hypocretins in pigeons (Columba livia)

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Regulatory Peptides 147 (2008) 9 – 18 www.elsevier.com/locate/regpep

Behavioral and metabolic effects of central injections of orexins/hypocretins in pigeons (Columba livia) Eduardo Simão da Silva a , Thiago Viçoso dos Santos a , Alexandre Ademar Hoeller a , Tiago Souza dos Santos a , Gustavo Vieira Pereira b , Cristiane Meneghelli a , Ana Isabel Pezlin a , Murilo Marcos dos Santos a , Moacir Serralvo Faria a , Marta Aparecida Paschoalini a , José Marino-Neto a,b,⁎ b

a Department of Physiological Sciences, CCB, Federal University of Santa Catarina, 88049-900 Florianópolis SC, Brazil Institute of Biomedical Engineering, EEL-CTC, Federa l University of Santa Catarina, 88049-900 Florianópolis SC, Brazil

Received 4 July 2007; received in revised form 8 November 2007; accepted 9 December 2007 Available online 26 December 2007

Abstract In the present study, the acute behavioral and ingestive effects of ICV injections of mammalian orexin-A (ORXA; vehicle, 0.2, 0.6 or 2 nmol) and of orexin-B (ORXB; vehicle, 0.2, 0.6 or 2 nmol), as well as possible long-term effects (through 24 h of continuous intake monitoring after 0.6 nmol of ORXA or ORXB) of these treatments in food/water intake and in blood levels of metabolic fuels (free fatty acids and glucose, after 0.2 or 0.6 nmol of ORXA) were examined in adult male pigeons. Both ORXA and ORXB treatments failed to produce acute (1–3 h) or long-term effects on feeding and drinking behaviors, and did not change blood free fatty acids and glucose 15 and 30 min after treatments, as compared to vehicle-treated animals. However, ORXA (but not ORXB) treatments evoked a dose-related, intense increase in exploratory behaviors, associated to reduced time spent in alert immobility and sleep-typical postures. These data substantiate the lack of orexigenic effects of ORXs in avian species, and suggest that an important role in vigilance control may represent a conserved functional attribute of orexinergic circuits in vertebrates. © 2007 Elsevier B.V. All rights reserved. Keywords: Food intake; Arousal; Avian; Hypothalamus; Sleep

1. Introduction Orexin-A (ORXA, a cyclic 33-amino acid peptide also known as hypocretin-1) and orexin-B (ORXB, or hypocretin-2, a linear, 28-amino acid peptide) are produced from the precursor protein prepro-orexin in neurons of the lateral hypothalamus of mammals [1,2]. ORX neurons have extensive projections to other hypothalamic nuclei and to diverse forebrain and brainstem regions [3] that actuate through two closely related G-protein-coupled receptors (termed the orexin-1 and orexin-2 receptors), which show a widespread distribution throughout the ⁎ Corresponding author. Department of Physiological Sciences, CCBUniversidade Federal de Santa Catarina, 88.040-900. Florianópolis, SC, Brazil. Fax: +55 48 331 9672. E-mail addresses: [email protected], [email protected] (J. Marino-Neto). 0167-0115/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2007.12.003

neuraxis [1,2,4,5]. These orexinergic circuits appear to represent a key mechanism for the coordination of sleep/wakefulness and energy homeostasis. Orexins have been implicated in appetite regulation and energy homeostasis. In mammals, fasting and hypoglycemia increase ORX production [6–8], while systemic, intracerebroventricular (ICV) or intrahypothalamic administration of exogenous ORX stimulates feeding [9,10] and activates neurons in a variety of neuronal groups that are involved in appetite regulation (e.g., [11]). ORX has also been implicated in the regulation of the sleep/wake cycle [12,13]. Prepro-orexin and OX2 receptor deficits are associated with narcolepsy [14,15]. Central injections of ORXA evoke behaviors indicative of an increased state of arousal, including increased locomotor activity and grooming [16–19] and decreased time spent in both slow wave and paradoxical sleep [20]. Feeding and activity

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effects of ORXA treatments are dissociated, and ORXAinduced feeding is unlikely to be a mere consequence of the enhanced arousal [10]. Furthermore, it has been shown that orexin administration increases body temperature, heart rate, blood pressure and renal sympathetic nerve activity [21–24]. An increase in circulating lipids [25] and hypoglycemia [26,27] activates hypothalamic ORX neurons, possibly contributing to hyperphagic responses. In line with this, ORXA treatments evoke a sympathetically-mediated increase in energy expenditure [28,29]. This increased sympathetic outflow, associated with enhanced feeding and arousal, may support attentive bodily states relevant to, for example, foraging and food seeking behaviors [5,13]. Orexins seem to be highly conserved among the different mammalian species so far examined and strong correspondence was observed also among the mammalian ORX sequences [1,2] and those of teleost fishes (zebrafish and pufferfish; [30], amphibians (Xenopus [31]) and avian (chicken, Gallus domesticus, [32]). Chicken prepro-orexin cDNA has been cloned, sequenced and characterized. ORXA and ORXB in this species showed approximately 85% and 65% (respectively) similarities with the corresponding mammalian sequence at the amino acid level. A chicken ORX receptor (ORXr) cDNA has been cloned and the distribution of orexin receptor mRNA in chickens was examined and shown to be abundant in the cerebrum, hypothalamus and optic tectum [33]. This chicken ORXr showed high similarity with mammalian orexin-2 receptors (that present similar affinity for ORXA and ORXB). Orexin-like immunoreactive cell bodies have been found in the hypothalamus of fishes [30], amphibians [31,34,35], reptiles [36], as well as in avian species (chickens: [32]; Japanese quails: [37]; finches [38]). Furthermore, an in situ hybridization signal of 35S end-labeled oligonucleotides for chicken orexin mRNA was observed in the periventricular hypothalamic nucleus and in the lateral hypothalamic area of the chicken brain, further reinforcing the notion that these highly conserved proteins may possibly play a fundamental regulatory role in vertebrates [32]. Despite these similarities, ICV injections of either ORXA or ORXB failed to affect total food intake in free-feeding or 3-h-fasted neonatal (2-day old) broiler chicks, 1 or 2 h after treatments [39]. In adult Japanese quails, in situ hybridization revealed that cell bodies expressing ORX were concentrated in a medial posterior hypothalamic nucleus, from with they spread laterally into the lateral hypothalamic area. While fasting increased the expression of a feeding-related peptide (the agouti-related peptide mRNA), it did not affect preproorexin mRNA levels [37]. Cerulenin is a natural fatty acid synthase (FAS) inhibitor that affects metabolic rate, feeding and body weight by changing metabolism in hypothalamic neurons. Cerulenin mRNA and protein are expressed in the hypothalamus of broiler chickens [40], and this expression is decreased by fasting. Systemic injections of cerulenin reduce food intake and downregulated FAS and melanocortin receptor gene expression in the chicken hypothalamus, but did not affect ORX or orexin receptor mRNA levels [40]. These data strongly suggest that ORX neurons may not be important to feeding control in birds.

