this system. Motivated behaviours are characterized by behavioural arousal, although it is not known which AAS nuclei are active during a motivated behaviour. To address this issue, rats were rendered motivated for food by fasting them for 1 day and then were enticed with food that they could not obtain for varying periods of time. We studied the level of arousal by polysomnography or radiotelemetry, and Fos-ir in the AAS, during food enticing. We found a strong arousal and an early increase in Fos-ir in the histaminergic neurons from the tuberomammillary nucleus, after 30 min of enticing, followed by increased Fos-ir in the whole AAS if food enticing was prolonged to 1 or 2 hours. In contrast, food presentation to non-motivated rats did not increase arousal or Fos-ir in the tuberomammillary nucleus. As opposed to the active arousal of the motivated rats, passive arousal induced by sensory stimulation was associated with increased Fos-ir in the locus coeruleus and the orexin neurons, but not with increased Fos-ir in the tuberomammillary nucleus or in the other nuclei of the AAS. We conclude that the arousal during feeding-related motivated behaviour is associated primarily with the activation of the tuberomammillary nucleus, while the other arousal-related nuclei become active later on.

Introduction The search for food, water, sexual partner or shelter are examples of 2004) acting on raphe nuclei neurons. Several authors (Stricker, 1990; motivated behaviours which are essential for the survival of the Robbins & Everitt, 1995; Pfaff et al., 2002; Garey et al., 2003) have individual and the species. All motivated behaviours are characterized argued that motivated behaviours have an arousal component that is by a behavioural arousal, particularly during the appetitive phase. In particularly notorious during the appetitive phase. An open question is mammals, arousal results primarily from the concerted activity of how the different nuclei of the AAS contribute to the increased arousal waking-active neurons (Steriade & McCarley, 1990; PaceSchott & that underlies the seeking or appetitive phase of a motivated behaviour Hobson, 2002; Jones, 2003) collectively named the ascending arousal such as feeding. system (AAS) (Saper et al., 2001). The AAS neurons reside in several It has been shown that as well as orexin neurons from the subcortical regions that include the locus coeruleus, the raphe nuclei, lateral hypothalamic–perifornical area, but not other neurons from the the cholinergic basal forebrain and pontomesencephalic nuclei, the AAS, become active in close temporal relation to the behavioural lateral hypothalamic area and the tuberomammillary nucleus. activation that goes with the anticipation of a meal, when the animals While the different components of the AAS contribute to wakeful- 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, which is somehow triggered by signals from a hypothetical specific way the performance in behavioural tests. In addition to these food-entrainable circadian oscillator (Stephan, 2002). We decided to subtle differences in the effects of AAS on use a simpler and more direct food-searching paradigm not involving 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 neurons are the first components of the AAS that become active at the acting on the locus coeruleus (Aston-Jones et al., 2000) or by changes onset of a food-searching behaviour. The stimulus used to trigger the in physiological parameters, such as an increase in CO2 (Richerson, behaviour was the presentation of food. We evaluated the arousal level during this behaviour in motivated (fasted) vs. non-motivated (fed) rats, and quantified Fos-ir as a marker of neuronal activation (Cirelli & Tononi, 2000) in AAS nuclei. To test whether the activation pattern of Correspondence: Dr Fernando Torrealba, 2Facultad de Ciencias Biologicas, as above. the AAS was related to the development of a motivated behaviour, as E-mail: [email protected] opposed to passive arousal, we mapped Fos expression in rats forced Received 13 October 2004, revised 4 January 2005, accepted 17 January 2005 to stay awake by experimenters tapping on their cages. doi:10.1111/j.1460-9568.2005.04013.x 1932 J. L. Valde´s et al.