It should be noted, however, that ICV injections of ORXB in the sheep have been shown to increase food intake [41], while long-term changes in body weight, or food deprivation, do not affect the expression of hypothalamic prepro-orexin mRNA in this species [42]. These data suggest that a challenged intakeinduced expression of ORX peptides or receptors (or its absence) does not always predict the behavioral effects of these peptides. Furthermore, poultry (such as quails and chicken) have been submitted to intense artificial selection directed at growth (in broilers) or at oviposition (in layers), that appears to result in important changes in the circuitry mediating metabolic and behavioral responses to energy demands or to drugs affecting feeding behavior [43,44]. Thus, a number of neuroactive substances that have similar orexigenic (such as ICVinjected noradrenaline) or anorexigenic effects (such as ICVinjected serotonin) in both mammals and in the pigeon (an avian species that was not submitted to feeding-directed artificial selection) [45–47], appear to have different feeding effects depending on the chicken lineage used [48–50]. Until now, the possible long-term effects of ORX treatments on ingestive behaviors as well as the potential role of ORXs in fuel homeostasis and in arousal regulation have not been investigated in birds. In the present paper we further investigate possible roles of ORX in avian species, by examining the acute behavioral effects of ICV injections of ORXA and ORXB in adult pigeons, as well as possible long-term effects of these treatments on food and water intake. The acute changes in blood levels of metabolic fuels (free fatty acids and glucose) after ORXA treatments were also studied. To our knowledge, this is the first report on the effects of orexins on the ingestive (beyond food intake) and arousal-related behaviors in a non-mammalian vertebrate. 2. Materials and methods 2.1. Animals and surgeries All the experimental procedures described below were conducted in strict adherence to the recommendations found in the “Principles of animal care” (NIH, 1985) and were approved by the local Committee of Ethics in Animal Research (CEUA-UFSC). Adult domestic pigeons (male Columba livia, 400–500 g body weight), maintained in individual cages, at a temperature of 22–24 °C, on a 12:12 light–dark cycle (lights on at 7:00 am) and with free access to food and water were used throughout the experiments. At least 9 days before experiments 1, 2 and 3, each animal was anesthetized (with 0.05 ml/100 g bw xylazine and 0.15 ml/100 g bw ketamine, i.m., in the pectoral muscle), and stereotaxically implanted with a stainless steel (G28) guide cannula aimed at the right lateral ventricle, according to coordinates (1.0 mm lateral to the midline, 6.0 mm anterior to the interaural line and 6.0 mm below the surface of the brain) derived from the brain atlas of the pigeon [51]. The cannula, anchored to the skull with jeweler's screws and fixed with dental cement, was maintained patent between experiments by an inner removable stylet.

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For repeated blood sampling, the brachial artery was cannulated in a group of 18 pigeons at least 7 days after their ICV cannulation surgery. This procedure was carried out under general anesthesia, 2 days before experiment 3. To avoid blood clotting and bacterial contamination, the arterial cannula was filled with a polyvinylpirrolidone solution (PVP: K25, 1.5 g/ml in pyrogen-free isotonic NaCl) containing heparin (500 IU/ml), streptomycin (2 mg/ml) and penicillin-G (4800 IU/ml). Complete recovery of presurgical patterns of food and water intake, locomotion and defecation were observed 24 h after surgery in all animals used in the experiments. 2.2. Drugs and injections ICV injections were made through an inner cannula (stainless steel, G30), connected by polyethylene tubing to a Hamilton microsyringe (10 µl), extending 1 mm beyond the tip of the guide cannula. The injected volume (5.0 µl) was administered over a period of 120 s and a further 120 s was allowed for the solution to diffuse from the cannula. Orexins (ORXA: synthetic human, rat, mouse orexin-A, catalogue no O-6012; ORXB: human orexin-B, catalogue no O-6137; Sigma-Aldrich, St Louis, MO) were freshly dissolved in sterile pyrogen-free 0.85% NaCl solution (used as control solution). 2.3. Experimental procedures 2.3.1. Experiment 1: acute behavioral effects of ICV injections of ORXA and ORXB Experiment 1 was designed to examine the effects of ICV injections of different doses of ORXA (vehicle, 0.2, 0.6 or 2 nmol, n = 8), or ORXB (vehicle, 0.2, 0.6 or 2 nmol, n = 6) on food and water intake in two groups of experimentally naive free-feeding pigeons. Each animal within a group (ORXA or ORXB group) was tested with vehicle (sterile pyrogen-free 0.85% NaCl solution) and all doses of the group's ORX, assigned to each animal according to a Latin-squared design. Experimental sessions were separated from each other by a minimum 7-day interval and began always between 9:00 and 11:00 am. Immediately after the injection, the animal was returned to its home cage and the behavioral recording started. During the first hour after drug injection, digital video recordings (Sony Handycam MiniDV DCR-HC15) were taken from the bird's home cage, and the latency to the first event, as well as the total duration (sum of the duration of all events during the recording session) of drinking, feeding, preening, locomotor, exploratory, alert immobility and sleeplike behaviors were scored. Feeding was defined as a bout of pecking movements directed at the feeder, and included brief interpecking intervals (b3 s), during which the animal adopted an upright posture, showed swallowing and beak movements, and then started pecking again. Pecking at the feeder starting after four or more seconds after the end of the last pecking was recorded as a new feeding bout. Pigeons drink by suction of water through the beak; a drinking bout was recorded for the interval between each beak immersion in and its removal from the water

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reservoir's spout. A sleeping pigeon might sit or stand on one or both legs, with chest and neck plumage puffed up and one or both eyelids closed. Sleep-like behavior was recorded when the pigeon puffed up its feathers with at least one eye closed. Locomotion (at least a complete hind limb step), exploratory behavior (ballistic and angular head movements directed to every direction, preceded by a short movement in the opposite direction), alert immobility (head, body and legs–wings immobility with only respiratory and/or ocular and rapid eyelid movements visible) and preening (rubbing the beak or limbs over or between the feathers) were also scored. These behavioral units were defined and used in previous reports [46,47,52] and are showed in a movie clip associated to the present paper. Food and water intake were recorded 1, 2, 3 and 24 h after the injections. Food pellets were delivered in plastic cups with three holes of 6 cm each at the top, providing easy access to food, little spillage of the pellets, and reliable food weighing. Water was provided in plastic bottles which were closed at the top, with a spout that projected through the cage wires. At the end of the recording period, any food pellets that eventually spilled on the cage floor were recovered and weighed with the food that remained on the feeder. The difference between food or water weight at the beginning and at the end of the recording period was taken as the amount of food or water consumed, and these are expressed in grams per 100 g of body weight. 2.3.2. Experiment 2: long-term (24 h) ingestive effects of ICV injections of ORXA and ORXB In experiment 2 we recorded continuously food and water intake of 5 experimentally naive pigeons before and after ICV injections of ORXA (0.6 nmol), ORXB (0.6 nmol) or vehicle. These long-term and continuous ingestive recordings were performed using an instrumented system for automatic acquisition of feeding and drinking data developed in our laboratory [53], that allowed continuous monitoring of the weight of food pans and water bottles, through charge cells connected to a PCclone computer. Weight from each charge cell was acquired at 5 samples/s, then stored and viewed through locally developed software, and the raw data files were processed to produce hourly accounts of food removed from the containers. Animals were adapted to the recording chamber (with dimensions similar to those of the home cage) for at least 5 days before starting the experiments. Pretreatment recordings started at 9:00 am: 24 h later the drug was injected and the recording continued for an additional period of 24 h. Each animal received all treatments with at least a 7-day interval. Food spillage was controlled only at the end of a 24-h recording period and never exceeded 3% of the total amount of food removed from the containers. 2.3.3. Experiment 3: effects of ICV injections of ORXA on blood glucose and free fatty acids Experiment 3 was designed to examine the effects of ICV injections of ORXA on plasma glucose and FFA concentrations in free-feeding pigeons. Blood samples (0.5 ml) were collected immediately before (time 0), then 15 and 30 min after ICV

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Table 1 Ingestive effects of ICV injections of ORXA, ORXB or vehicle in free-feeding pigeons, 1, 2, 3 and 24 h after treatment Vehicle 1h ORXA n = 8/dose Food intake

0.1 ± 0.0 Water intake 0.3 ± 0.1 ORXB n = 6/dose Food intake

0.4 ± 0.1 Water intake 0.7 ± 0.2

0.2 nmol

0.6 nmol 2h

3h

24 h

1h

2.0 nmol

2h

3h

24 h 1 h

2h

3h

24 h

1h

2h

3h

24 h

0.3 ± 0.0 0.5 ± 0.1

0.3 ± 0.1 0.3 ± 0.1

4.6 ± 0.2 ± 0.0 0.2 ± 0.0 0.2 ± 0.0 0.3 7.5 ± 0.6 ± 0.1 0.4 ± 0.1 0.2 ± 0.1 0.7

0.2 ± 0.0 0.4 ± 0.1

0.3 ± 0.1 0.7 ± 0.2

5.9 ± 0.3 ± 0.1 0.3 ± 0.1 0.3 ± 0.1 5.7 ± .6 0.3 ± 0.1 0.1 ± 0.0 0.1 ± 0.0 5.6 ± 0.6 0.3 ± 0.1 0.2 ± 0.0 0.3 ± 0.1 6.8 ± 0.5 0.6 9.6 ± 0.5 ± 0.1 0.5 ± 0.1 0.6 ± 0.2 12.7 ± 2.4 0.5 ± 0.3 0.3 ± 0.1 0.3 ± 0.0 11.3 ± 1.6 0.8 ± 0.2 0.6 ± 0.1 0.4 ± 0.2 17.1 ± 5.3 1.2

5.5 ± 0.9 0.5 ± 0.1 0.3 ± 0.1 0.4 ± 0.0

4.4 ± 0.5 0.3 ± 0.1 0.3 ± 0.0 0.3 ± 0.1

4.3 ± 0.3

9.6 ± 0.8 0.5 ± 0.1 0.4 ± 0.1 0.6 ± 0.2

6.8 ± 0.8 0.8 ± 0.2 0.3 ± 0.1 0.5 ± 0.1

7.6 ± 0.6

Data are expressed as mean ± SEM of food or water intake (in g/100 g bw).

injections of ORXA (0, 0.2 or 0.6 nmol, N = 7, 6 and 5 per dose, respectively). The blood samples were centrifuged and aliquots of plasma were stored at − 20 °C until the time for the glucose and FFA assays. FFA levels were measured by the method of [54], adapted to the use of small (200 µl) amounts of plasma, and plasma glucose concentration was measured by the oxidase method (Analisa Diagnostica, Belo Horizonte, Brazil). At least 1 h before the beginning, and during the experiments, food was removed from the cage.

2.4. Histological and data analysis At the end of the experiments, the pigeons were deeply anesthetized and Evans blue dye (1% in water, 1 µl) was injected through the guide cannula, to confirm successful placement inside the lateral ventricle. The brains were removed and cut in a transverse plane so that the success of the cannula placement could be verified by the presence of dye in the ventricular system.

Table 2 Duration (in seconds), latency to the 1st episode (in seconds) and frequency of behavioral parameters of feeding, drinking, alert immobility, locomotor, preening, sleep-like and exploratory behaviors, 1 h after ICV injections of ORXA, ORXB or vehicle in free-feeding pigeons

Feeding Duration Latency Frequency Drinking Duration Latency Frequency Immobility Duration Latency Frequency Locomotion Duration Latency Frequency Preening Duration Latency Frequency Sleep-like Duration Latency Frequency Exploratory Duration Latency Frequency