Materials and methods Experimental protocols Subjects Rats were subjected to a feeding protocol of one day ad libitum, one day Subjects were 59 male Sprague-Dawley rats weighing 270–350 g. of fast (motivated rats, n ¼ 9) or ad libitum (non-motivated rats; n ¼ 5) They were kept in individual cages, under 12 : 12-h cycles of and a day of food presentation. Placing a closed wire-mesh box full of light : darkness, at a constant temperature of 22 C and with ad libitum food pellets on top of the rat cage, for 2 h, induced the appetitive phase provision of water and rat chow. All experiments were carried out in of feeding behaviour. The wire box measured 10 cm on each side and accordance to the NIH Guide for the Care and Use of Laboratory the size of the mesh squares was 0.4 cm, so that the rats could smell, Animals (NIH Publications no. 80–23, revised 1996). The surgery to touch and see the food but could not get it out of the box or gnaw the implant electrodes for polysomnographic or the telemetric sensors was pellets through the mesh. To assess the temporal pattern of Fos performed under aseptic conditions. The rats were anaesthetized with expression in the different nuclei of the AAS, three other groups of ketamine (50 mg ⁄ kg; ImalgeneTM, Rhodia Merieux, Santiago, Chile) fasted rats were presented with the wire box with food for 0 min (n ¼ 5 and xylazine (10 mg ⁄ kg; i.p.; RompunTM, Bayer, Santiago, Chile) i.p. rats), 30 min (n ¼ 6 rats) or for 1 h (n ¼ 5). Two additional groups of At the end of the surgical procedure the rats received an antibiotic rats were used as controls for arousal that was independent of food- (BaytrilTM 5mg⁄ kg, i.p.; Bayer) and an analgesic (KetofenTM, seeking behaviour. One group consisted of five naı¨ve rats under baseline 0.2 mg ⁄ kg, s.c.) conditions of ad libitum feeding; this group was used to assess basal Fos-ir expression between 10.00 and 12.00 h. The other control group (fast + forced waking; n ¼ 5) corresponded to fasted rats that were kept awake for 1 h by gently tapping the rat cage every 5 min. To control for Polysomnographic recordings the Fos-ir expression that is related to the act of eating, we studied a Two cranial epidural electrodes (stainless steel screws) were placed as group of five rats that had food for 2 h after a 24-h fast. Table 1 described elsewhere (Ocampo-Garces & Vivaldi, 2002). Two stainless summarizes the rats used to study Fos-ir expression in the AAS. steel neck muscle electrodes were sutured for EMG recording. A All the rats were killed between 10.00 and 12.00 h. They were piezoelectric device at the base of the animal’s cage detected the deeply anaesthetized with 7% chloral hydrate (350 mg ⁄ kg) and horizontal locomotor activity. After 10 days of postoperative recovery, perfused through the left ventricle with a saline flush (100 mL) animals (n ¼ 12) were connected to a flexible, counter-balanced cable followed by 500 mL of 4% paraformaldehyde in 0.1 m phosphate attached to a slip ring and maintained under a stable 12 : 12-h light buffer (PB; pH 7.4). The brains were removed, postfixed in the same dark cycle in recording cages (30 · 40 · 30 cm) contained in fixative for 2 h, transferred to 30% sucrose in phosphate-buffered saline individual isolation chambers (65 · 60 · 60 cm). Ambient tempera- (PBS) with 0.02% sodium azide for 2 days and blocked in the coronal ture was maintained at 21–23 C, with food and water ad libitum. plane to be subsequently cut under dry ice using a sliding microtome. 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 250 Hz after analogue filtering and We assessed Fos-ir expression in seven nuclei and areas of the AAS. conditioning of signals. An on-line display allowed for quality control. The tuberomammillary nucleus (TMN) was identified by immunocyto- Data were stored in 15-s epochs (240 epochs ⁄ h). Two independent chemistry against the marker enzyme adenosine deaminase (ADA) scorers assigned epochs to wakefulness, rapid eye movement (REM) (Senba et al., 1985). The ventral tegmental area (VTA), and the locus sleep or non-REM (NREM) sleep by off-line polysomnographic visual coeruleus (LC) were identified by tyrosine hydroxylase (TH) immu- analysis. Wakefulness was identified by the presence of a desynchro- noreactivity, and the orexin neurons of the lateral hypothalamic area nized EEG and phasic EMG activity. NREM sleep consisted of a high- amplitude slow-wave EEG together with a low EMG tone relative to waking. REM sleep was identified by the presence of regular theta Table 1. Summary of the control and experimental groups of rats used to activity coupled with low EMG tone relative to NREM sleep. Epochs study Fos-ir expression in the AAS scored as wakefulness were further analysed to determine active waking or quiet waking by means of visual inspection of locomotor Groups n Description of treatments activity. The presence of horizontal movement defined active waking. Epochs with artifacts were excluded from analysis. Hours containing Experimental (motivated) > 10% of damaged data were excluded from statistics. The scoring Fast + 0 min food presentation 5 24 h fast Fast + 30 min food presentation 6 24 h fast followed by 30 min procedure assigned each epoch to the state that occupied > 50% of that presentation of boxed food epoch. The brains of these rats were not used for Fos immunohist- Fast + 1 h food presentation 5 24 h fast followed by 1 h ochemistry. presentation of boxed food Fast + 2 h food presentation 11 24 h fast followed by 2 h presentation of boxed food Control Locomotor activity and core temperature recordings Naı¨ve (baseline) 5 Ad lib feeding Fourteen rats were implanted with radiotelemetric sensors (PDT-4000 Non-motivated 5 Ad lib feeding followed by 2 h E-mitter sensor from MiniMitter, Sun River, OR, USA) in the presentation of boxed food Fast + forced waking 5 24 h fast followed by waking peritoneal cavity, after a small midline abdominal incision. The rats up once every 5 min for 1 h had a week to recover from surgery before the continuous recordings Fast + 2 h feeding 5 24 h fast followed by 2 h started. Core body temperature and locomotor activity data were feeding acquired using the VitalView software (Mini Mitter). After 2 h of food Total 47 presentation, these 14 rats were anaesthetized and their brains processed for Fos-immunoreactive (-ir) immunohistochemistry. n, number of rats.