ORXA vehicle

ORXA 0.2 nmol

ORXA 0.6 nmol

n=8

n=8

n=8

ORXA 2.0 nmol

ORXB vehicle

ORXB 0.2 nmol

ORXB 0.6 nmol

ORXB 2.0 nmol

39 ± 17.6 903 ± 446 2.3 ± 0.4

92.3 ± 39.2 1109 ± 391 5.8 ± 2.5

119 ± 35.1 773 ± 368 9.7 ± 3.4

77.8 ± 28.0 1368 ± 514 7.0 ± 2.7

43.3 ± 12.9 525 ± 315 3.8 ± 0.7

28.2 ± 8.3 1214 ± 462 3.6 ± 1.0

47.4 ± 17.8 1368 ± 497 3.0 ± 1.0

42.0 ± 6.2 647 ± 184 3.8 ± 0.6

4.6 ± 1.1 1428 ± 557 1.1 ± 0.3

28.3 ± 11.3 753 ± 272 3.0 ± 0.9

13.5 ± 5.1 1118 ± 412 3.2 ± 1.5

27.0 ± 7.7 1124 ± 521 3.7 ± 1.2

24.6 ± 12.0 1213 ± 505 2.7 ± 0.8

21.2 ± 7.5 1392 ± 402 2.4 ± 0.8

8.2 ± 4.4 1711 ± 448 1.6 ± 1.0

38.3 ± 11.9 1325 ± 88 4.8 ± 1.7

956 ± 216 375 ± 225 18.6 ± 1.8

593 ± 143 1238 ± 364 10.1 ± 1.6⁎

404 ± 240 2061 ± 585⁎ 8.8 ± 4.4⁎

216 ± 140 2239 ± 497⁎ 2.8 ± 1.2⁎

1272 ± 175 261 ± 85 16.5 ± 1.9

1102 ± 207 359 ± 128.7 14.7 ± 2.4

1310 ± 252 770 ± 307 11.2 ± 1.7

998 ± 265 503 ± 166 10.5 ± 1.8

26.1 ± 7.8 523 ± 283 9.0 ± 2.4

74.7 ± 25.0 409 ± 177 18.8 ± 5.2

75.6 ± 32.6 213 ± 64 21.3 ± 7.9

150.1 ± 67.9 128 ± 40 29.1 ± 7.6

74.6 ± 5.9 260 ± 43 14.7 ± 3.7

97.6 ± 46.3 366 ± 151 16.6 ± 5.6

75.3 ± 27.9 377 ± 165 13.5 ± 5.7

113.6 ± 40.9 703 ± 579 26.0 ± 6.7

668 ± 164 147 ± 29 16.6 ± 1.3

896 ± 112 285 ± 85 21.0 ± 3.8

550 ± 188 517 ± 229 19.8 ± 4.0

645 ± 113 148 ± 68 32.8 ± 5.1⁎

361 ± 84 238 ± 69 11.8 ± 2.2

570 ± 111 339 ± 150 13.7 ± 2.5

664 ± 103 311 ± 76 14.0 ± 2.8

441 ± 81 151 ± 23 15.0 ± 1.8

871 ± 186 891 ± 459 10.7 ± 2.7

369 ± 164⁎ 2443 ± 468⁎ 3.3 ± 1.3⁎

329 ± 180⁎ 2530 ± 459⁎ 5.2 ± 2.8⁎

0.7 ± 0.7⁎ 3483 ± 116⁎ 0.1 ± 0.1⁎

602 ± 180 1208 ± 298 7.7 ± 1.8

611 ± 183 1234 ± 301 7.4 ± 1.5

430 ± 115 1444 ± 375 5.4 ± 1.0

394 ± 16 2011 ± 564 5.3 ± 0.4

965 ± 138 3.6 ± 2.8 32.7 ± 3.9

1545 ± 269 24.7 ± 24.7 46.8 ± 8.6

2107 ± 297⁎ 0.0 ± 0.0 61.2 ± 9.9⁎

2482 ± 167⁎ 0.5 ± 0.5 70.5 ± 11.3⁎

1221 ± 184 26.2 ± 85.2 30.9 ± 4.2

1168 ± 258 11.1 ± 7.6 34.5 ± 6.4

1127 ± 222 8.4 ± 8.4 31.5 ± 6.8

1922 ± 274 21.5 ± 17.6 45.6 ± 5.9

Values are expressed as mean ± SEM. (⁎) p b 0.05 as compared to vehicle-injected animals.

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In experiment 1, food and water intake as well as behavioral data in each group (ORXA or ORXB) were examined by oneway ANOVA. In experiment 2, possible differences between vehicle, ORXA (0.6 nmol) and ORXB (0.6 nmol) injections were tested by a two-way ANOVA with time and treatment as factors. The same procedures were used for comparisons between pre- and post-injection data, using day time and recording period as factors. Data of plasma glucose and FFA produced in experiment 3 are expressed as mean ± SEM of the differences between basal and post-treatment values, and were analyzed by two-way ANOVA with treatment and sample time as factors. These procedures were followed by the post-hoc

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Duncan test, and a value of p b 0.05 was accepted as being statistically significant in all these tests. 3. Results Data from experiment 1 indicate that both ORXA and ORXB failed to affect food intake and water intake in any of the first 3 h or 24 h after treatments (Table 1), and did not change drinking and feeding behavioral parameters 1 h after injections (Table 2). Analysis of cumulative food or water intake in the first 3 h also indicated similar absence of ingestive effects (data not shown). Both ingestive and non-ingestive behavioral indexes remained

Fig. 1. Sleep-like and exploratory behavior parameters 1 h after ICV injection of ORXA (0.2, 0.6 or 2.0 nmol) or vehicle (sterile pyrogen-free 0.85% NaCl solution) in free-feeding pigeons. All data are expressed as mean ± SEM. (⁎) p b 0.05 as compared to vehicle-injected animals.

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unchanged after ORXB injections (Table 2). However, ORXA significantly changed time expended in exploratory behaviors and in sleeping-like behaviors (Fig. 1). Dose-related decreases in sleep duration [F(3,28) = 5.4; p = 0.02] and frequency [F(3,28) = 4.5; p = 0.02], as well as increases in latency to the first sleep-like episode [F(3,28) = 7.0; p = 0.01] were observed (Fig. 1). The duration [F(3,28) = 6.1; p = 0.02] and frequency [F(3,28) = 6.9; p = 0.01] of the episodes of alert immobility also decreased, and the latency to its first occurrence was increased [F(3,28) = 9.2; p = 0.004] (Table 2). These changes were associated with an increased duration [F(3,28) = 8.43; p = 0.002] and frequency [F(3,28) = 3.46; p = 0.03] of exploratory behavior (Fig. 1) and with an also increased frequency of preening [F(3,28) = 3.41; p = 0.009] (Table 2). Locomotor behavior showed a nonsignificant trend (p = 0.07) to increase in frequency and duration (Table 2). Beyond the above mentioned effects, the 0.6 and 2.0 nmol ORXA doses evoked frequent, frantic and coordinated flapping movements of both wings, which resemble wing-flapping flight movements and are rarely observed in the experimental set in untreated pigeons or after vehicle injections. The results of experiment 2 essentially confirmed the results of experiment 1, indicating an absence of long-term effects of ICV injections of ORXA and of ORXB on feeding (Fig. 2) or drinking (not shown). The hourly gathered food and water intake were statistically indistinguishable from those of the same hour in the pre-injection period and of the same hour in vehicle experiments. An exception was the food intake in the fourth hour after ORXA injection, which was significantly different (p = 0.04) from the vehicle data for the same hour (but not from the same hour in the pre-injection period). Analysis of the raw data indicates that this difference was due to an increased intake at this time, observed in 2 pigeons (out of 5). In experiment 3, the ICV injections of ORXA also failed to significantly affect blood glucose and FFA levels 15 and 30 min after treatment (Fig. 3).

4. Discussion The present data indicate that ICV injections of mammalian ORXA and ORXB, in a range of doses that can evoke prompt hyperphagic responses in rodents and teleost fishes (see below), are unable to affect either food/water intake or feeding/drinking behaviors in adult pigeons. These findings confirm, through direct behavioral observation, early proposals by which avian orexin circuits are not important to avian feeding control. These proposals were based on food intake effects of ICV ORXA and ORXB injections in neonatal chicken and on in situ hybridization experiments indicating no cerulenin- or fasting-induced changes in ORX or ORX receptor mRNA levels [37,39,40]. Additional support for the notion that central ORXA circuits in the pigeon may have poor, if any, role in energy balance, comes from the results of experiment 3. These treatments also failed to change the blood levels of glucose and FFA in the pigeon. The absence of noticeable ORXB effects either on feeding or on arousal-related behaviors in the present study is particularly puzzling. A relevant phylogenetic conservation of ORXA and ORXB amino acid sequences in different vertebrate classes including chicken has been noted [1,31,32]. However, the cloned cDNA for the chicken (cORXr) corresponds to the mammalian (rat, dog and human) type 2 ORX receptor, showing nearly 80% similarity at the amino acid level to those of mammals [33]; in this study, no evidence was found for a gene encoding a receptor similar to the mammalian type 1 ORXr. Interestingly, mammalian ORX2r binds ORXA and -B with similar affinity, while ORXA binds to type 1 ORXr with an affinity 10 times higher than that of ORXB [1]. Thus, it was reasonable to expect at least qualitatively similar effects of both peptides. While we cannot presently provide any explanation for the absence of human ORXB peptide effects in the pigeon, our data indicate that at least the mammalian ORXA peptide may interact with the avian-type ORXr, to evoke intense arousing effects.

Fig. 2. Food intake (total for each hour during 48 consecutive hours) before and after ICV injections of ORXA (0.6 nmol) or vehicle (sterile pyrogen-free 0.85% NaCl solution) in free-feeding pigeons. The arrow indicates the moment of the injections. All data are expressed as mean ± SEM. (⁎) p b 0.05 as compared to vehicle-injected animals at the same hour.

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Fig. 3. Effects of ICV injections of ORXA (0.2 or 0.6 nmol) or vehicle (sterile pyrogen-free 0.85% NaCl solution) on blood glucose and free fatty acids (FFA) in free-feeding pigeons. All data are the mean ± SEM of the difference from baseline (pre-injection) values. Baseline values for blood glucose (mg/100 ml): vehicle (n = 7) = 296 ± 6; ORXA (0.2 nmol, n = 6) = 293 ± 5; ORXA (0.6 nmol, n = 5) = 301 ± 21. Baseline FFA values (μmol/ml): vehicle (n = 7) = 1.25 ± 0.10; ORXA (0.2 nmol, n = 6) = 0.93 ± 0.06; ORXA (0.6 nmol, n = 5) = 1.88 ± 0.38. (⁎) p b 0.05 as compared to vehicle-treated animals.

Our data run opposite to the intense hyperphagic responses observed when ORXA is administered centrally to rodents (e.g., [1,8]) and to teleost fish (goldfish; [55,56]). These dissimilar results could be due to differences in pigeon's ORX receptors affecting their responses to mammalian ORXA. Although we cannot completely exclude this possibility, it should be noted that there are strong correspondences between the cloned chicken prepro-orexin cDNA, the ORXA and ORXB peptides and the cloned chicken ORX receptor cDNA with their mammalian counterparts [33]. Summed up to the intense hyperphagic effects of mammalian ORXA observed in a taxon phylogenetically far distant from mammals (as the teleost fishes), and the powerful arousing effects of mammalian ORXA in the pigeon (similar to those found in fishes and mammals, see

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below), these data make the pharmacological and biochemical differences between avian and mammalian ORX systems less tenable as explanations for the absence of ORX-evoked hyperphagy in birds. In this line, it is interesting to note that, even in rodents, the involvement of ORX systems on feeding regulation is apparently weaker than that of other orexigenic peptides (e.g., [57]), ephemeral (evoking no change on cumulative 24-h food intake;) and to be significantly dependent on the nutritional state, age and time of the light–dark cycle [8,57,58]. ICV injections of ORX in rats can evoke a thermogenic-associated hypophagic effect in fasting rats, if it is administrated 6 h before food presentation [59]. It is also worth noting that 48-h food deprivation does not change ORXA levels in the cerebrospinal fluid of dogs [60], and that fasting or long-term alterations in the body weight do not affect on expression of mRNA for preproorexin in the hypothalamus of the sheep [42], although ICV (but not i.v.) injections of ORXB in the sheep have been shown to increase feeding [41]. ICV injections of ORXA decreased food intake at 2, 4 and 8 h in adult male rhesus monkeys, after peptide doses that are hyperphagic in rodents [61]. The absence of acute changes in circulating glucose and FFA after ORX treatments in the present study further underscores an apparently poor association between ORX circuits and energy homeostasis-related signals in the pigeon, as opposed to the observed in mammals. Even in the lack of comparable behavioral analyses of ORX effects in amphibians and reptiles (and although their brains contain ORX immunoreactive neurons, see Introduction), these data suggest that a role for ORX in feeding behavior control is not a functional character common to (or fundamental in) all vertebrates and that ORXmediated feeding may represent a species-specific functional character even among mammals. This proposal clearly demands further and systematic behavioral studies in a broader range of vertebrate forms. Conversely, the present data provide the first demonstration that ICV injections of mammalian ORXA induce to potent, rapid, and dose-dependent arousal-promoting effects in birds. ORXA injections evoked a dose-related decrease in the incidence and duration of sleep-like postures and of alert immobility. These changes were accompanied by a remarkable increase in head and neck exploratory movements, as well as an increased incidence of wing-flapping behavior. Paradoxically, ORXA treatments only weakly affected body displacement (locomotion) and preening; these data may suggest that behavioral activation produced by ORXA is more related to a specific increase in vigilance state (and thus intense exploratory behavior toward environment) than to an unspecific increase in motor activity. The appearance of the wing-flapping behavior after the two higher doses of ORXA adds further support to this suggestion. Wing-flapping was suggested to be a stress-related behavior [62] or an index of locomotor behavior in chickens [63] and was elicited by ICV injections of ACTH in the pigeon [64] and in the chicken [65]. In the latter study, the behavioral effects of ACTH (1–24) ICV injections in chicks suggested the presence of a state of arousal that resembles fear/anxiety. ICV injection of OT [66] and of the brain–gut peptide neuromedin U [67] in chicks