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(LHA) were identified by using an antiorexin A antibody. We used VTA or the LC, or orexin-ir neurons in the LHA. The second cytoarchitectonic criteria (see below) to identify the dorsal raphe (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 We obtained three alternate series of 50-lm sections. One series was cytoplasmic precipitate that contrasted with the dark violet nuclear stained with Cresyl Violet and the other two series were used for DAB–nickel labelling of the Fos-ir. The antisera used were anti-ADA immunohistochemistry. Free-floating sections were incubated in 0.3% (polyclonal, raised in rabbit, diluted 1 : 10,000, from Chemicon, CA, H2O2 in PBS for 30 min, rinsed in PBS and transferred to the blocking USA); anti-TH (rabbit polyclonal, 1 : 10,000, Chemicon); antiorex- solution (0.4% Triton X-100, 0.02% sodium azide and 3% normal in A (rabbit polyclonal, 1 : 2000; from Phoenix Pharmaceutical Inc., goat serum in PBS) for 1 h. The sections were then transferred to the CA, USA). primary antibody incubation solution and left there overnight at room 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 : 20 000 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 brain tissue. IgG (H + L) from Jackson ImmunoResearch, PA, USA; diluted 1 : 1000 in 0.4% Triton X-100 and 1.5% normal goat serum in PBS]. After rinsing for 40 min, the sections were incubated for 1 h in Quantification of Fos-ir expression Vectastain ABC Elite kit (Vector Laboratories, CA, USA) diluted The activation of the different nuclei of the ascending arousal system 1 : 500 in PBS, rinsed and incubated in a 0.05% 3–3¢ diaminobenzi- was assessed by counting Fos-ir neurons bilaterally in three coronal dine hydrochloride (DAB) solution containing 0.003% H2O2 and sections from each nucleus, per rat. In the TMN, the LHA and the 0.05% nickel chloride to get a dark blue reaction product. Selected VTA we counted neurons doubly labelled for Fos-ir and either ADA, sections from both series, already immunostained for nickel-enhanced orexin or TH, respectively. For the LC, LDT, SI and DR we used grids 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 neurons are shown in Fig. 1.

Fig. 1. Photomicrographs of representative coronal sections through the AAS nuclei analysed in the present study (open rectangles). We counted Fos-ir neurons in three sections from each nucleus, and the pictures show the intermediate section. (a) ADA-ir neurons from the ventral TMN (level )4.2 mm AP; Swanson, 1998). (b) Orexin-ir neurons in the LHA–perifornical area, at a level )2.85 mm. (c) The VTA as outlined by TH-ir, level )5.2 mm. (d) The LC was identified by the presence of TH-ir neurons; the )9.8 mm level is shown. The overlying rectangle of 100 · 450 lm delimits the LC area where we counted Fos-ir neurons. (e) The LDT and the DR at )8.6 mm; the 250-lm square used to count Fos-ir neurons in the LDT and the 200 · 600 lm rectangle used to count neurons in the DR are shown. (f) The 500-lm square used for the same purpose, but in the SI. The photomicrograph shows a )0.11-mm level, just ventral to the temporal limb of the anterior commissure. Abbreviations: aco, anterior commissure, olfactory limb; act, temporal limb of the anterior commissure; AQ, cerebral aqueduct; CP, caudate– putamen; f, fornix; fr, fasciculus retroflexus; ME, median eminence; mlf, medial longitudinal fascicle; MM, medial mammillary nucleus; mp, mammillary peduncle; NDB, nucleus of the diagonal band; och, optic chiasm; PCG, pontine central grey; pm, principal mammillary tract; scp, superior cerebellar peduncle; SNc, substantia nigra, pars compacta; SUV, superior vestibular nucleus; V3, third ventricle; V3m, third ventricle, mammillary recess; V4, fourth ventricle. Scale bar, 0.5 mm.

ª 2005 Federation of European Neuroscience Societies, European Journal of Neuroscience, 21, 1931–1942 1934 J. L. Valde´s et al.