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increased locomotor activity as measured by a greater incidence of wing-flapping (among other active behaviors), possibly acting through corticotrophin releasing hormone (CRH) and/or arginine-vasotocin (AVT, a non-mammalian equivalent of arginine-vasopressin) hypothalamic circuits. It is thus possible that ORX circuits in birds are involved also in fear- or stressrelated behaviors, besides (or associated to) their role in arousal or vigilance. In house finches (C. mexicanus), high densities of orexin-immunoreactive fibers were observed in most parts of the diencephalon (particularly in the ventral and medial hypothalamic, preoptic and basal prosencephalic regions) and, with less intensity, also in most of the nidopallial and mesopallial telencephalic regions. In addition, brainstem regions comparable to mammalian nuclei involved in arousal, vigilance and in the control of sleep–waking cycle, such as the locus coeruleus, the periaqueductal gray and the pars compacta of the substantia nigra, are intensely innervated by orexin-ir fibers [38]. These connections put orexinergic circuits in a position suitable to influence widespread mechanisms controlling arousal states and species-typical behaviors in birds. Heightened arousal, increased wakefulness, locomotion and exploratory behaviors and suppression of sleep-related signals are among the most prominent effects of central injections of orexins in mammals [5,8,12,13]. Orexins have been reported to induce vigilance, locomotor, grooming and burrowing, as well as stress-related behaviors in rats (e.g., [4,17]) that are possibly mediated by ORX-induced CRH secretion [68]. Furthermore, ORXA was observed to be increased in the CSF of dogs after sleep deprivation and exercise [60]. Numerous studies suggest that orexin neurons are active during wakefulness and inactive during sleep in rodents and that orexin neuron deficit in humans and dogs is associated to narcolepsy, which is characterized by poor maintenance of wakefulness and intrusions of rapid eye movement sleep-like phenomena into wakefulness [12]. These data indicate that ORX circuits contribute importantly to the stability of vigilance states in mammals. ICV injections of mammalian ORX in the goldfish (C. auratus) resulted both in increased feeding and in increased locomotor behavior, while fasting or glycemic changes modulate the ORXlike immuoreactivity in the hypothalamus [55,56,69], indicating that ORX circuits in teleosts may coordinate feeding and arousalrelated mechanisms in a way which resembles that repeatedly observed in rodents. While a clear picture of the distribution of ORX functional attributes in vertebrates must wait for studies in a more comprehensive range of animals from each taxonomic category, it is apparent that alertness-related functions are more widely disseminated among the vertebrate species so far examined than the functions associated to ingestive behaviors. The present data substantiate the lack of orexigenic effects of ORXs in avian species and suggest that an important role in vigilance control may represent a conserved functional attribute of orexinergic circuits in vertebrates. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.regpep.2007.12.003.

References [1] Sakurai T, Amemiya A, Ishii M, Matsuzaki I, Chemelli RM, Tanaka H, Williams SC, Richarson JA, Kozlowski GP, Wilson S, Arch JP, Buckingham RE, Haynes AC, Carr SA, Annan RS, McNulty DE, Liu WS, Terret JA, Elshourbagy NA, Bergsma DJ, Yanagisawa M. Orexins and orexin receptors: a family of hypothalamic neuropeptides and G-proteincoupled receptors that regulate feeding behavior. Cell 1998;92:573–85. [2] De Lecea L, Kilduff TS, Peyron C, Gao X, Foye PE, Danielson PE, Fukuhara C, Battenberg EL, Gautvik VT, Bartlett FS, Frankel WN, van den Pol AN, Bloom FE, Gautvik KM, Sutcliffe JG. The hypocretins: hypothalamus-specific peptides with neuroexcitatory activity. Proc Natl Acad Sci USA 1998;95:322–7. [3] Peyron C, Tighe DK, van den Pol AN, de Lecea L, Heller HC, Sutcliffe JG, Kilduff TS. Neurons containing hypocretin (orexin) project to multiple neuronal systems. J Neurosci 1998;18:9996–10015. [4] Date Y, Ueta Y, Yamashita H, Yamaguchi H, Matsukura S, Kangawa K, Sakurai T, Yanagisawa M, Nakazato M. Orexins, orexigenic hypothalamic peptides, interact with autonomic, neuroendocrine and neuroregulatory systems. Proc Natl Acad Sci USA 1999;96:748–53. [5] Rodgers RJ, Ishii Y, Halford JCG, Blundell JE. Orexins and appetite regulation. Neuropeptides 2002;36:303–25. [6] Cai XJ, Widdowson PS, Harrold J, Wilson S, Buckingham RE, Arch JRS, Tadayyon M, Clapham JC, Wilding J, Williams G. Hypothalamic orexin expression: modulation by blood glucose and feeding. Diabetes 1999;48:2132–7. [7] Yamamoto Y, Ueta Y, Serino R, Nomura M, Shibuya I, Yamashita H. Effects of food restriction on the hypothalamic prepro-orexin gene expression in genetically obese mice. Brain Res Bull 2000;51:515–21. [8] Kotz CM. Integration of feeding and spontaneous physical activity: role for orexin. Physiol Behav 2006;88:294–301. [9] Dube MG, Kalra SP, Kalra PS. Food intake elicited by central administration of orexins/hypocretins: identification of hypothalamic sites of action. Brain Res 1999;842:473–7. [10] Kotz CM, Teske JA, Levine JA, Wang CF. Feeding and activity induced by orexin A in the lateral hypothalamus in rats. Regul Pept 2002;104:27–32. [11] Mullet MA, Billington CJ, Levine AS, Kotz CM. Hypocretin 1 in the lateral hypothalamus activates key feeding-regulatory brain sites. NeuroReport 2000;11:103–8. [12] De Lecea L, Sutcliffe JG. The hypocretins and sleep. FEBS J 2005;272(22):5675–88. [13] Sakurai T. Roles of orexin/hypocretin in regulation of sleep/wakefulness and energy homeostasis. Sleep Med Rev 2005;9(4):231–41. [14] Chemelli RM, Willie JT, Sinton CM, Elmquist JK, Scammell T, Lee C, Richardson JA, Williams SC, Xiong Y, Kisanuki Y, Fitch TE, Nakazato M, Hammer RE, Saper CB, Yanagisawa M. Narcolepsy in orexin knockout mice: molecular genetics of sleep regulation. Cell 1999;98:437–51. [15] Nishino S, Ripley B, Overeem S, Lammers GJ, Mignot E. Hypocretin (orexin) deficiency in human narcolepsy. Lancet 2000;355:39–40. [16] Hagan J, Leslie RA, Patel S, Evans ML, Wattam TA, Holmes S, Benham CD, Taylor SG, Routledge C, Hemmati P, Munton RP, Ashmeade TE, Shah AS, Hatcher JP, Hatcher PD, Jones DNC, Smith MI, Piper DC, Hunter AJ, Porter RA, Upton N. Orexin A activates locus coeruleus cell firing and increases arousal in the rat. Proc Natl Acad Sci USA 1999;96:10911–6. [17] Ida T, Nakahara K, Katayama T, Murakami N, Nakazato M. Effect of lateral cerebroventricular injection of the appetite-stimulating neuropeptide, orexin and neuropeptide Y, on the various behavioral activities of rats. Brain Res 1999;821:526–9. [18] Piper DC, Upton N, Smith MI, Hunter AJ. The novel brain neuro-peptide, orexin-A, modulates the sleep–wake cycle of rats. Eur J Neurosci 2000;12:726–30. [19] Duxon MS, Stretton J, Starr K, Jones DNC, Holland V, Riley G, Jerman J, Brough S, Smart D, Johns A, Chan W, Porter RA, Upton N. Evidence that orexin-A-evoked grooming in the rat is mediated by orexin-1 (OX1 ) receptors, with downstream 5-HT2C receptor involvement. Psychopharmacology 2001;153:203–9. [20] Bourgin P, Huitron-Resendiz S, Spier AD, Fabre V, Morte B, Criado JR, Sutcliffe JG, Henriksen SJ, DeLecea L. Hypocretin-1 modulates rapid eye