For the TMN, the intermediate rostrocaudal section (Fig. 1a) nuclei and under different treatments was analysed using the Kruskal– corresponded to the level of the mammillary recess of the third Wallis one-way anova followed by Dunn’s multiple pairwise ventricle (approximate level )4.2 of the atlas of Swanson, 1998). The comparison method. other two sections were 150 lm 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 landmark the anterior pole of the median eminence; the caudal section Effect of food presentation on arousal 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 ized by a desynchronized and fast EEG, increased locomotor activity sections, corresponded to level )2.85 of the atlas of Swanson (1998). and a rise in core body temperature. EEG and locomotor activity signs For the VTA, the intermediate rostrocaudal section (Fig. 1c) was of arousal increased from the onset of food presentation and lasted for identified by the caudal tip of the mammillary body and corresponded nearly 1 h, while the temperature increase was delayed by about to level )5.2 mm of the atlas of Swanson (1998). At this level, the 20 min and also lasted for 1 h. mammillary peduncle clearly separated the VTA from the TH-ir The temporal profile of wake and sleep states in the 09.00–12.00 h substantia nigra. The other two sections were 300 lm apart. interval was studied for 3 consecutive days, including one day under In the LC (Fig. 1d) the TH-ir neurons were so tightly packed that it 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 day when food was presented (at 10.00 h). Values obtained during the counted Fos-ir neurons within a rectangle of 450 lm by 100 lm first 2 days were averaged to obtain the baseline of the sleep-state (0.045 mm2) 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), that is, just caudal to the parabrachial nucleus. The other two preceding food presentation and to the first hour of food presentation. sections were 300 lm 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; P ¼ 0.02, paired t-test) and less time in magnocellular cell group forming a round nucleus near the aqueduct. NREM sleep (42.9 ± 3.7%; P ¼ 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 )8.6 mm of the atlas of Swanson (1998), where the LDT abuts the the 10.00–11.00 h interval, food presentation to fasted rats increased superior cerebellar peduncle. We used a 250-lm square (0.0625 mm2) the proportion of time spent in active waking to 72.1%, mainly at the 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 section was 300 lm rostral and the third section 600 lm caudal to the spent in REM sleep (4%). These changes were significant (paired intermediate level. t-test; P < 0.001 for active waking and NREM sleep; P ¼ 0.002 for The rostral DR was identified in Nissl-stained sections at the same 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 (0.12 mm2), 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, 600 lm apart from the other two sections, is shown in Fig. 1e, second hour of food presentation, the rats did not show significant and it was from the same )8.6 mm level used for the LDT. differences in sleep–wake profile relative to baseline days. In the SI we used a 500-lm square (0.25 mm2) as a counting grid Once we were certain that food presentation induced a strong arousal (Fig. 1f). The intermediate section is depicted in Fig. 1f and was in fasted rats, as demonstrated by the large increase in active waking, 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 mm of the atlas of Swanson (1998). At this level, the counting in motivated (fasted) and non-motivated (fed) rats. We continuously square was placed just below the central portion of the temporal limb measured locomotor activity and core temperature for 3 days in two of the anterior commissure. The lateral edge of the square abutted the groups of rats. Nine rats spent one day under ad libitum feeding and fundus of the . The rostral SI section corresponded to the most one day under fasting, and then were presented with the boxed food on caudal section where the anterior commissure displayed no temporal the morning of the third day. Five rats spent 2 days under ad libitum limb (level +0.10 mm). Here the counting square was placed just feeding and then were presented with the boxed food. The recordings below the anterior commissure. The caudal section corresponded to performed during the 2 days before food presentation showed normal the most anterior section with a fused anterior commissure (atlas level circadian rhythms of locomotor activity and of core temperature in both )0.26 mm), and here the counting square was centred along a vertical 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– After 24 h of fasting, the rats showed a fall in core temperature (arrow putamen; the upper edge of the square abutted the internal capsule. in Fig. 3) relative to the same clock time under ad libitum feeding. In the 06.00–10.00 h interval, fasted rats had a mean ± SEM temperature of 37.1 ± 0.03 C, while the same rats under ad lib feeding condition Statistical analysis had a core temperature of 37.4 ± 0.04 C(P ¼ 0.005, Mann–Whitney We used the SigmaStat 3.0 software (SPSS Inc., Chicago, IL, USA). rank-sum test). A fall in core temperature after a 24-h fast has been Accumulated locomotor activity and the pattern of sleep–wake cycles described previously (Nagashima et al., 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 locomotor activity of a single animal during the ad libitum day, 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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Fig. 2. Percentage of time spent in different states of the sleep–wake cycle, based on polysomnographic recordings (n ¼ 12 rats), (a) during the hour preceding food presentation and (b) during the first hour of food presentation. Asterisks indicate a significant difference with respect to the baseline condition (P<0.05, paired t-test). AW, active waking; NREM, slow-wave sleep; QW, quiet waking; REM, rapid eye movement sleep. *P < 0.05.