E.S. da Silva et al. / Regulatory Peptides 147 (2008) 9–18

[21]

[22]

[23]

[24]

[25]

[26]

[27]

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

[40]

movement sleep through activation of locus coeruleus neurons. J Neurosci 2000;20:7760–5. Monda M, Viggiano A, Fuccio F, De Luca V. Clozapine blocks sympathetic and thermogenic reactions induced by orexin A in rat. Physiol Res 2004;53:507–13. Shirasaka T, Nakazato M, Matsukura S, Takasaki M, Kannan H. Sympathetic and cardiovascular actions of orexins in conscious rats. Am J Physiol 1999;277:R1780–5. Yoshimichi G, Yoshimatsu H, Masaki T, Sakata T. Orexin-A regulates body temperature in coordination with arousal status. Exp Biol Med 2001;226:468–76. Berthoud HR, Patterson LM, Sutton GM, Morrison C, Zheng H. Orexin inputs to caudal raphé neurons involved in thermal, cardiovascular, and gastrointestinal regulation. Histochem Cell Biol 2005;123:147–56. Chang GQ, Karatayev O, Davydova Z, Leibowitz SF. Circulating triglycerides impact on orexigenic peptides and neuronal activity in hypothalamus. Endocrinology 2004;145:3904–12. Cai XJ, Evans ML, Lister CA, Leslie RA, Arch JR, Wilson S, Williams G. Hypoglycemia activates orexin neurons and selectively increases hypothalamic orexin-B levels: responses inhibited by feeding and possibly mediated by the nucleus of the solitary tract. Diabetes 2001;50(1):105–12. Burdakov D, Gerasimenko O, Verkhratsky A. Physiological changes in glucose differentially modulate the excitability of hypothalamic melanin-concentrating hormone and orexin neurons in situ. J Neurosci 2005;25:2429–33. Wang J, Osaka T, Inoue S. Energy expenditure by intracerebroventricular administration of orexin to anesthetized rats. Neurosci Lett 2001;315:49–52. Lubkin M, Stricker-Krongrad A. Independent feeding and metabolic actions of orexins in mice. Biochem Biophys Res Commun 1998;253:241–5. Kaslin J, Nystedt JM, Ostergard M, Peitsaro N, Panula P. The orexin/ hypocretin system in zebrafish is connected to the aminergic and cholinergic systems. J Neurosci 2004;24:2678–89. Shibahara M, Sakurai T, Nambu T, Takenouchi T, Iwaasa H, Egashira SI, Ihara M, Goto K. Structure, tissue distribution, and pharmacological characterization of Xenopus orexins. Peptides 1999;20:1169–76. Ohkubo T, Boswell T, Lumineau S. Molecular cloning of chicken preproorexin cDNA and preferential expression in the chicken hypothalamus. Biochim Biophys Acta 2002;1577:476–80. Ohkubo T, Tsukada A, Shamoto K. cDNA cloning of chicken orexin receptor and tissue distribution: sexually dimorphic expression in chicken gonads. J Mol Endocrinol 2003;31(3):499–508. Galas L, Vaudry H, Braun B, van den Pol AN, de Lecea L, Sutcliffe JG, Chartrel N. Immunohistochemical localization and biochemical characterization of hypocretin/orexin-related peptides in the central nervous system of the frog (Rana ridibunda). J Comp Neurol 2001;429:242–52. Singletary KG, Delville Y, Farrell WJ, Wilczynski W. Distribution of orexin/hypocretin immunoreactivity in the nervous system of the green treefrog, Hyla cinerea. Brain Res 2005;1041:231–6. Farrell WJ, Delville Y, Wilczynski W. Immunocytochemical localization of orexin in the brain of the green anole lizard (Anolis carolinensis). Soc Neur Abstr 2003;33:828–34. Phillips-Singh D, Li Q, Takeuchi S, Ohkubo T, Sharp PJ, Boswell T. Fasting differentially regulates expression of agouti-related peptide, pro-opiomelanocortin, prepro-orexin, and vasoactive intestinal polypeptide mRNAs in the hypothalamus of Japanese quail. Cell Tissue Res 2003;313(2):217–25. Singletary KG, Deviche P, Strand C, Delville Y. Distribution of orexin/ hypocretin immunoreactivity in the brain of a male songbird, the house finch, Carpodacus mexicanus. J Chem Neuroanat 2007;33:101–9. Furuse M, Ando R, Bungo T, Ao R, Shimojo M, Masuda. Intracerebroventricular injection of orexins does not stimulate food intake in neonatal chicks. Br Poult Sci 1999;40(5):698–700. Dridi S, Ververken C, Hillgartner FB, Arckens L, Van der Gucht E, Cnops L, Decuypere E, Buyse J. FAS inhibitor cerulenin reduces food intake and melanocortin receptor gene expression without modulating the other (an)

[41]

[42]

[43] [44] [45]

[46]