Fig. 3. Circadian rhythms of mean locomotor activity and core temperature (including the SEM in grey) from the group of nine rats that were presented with food for 2 h. In open background is the day under ad libitum feeding (day d1 of Fig. 4a), and in grey backgroundis shown the day of fasting (from 10.00 h of day d2, as in Fig. 4a). The arrow points to the fall in temperature caused by the overnight fast. The empty and filled bars below the graph indicate the lights-on and lights-off intervals, respectively. during the dark hours (Fig. 4a) and a minimum during the day. The tion (Fig. 4c) was almost twice as much as the baseline days (P<0.001, presentation of food for 2 h in the morning of experimental day 3 (bar paired t-test). The motor activity returned to baseline values during the in Fig. 4a) induced a large increase in locomotor activity compared to second hour of food presentation (P ¼ 0.39, paired t-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 C at time zero (10.00 h) to 37.54 ± 0.2 C after 50 min The analysis of the 10.00–12.00 h interval for the group of nine rats of food presentation (Fig. 4d). The two-way repeated-measures that fasted (Fig. 4b) confirmed that they were much more active during anova showed a time-dependent change in temperature food presentation. The ambulatory activity during the presentation of (P<0.001). The Holm–Sidak method showed that at the times food on day 3 (s in Fig. 4b) was higher than the previous days (d), and indicated by asterisks in Fig. 4d the temperatures were higher than remained so at least for another 25 min. A two-way repeated-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 locomotor activity (P<0.001), that the locomotor activity was higher 20 min, and lasted about half an hour, to slowly decline thereafter. in the food presentation condition (P ¼ 0.011), and that there was a We detected no differences between baseline and food presentation significant interaction (P<0.001) between time and treatment. The (P ¼ 0.651), probably because the presentation curve began at a lower multiple-comparison procedure (Holm–Sidak method; P < 0.05) temperature due to the prior fasting. showed that, at the times indicated by the asterisks in Fig. 4b, the In contrast, food presentation to non-motivated (fed) rats induced a experimental rats had more locomotor activity than during the baseline weak locomotor activation (Fig. 5a; P ¼ 0.014, two-way repeated- days and than at the time 5 min before food presentation. The measures anova, followed by a multiple pair-wise comparison, cumulative ambulatory activity during the first hour of food presenta- Holm–Sidak method, P < 0.05), that did not increase the cumulative

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Fig. 4. Effects of food presentation on locomotor activity and core temperature in motivated rats. (a) Actogram showing the locomotor activity of a single rat recorded through 3 consecutive days (d1–d3) under 12 : 12 h of light : dark cycle (indicated in the first row); the bar in d3 indicates the 10.00–12.00 h interval during which the rat was presented with a wire box full of rat chow. The rat was under ad libitum food during d1, and food was removed at 10.00 h of d2. The quantitative analysis of locomotor activity and of core temperature was centred in the time interval spanning the 2 h from 10.00 to 12.00 h. The baseline condition was obtained by averaging the activity of the 10.00–12.00-h interval during days d1 and d2. (b) Mean locomotor activity of nine motivated rats during the 10.00– 12.00 h 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 cumulative activity during the first hour (10.00–11.00 h) and second hour (11.00–12.00 h) of enticing with food (open bars) was compared to the activity at the same time during days d1–d2 (baseline condition, dark bars). The activity was significantly higher during the first hour of presentation to food (*P < 0.05, Student’s t-test). (d) The average core temperature response to food presentation (s), compared to the temperature during the baseline condition (d). The asterisks indicate significant differences (*P<0.05; two-way anova for repeated measurements) relative to time 5 min before food presentation. locomotor activity (Fig. 5a, inset; P ¼ 0.38 for the first hour and Fos-ir above the levels found in naı¨ve rats only in the LDT and the LC P ¼ 0.75 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; P ¼ 0.99, two-way repeated-measures anova) relative to method for multiple comparisons), but not in the TMN, the orexin baseline days. neurons in the LHA, the DR, the SI or the VTA. After 30 min of food presentation, fasted rats showed a significant increase in the number of Fos-ir neurons in the TMN (P<0.001, Kruskal–Wallis one-way Time course of Fos-ir expression in the AAS in fasted rats anova; P < 0.05, Dunn’s method for multiple comparisons), invol- enticed by food ving 15% of the ADA-ir neurons (Fig. 6a and c). The other AAS The AAS nuclei showed very few Fos-ir neurons in the naı¨ve rats nuclei did not show this early increase in the number of Fos-ir neurons during the 10.00–12.00 h time interval (Fig. 6a and b), as has been relative to the naı¨ve rats after 30 min of presentation (Fig. 6). The described (Ko et al., 2003), indicating a low level of neuronal situation changed after 60 min of food presentation. The TMN showed activation. In the rats killed at time zero of food presentation, we found increased levels of Fos-ir while the orexin neurons in the LHA

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Fig. 5. Locomotor activity and body core temperature during food presentation to non-motivated (fed) rats. (a) Mean locomotor activity of five non-motivated rats during the 10.00–12.00 h of day d3. The asterisks indicate the values that were statistically different from the baseline (*P<0.05; two-way anova for repeated measurements). The inset shows the total locomotor activity during the first and second hour of food presentation, compared to the baseline condition for the same rats. (b) The average core temperature response to food presentation (s), compared to the temperature during the baseline condition (d). There were no significant differences between the two curves.

(Fig. 6a) as well as the LDT, SI, DR and LC (Fig. 6b) had significantly kept forcibly awake for 60 min by tapping the rat cage every 5 min. more Fos-ir neurons than the naı¨ve controls (P<0.001, Kruskal– They spent the hour with their eyes open, but motionless, in a state of 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 120 min 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 actively trying to get the food from the wire box for 1 h. We found that naı¨ve controls. The photomicrographs in Fig. 6c–h illustrate the keeping the rats forcibly awake for 1 h induced a significant increase increase in the number of Fos-ir neurons, in response to 30 or 120 min 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 of the LHA–perifornical area. P < 0.05, Dunn’s method for multiple comparisons). In marked The weak behavioural activation of the non-motivated rats by food contrast to the rats killed after 1 h of active arousal (Fig. 6a and b), the presentation was reflected in a dramatically different activation pattern rats kept forcibly awake showed no increase in the number of Fos-ir of the AAS than that observed in motivated rats. The marked neurons in the TMN, the DR, the LDT, VTA or the SI. The number of activation of the TMN, the DR and the orexin-ir neurons, and the orexin neurons with Fos-ir was significantly lower in the forced- milder activation of LC, SI and of VTA, observed in the motivated rats waking group than in the 60-min food-presentation group (P<0.05). after 120 min of food presentation completely disappeared in the non- 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 naı¨ve controls (P<0.001, Kruskal–Wallis one-way rats that had food available for 2 h after a 24-h fast (n ¼ 5). They ate anova, followed by Dunn’s method for multiple comparisons, 5.2 ± 0.7 g of chow during that time. These rats displayed activation P < 0.05), but not different from the increase observed after 2 h of of every AAS nucleus studied except the VTA (Fig. 8). We used the food presentation (Fig. 6b) to motivated rats. Kruskal–Wallis one-way anova, and Dunn’s method for multiple To better understand the relationship between Fos-ir expression and comparisons. In the TMN 8% of the ADA-ir neurons were also increased locomotor activity as a manifestation of arousal, we carried Fos-ir. While this percentage was significantly higher that naı¨ve out a correlation study between these variables during food presen- controls values (P<0.05), it was not significantly different from the tation. The results, shown in Table 2, indicate that there was a positive 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 naı¨ve 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 in locomotor 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<0.05 relative to naı¨ve controls), but only the LC and the could have been related to the waking-up of the rats caused by the LDT had significantly more Fos-ir neurons following feeding than sensory stimulation that necessarily went with the placing of the wire following 120 min of food presentation (P<0.05). box of food. To assess how specific was the pattern of activation of the AAS relative to the active seeking of food in motivated rats, as opposed to a passive arousal due to sensory stimulation, we analysed a Discussion group of five rats that had fasted for the previous 24 h. These The present study shows that food-seeking behaviour has an important experiments were also conducted from 10.00 to 12.00 h. The rats were arousal component that is related to a specific temporal pattern of Fos-ir awakened by placing the empty wire box on top of their cages, and of the AAS nuclei, where the TMN is the first nucleus of the AAS to

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Fig. 6. Activation of the different nuclei of the AAS, assessed by the expression of Fos-ir, during the presentation of food to motivated rats. We counted the number of Fos-ir neurons in rats under ad libitum feeding (naı¨ve controls) that were killed, as the experimental rats, in the 10.00–12.00 h interval. The experimental rats were killed after 0, 30, 60 or 120 min of food presentation. (a) Mean ± SEM percentage of ADA-ir that also expressed Fos-ir in the TMN, of orexin-ir neurons with Fos- ir in the LHA, and of TH-ir neurons in the VTA that also had Fos-ir. The asterisks indicate the time points when these nuclei had more Fos-ir neurons than the naı¨ve rats (P<0.05; one-way anova). (b) Number of Fos-ir neurons ⁄ mm2 in the LC, DR, LDT and SI. Labels as in (a). (c–h) Photomicrographs of Fos-ir neurons in (c and f) the ventral division of the tuberomammillary nucleus (TMN), doubly immunostained for adenosine deaminase (ADA) -ir, of (d and g) orexin-ir neurons in the LHA–perifornical area (LHA), and of (e and h) the locus coeruleus (LC) immunostained for tyrosine hydroxylase (TH). The rats were killed after (c–e) 30 min or (f–h) 120 min of food presentation. The blue arrows point to doubly labelled neurons. Scale bar, 50 lm. *P < 0.05. show Fos-ir during this behaviour. The other AAS nuclei become motivated rats, or than during a baseline day. The average time spent active nearly 30 min after the activation of the histamine neurons. The in active waking during the first hour of food 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. Arousal and core temperature often, but not always, covary non-motivated rats had lower levels of arousal and no TMN activation in healthy animals, as exemplified by the circadian rhythms of both during food presentation. Moreover, the arousal forced by strong variables and by the observation that arousal-inducing stimulants such sensory stimulation failed to activate the tuberomammillary nucleus as d-amphetamine (Pigeau et al., 1995a) or (Bonnet & Arand, and only activated the locus coeruleus. 