[47]

[48]

[49]

[50] [51] [52]

[53]

[54] [55]

[56]

[57]

[58]

[59] [60]

[61]

17

orexigenic neuropeptides in chickens. Am J Physiol Regul Integr Comp Physiol 2006;291(1):R138–47. Sartin JL, Dyer C, Matteri R, Buxton D, Buonomo F, Shores M, Baker J, Osborne JA, Braden T, Steele B. Effect of intracerebroventricular orexin-B on food intake in sheep. J Anim Sci 2001;79(6):1573–7. Iqbal J, Henry BA, Pompolo S, Rao A, Clarke IJ. Long-term alteration in bodyweight and food restriction does not affect the gene expression of either preproorexin or prodynorphin in the sheep. Neuroscience 2003;118(1):217–26. Richards MP. Genetic regulation of feed intake and energy balance in poultry. Poultry Sci 2003;82(6):907–16. Barbatto GF. Genetic control of food intake in chickens. J Nutr 1994;124:1341S–8S. Dario AJS, Lopes PRC, Freitas CG, Paschoalini MA, Marino-Neto J. Electrographic patterns of postprandial sleep after food deprivation or intraventricular adrenaline injections in pigeons. Brain Res Bull 1996;39:249–54. Hagemann LF, Costa CV, Zeni LZR, Freitas CG, Marino Neto J, Paschoalini MA. Food intake after adrenaline and noradrenaline injections into the hypothalamic paraventricular nucleus in pigeons. Physiol Behav 1998;64:645–52. Steffens SM, Casas DC, Milanez BC, Freitas CG, Paschoalini MA, Marino Neto J. Hypophagic and dipsogenic effects of central 5-HT injections in pigeons. Brain Res Bull 1997;44:681–8. Denbow DM, Van Krey HP, Cherry JA. Feeding and drinking response of young chicks to injections of serotonin into the lateral ventricle of the brain. Poultry Sci 1982;61:150–5. Denbow DM, Van krey HP, Lacy MP, Dietrick TJ. Feeding, drinking and body temperature of Leghorn chicks: effects of ICV injections of biogenic amines. Physiol Behav 1983;31:85–90. Denbow DM. Body temperature and food intake of turkeys following ICV injections of serotonin. Nutr Behav 1984;1:301–4. Karten HJ, Hodos WA. A stereotaxic atlas of the brain of the pigeon (Columba livia). Baltimore: Johns Hopkins Press; 1967. 193 pp. Da Silva A, Marino Neto J, Paschoalini MA. Feeding induced by microinjections of NMDA and AMPA-kainate receptor antagonists into ventral striatal and ventral pallidal areas of the pigeon. Brain Res 2003;966:76–83. Pereira GV, Bose R, Paschoalini MA, Marino Neto J. Instrumented system for automatic acquisition of ponderal and behavioral data of related to feeding and drinking behaviors in avian species. Anais do XXI Congresso da Associação Latino-americana de Ciências Fisiológicas, vol. 21. Ribeirão Preto, SP; 2003. p. 341. Dole P, Meinertz H. Microdetermination of long chain fatty acids in plasma and tissues. J Biol Chem 1960;235:2595–9. Nakamachi T, Matsuda K, Maruyama K, Miura T, Uchiyama M, Funahashi H, Sakurai T, Shioda S. Regulation by orexin of feeding behaviour and locomotor activity in the goldfish. J Neuroendocrinol 2006;18:290–7. Volkoff H. The role of neuropeptide Y, orexins, cocaine and amphetamine-related transcript, cholecystokinin, amylin and leptin in the regulation of feeding in fish. Comp Biochem Physiol A Mol Integr Physiol 2006;144(3):325–31. Akimoto-Takano S, Sakurai C, Kanai S, Hosoya H, Ohta M, Miyasaka K. Differences in the appetite-stimulating effect of orexin, neuropeptide Y and ghrelin among young, adult and old rats. Neuroendocrinology 2005;82(5–6):256–63. Takano S, Kanai S, Hosoya H, Ohta M, Uematsu H, Miyasaka K. OrexinA does not stimulate food intake in old rats. Am J Physiol Gastrointest Liver Physiol 2004;287(6):1182–7. Monda M, Viggiano A, De Luca V. Paradoxical effect of orexin A: hypophagia induced by hyperthermia. Brain Res 2003;961:220–8. Wu MF, John J, Maidment N, Lam HA, Siegel JM. Hypocretin release in normal and narcoleptic dogs after food and sleep deprivation, eating, and movement. Am J Physiol Regul Integr Comp Physiol 2002;283(5):1079–86. Ramsey JJ, Kemnitz JW, Newton W, Hagopian K, Patterson TA, Swick AG. Food intake in rhesus monkeys following central administration of orexins. Regul Pept 2005;124:209–14.

18

E.S. da Silva et al. / Regulatory Peptides 147 (2008) 9–18

[62] Debut M, Berri C, Arnould C, Guemene D, Sante-Lhoutellier V, Sellier N, Baeza E, Jehl N, Jego Y, Beaumont C, Le Bihan-Duval E. Behavioural and physiological responses of three chicken breeds to pre-slaughter shackling and acute heat stress. Br Poult Sci 2005;46:527–35. [63] Abbasnejad M, Jonaidi H, Denbow DM, Pour Rahimi AM. Feeding and locomotion responses to centrally injected nociceptin/orphanin FQ in chicks. Physiol Behav 2005;85:383–6. [64] Deviche P, Delius JD. Short-term modulation of domestic pigeon (Columba livia L.) behavior induced by intraventricular administration of ACTH. Z Tierpsychol 1981;55:335–42. [65] Panksepp J, Normansell L. Effects of ACTH (1–24) and ACTH/MSH (4–10) on isolation-induced distress vocalization in domestic chicks. Peptides 1990;11:915–9.

[66] Jonaidi H, Oloumi MM, Denbow DM. Behavioral effects of intracerebroventricular injection of oxytocin in birds. Physiol Behav 2003;79(4–5):725–9. [67] Kamisoyama H, Honda K, Saneyasu T, Sugahara K, Hasegawa S. Central administration of neuromedin U suppresses food intake in chicks. Neurosci Lett 2007;420:1–5. [68] Ida T, Nakahara K, Murakami T, Hanada R, Nakazato M, Murakami N. Possible involvement of orexin in the stress reaction in rats. Biochem Biophys Res Commun 2000;270:318–23. [69] Volkoff H, Bjorklund JM, Peter RE. Stimulation of feeding behavior and food consumption in the goldfish, Carassius auratus, by orexin-A and orexin-B. Brain Res 1999;846:204–9.