1992), but not modafinil (Pigeau et al., 1995b; Scammell et al., 2000), The rats motivated to get food had significantly more waking increase body temperature. Other states of high arousal, such as episodes, spent more time in active waking, had much more locomotor 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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Fig. 7. Fos-ir expression in the AAS nuclei of (a and b) non-motivated rats presented with food for 120 min, and of (c and d) rats kept forcibly awake for 60 min. The asterisks indicate the AAS nuclei that had significantly more Fos-ir neurons than the naı¨ve controls. They were the LDT in the non-motivated rats and the LC and the orexin neurons in the forcibly awake group (see text for details). presentation may be one consequence of the increased motor activity Table 2. Pearson product–moment correlation coefficients (CC) and P-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 different AAS nuclei against the amount of locomotor activity after 1 h of food physiological priming to obtain and consume a meal. presentation to motivated (n ¼ 7) versus non-motivated (n ¼ 5) rats. It has been shown that the different nuclei of the AAS increase their discharge of action potentials as soon as the animal is awakening TMN VTA SI LHA DR LC LDT (Trulson & Jacobs, 1979; Foote et al., 1980; Alam et al., 2002; Jacobs Change in LA 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 presentation we found Fos-ir expression above background in the LC CC 0.738 0.719 0.649 0.624 0.491 0.463 0.047 and the LDT of fasted rats. This increased Fos-ir in the LC and LDT of Total LAà fasted rats may reflect the relatively high frequency of active waking P 0.185 0.012 0.271 0.212 0.314 0.328 0.782 episodes we found during the hour preceding food presentation (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 LA, locomotor activity. This value is the difference betwen LA during food feeding ad libitum. à When the motivated rats were killed after 30 min of enticing, only presentation and baseline. Corresponds to LA during food presentation. 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 presentation most of the AAS nuclei were engaged, and they remained TMN activation may go undetected by Fos-ir expression. Fos active if the enticing with food was prolonged to 2 h. The relative expression may depend on a particularly high rate of action potentials 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 good tool for discerning small changes in neuronal activity (Cirelli & increase in Fos expression in the TMN suggests that arousal might

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Fig. 8. The effect of feeding for 2 h on the number of Fos-ir neurons in the AAS nuclei. When the fasted rats were allowed to eat, most of the AAS nuclei, except for the VTA, had significantly more Fos-ir neurons than the naı¨ve controls (*). Most nuclei had the same number of Fos-ir neurons after feeding as after food presentation for 2 h, except for the LC which had more Fos-ir neurons after feeding, and the VTA which had more Fos-ir neurons after food presentation (see text). trigger activity-dependent cascades of second messengers leading to activated in sequence during feeding, first induce arousal by acting on earlier changes in genes expression in this nucleus. Consistent with cortical and thalamic neurons and then contribute to satiety, an effect our finding that the TMN gets active first, there are examples of of histamine on ventromedial and paraventricular hypothalamic nuclei differential expression of Fos-ir in the AAS nuclei of rats kept awake that is driven by mastication (Sakata et al., 2003). by different treatments. In rats kept awake > 90% of the time with Our findings are consistent with the observation that histamine is intermediate doses of modafinil, Fos-ir was increased in the TMN and released during feeding. Histamine was released in the in orexin neurons but not in other AAS nuclei such as the LC, LDT or of fasted rats when they ate for 15 min (Itoh et al., 1991). DR (Scammell et al., 2000). In the same line, orexin neurons show Interestingly, histamine release increased not only during actual eating large increases in Fos-ir in association with high-arousal waking, but but also during food presentation for 15 min. These authors suggested not with spontaneous waking, suggesting that orexin neurotransmis- that release of histamine is enhanced during feeding, but also did not sion is responsive to high-arousal and not to waking per se (Espana exclude the possibility that behavioural excitation induced by the et al., 2003). It is possible that the early TMN activation may reflect a 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 because of the stress or, alternatively, because the rats are performing a pattern of Fos-ir in the AAS nuclei is better related to the procedure of motivated behaviour to survive. In these experiments, the control rats inducing arousal rather than to different kinetics of Fos-ir expression. that were just handled, and were presumably awake, showed no A straightforward explanation for the recruitment of the other AAS histamine release (Endou et al., 2001), suggesting that gentle sensory nuclei would be that the TMN contributed to the activation of the other 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 et al., 1996) and basal forebrain neurons (Jones, 2004; Ramesh et al., reported here may be considered one consequence of TMN activation, 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). close at hand, as during presentation. Under this view, the early TMN The cortical activation during early food presentation and the activation may reflect some endogenous signals that prepare the rats to increased sensory responsiveness and motor activation may also have obtain food by increasing the arousal level. In this sense, our results contributed to engaging the other AAS nuclei and so maintaining a support the suggestions that histaminergic neurons play a key role in level of arousal for a prolonged time. exploratory behaviour (Inoue et al., 1996) and in maintaining There is a complex, and not well-understood, relationship between 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 brain histamine has a maximum during the active phase, that is, 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 and in 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 locomotor activity, there is a possibility that the early TMN activation (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 related to increased motivation for food. The number of Fos-ir neurons decarboxylase (Fulop et al., 2003) are not hyperphagic, but show in the TMN, VTA, SI and LHA showed a positive and significant reduced responses to leptin. It is possible that different TMN neurons, correlation with the change in locomotor activity, but not with the

ª 2005 Federation of European Neuroscience Societies, European Journal of Neuroscience, 21, 1931–1942 Arousal system and feeding 1941 level of locomotor activity. However, this analysis cannot distinguish Azmitia, E.C. (1999) Serotonin neurons, neuroplasticity, and homeostasis of cause from effect. Further studies using a forced locomotion condition neural tissue. Neuropsychopharmacology, 21, 33S–45S. in the absence of increased food motivation are needed to elucidate Bonnet, M.H. & Arand, D.L. (1992) Caffeine use as a model of acute and chronic insomnia. Sleep, 15, 526–536. this point. Brown, R.E., Sergeeva, O.A., Eriksson, K.S. & Haas, H.L. (2002) Convergent There is evidence indicating that the LC and the raphe nuclei, which excitation of dorsal raphe serotonin neurons by multiple arousal systems are AAS nuclei, are primarily responsive to sensory stimulation and to (Orexin ⁄ hypocretin, histamine and noradrenaline). J. Neurosci., 22, 8850– altered brain tissue homeostasis, respectively. The LC neurons are 8859. Chen, C.T., Dun, S.L., Kwok, E.H., Dun, N.J. & Chang, J.K. (1999) Orexin responsive to sensory stimulation of various modalities, either because A-like immunoreactivity in the rat brain. Neurosci. Lett., 260, 161–164. the stimuli are intense (Foote et al., 1980) or because they are salient Cirelli, C. & Tononi, G. (2000) On the functional significance of c-fos to the animal as a result of conditioning (Aston-Jones et al., 1994). induction during the sleep-waking cycle. Sleep, 23, 453–469. Our results show that sensory stimulation that kept the rats awake for Constandil, L., Parraguez, V.H., Torrealba, F., Valenzuela, G. & Seron-Ferre, 1 h induced Fos-ir in the LC. Similarly, serotonin neurons may be M. (1995) Day–night changes in c-fos expression in the fetal sheep suprachiasmatic nucleus at late gestation. Reprod. Fertil. Dev., 7, 411–413. considered CO2 sensors that contribute to pH homeostasis in the brain Endou, M., Yanai, K., Sakurai, E., Fukudo, S., Hongo, M. & Watanabe, T. (Azmitia, 1999; Richerson, 2004). When activated, raphe neurons (2001) Food-deprived activity stress decreased the activity of the mediate respiratory responses but also motor, anxiety, arousing and histaminergic system in rats. Brain Res., 891, 32–41. cerebrovascular responses to altered CO and pH in the brain. We Ericson, H., Blomqvist, A. & Kohler, C. (1991) Origin of neuronal inputs to the 2 region of the tuberomammillary nucleus of the rat brain. J. Comp. Neurol., want to advance the hypothesis that, just as LC is primarily responsive 311, 45–64. to sensory stimulation and raphe nuclei to metabolic brain signals, the Espana, R.A., Valentino, R.J. & Berridge, C.W. (2003) Fos immunoreactivity in TMN is primarily activated by signals that reflect an internal decision hypocretin-synthesizing and hypocretin-1 receptor-expressing neurons: to engage in a motivated behaviour. These signals may arise from Effects of diurnal and nocturnal spontaneous waking, stress and hypocre- prefrontal cortex neurons (Robbins, 2000), probably from the tin-1 administration. Neuroscience, 121, 201–217. Fleckenstein, A.E., Lookingland, K.J. & Moore, K.E. (1994) Activation of infralimbic area which is the main source of cortical afferents to the noradrenergic neurons projecting to the diencephalon following central TMN (Wouterlood et al., 1987; Ericson et al., 1991), in addition administration of histamine is mediated by H1 receptors. Brain Res., 638, providing fairly strong inputs to the LHA–perifornical area and 243–247. weaker inputs to other AAS nuclei such as the DR, LC and LDT Foote, S.L., Aston-Jones, G. & Bloom, F.E. (1980) Impulse activity of locus coeruleus neurons in awake rats and monkeys is a function of sensory (Hurley et al., 1991). stimulation and arousal. Proc. Natl Acad. Sci. USA, 77, 3033–3037. In conclusion, food presentation to motivated rats induced a strong Fulop, A.K., Foldes, A., Buzas, E., Hegyi, K., Miklos, I.H., Romics, L., arousal characterized by a desynchronized EEG and increased Kleiber, M., Nagy, A., Falus, A. & Kovacs, K.J. (2003) Hyperleptinemia, locomotor activity and body core temperature. In parallel with the visceral adiposity, and decreased glucose tolerance in mice with a targeted intense arousal we found a sequential activation of the AAS, where the disruption of the histidine decarboxylase gene. Endocrinology, 144, 4306– 4314. TMN was the first nucleus that became active, and was followed by Garey, J., Goodwillie, A., Frohlich, J., Morgan, M., Gustafsson, J.A., Smithies, the activation of the other AAS nuclei. We speculate that the TMN O., Korach, K.S., Ogawa, S. & Pfaff, D.W. (2003) Genetic contributions to was activated by signals that reflect an internal decision to engage in a generalized arousal of brain and behavior. Proc. Natl Acad. Sci. USA, 100, motivated behaviour, and that this TMN activation induced the 11019–11022. Gerashchenko, D., Kohls, M.D., Greco, M., Waleh, N.S., SalinPascual, R., increased arousal we found during food-enticing of fasted rats. Kilduff, T.S., Lappi, D.A. & Shiromani, P.J. (2001) Hypocretin-2-saporin lesions of the produce narcoleptic-like sleep behavior in the rat. J. Neurosci., 21, 7273–7283. Acknowledgement Hebb, D.O. (1955) Drives and the C.N.S. (Conceptual Nervous System). Psychol. Rev., 62, 243–254. Financed by Fondecyt grant no. 1020718. Hurley, K.M., Herbert, H., Moga, M.M. & Saper, C.B. (1991) Efferent projections of the infralimbic cortex of the rat. J. Comp. Neurol., 308, 249– 276. 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