Orexin A enhances locomotor activity and induces anxiogenic-like action in the goldfish, Carassius auratus

Hormones and Behavior 66 (2014) 317–323

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Regular article

Orexin A enhances locomotor activity and induces anxiogenic-like action in the goldfish, Carassius auratus Tomoya Nakamachi a,1, Haruki Shibata a,1, Atsushi Sakashita a, Naoto Iinuma a, Kohei Wada a, Norifumi Konno a, Kouhei Matsuda a,b,⁎ a b

Laboratory of Regulatory Biology, Graduate School of Science and Engineering, University of Toyama, 3190-Gofuku, Toyama 930-8555, Japan Laboratory of Regulatory Biology, Graduate School of Innovative Life Science, University of Toyama, 3190-Gofuku, Toyama 930-8555, Japan

a r t i c l e

i n f o

Article history: Received 7 March 2014 Revised 5 June 2014 Accepted 6 June 2014 Available online 14 June 2014 Keywords: Goldfish Orexin A ICV administration Locomotor activity Psychomotor activity Anxiogenic-like action

a b s t r a c t Orexin acts as an orexigenic factor for the regulation of appetite and rhythmicity in rodents. In goldfish, intracerebroventricular (ICV) administration of orexin A has been shown to affect not only food intake, but also locomotor activity. However, as there is still no information regarding the effect of orexin A on emotional behavior in goldfish, we investigated the effect of orexin A on psychomotor activity in this species. Intracerebroventricular administration of synthetic orexin A at 2 and 4 pmol/g body weight (BW) enhanced locomotor activity, and this enhancement by orexin A at 4 pmol/g BW was attenuated by treatment with the orexin receptor 1 antagonist, SB334867, at 10 pmol/g BW. Since intact goldfish prefer a black to a white background area, or the lower to the upper area of a tank, we used two types of preference tests (black/white and upper/lower tests) for measuring anxiety-like behavior in goldfish. Intracerebroventricular administration of orexin A at 4 pmol/g BW shortened the time spent in the white background area, and increased the time taken to move from the lower to the upper area. This action of orexin A mimicked that of the central-type benzodiazepine receptor inverse agonist, FG-7142 (an anxiogenic agent), at 4 pmol/g BW. The anxiogenic-like effect of orexin A was abolished by treatment with SB334867 at 10 pmol/g BW. These results indicate that orexin A potently affects psychomotor activity in goldfish. © 2014 Elsevier Inc. All rights reserved.

Introduction Orexin (also termed hypocretin) is a neuropeptide that was first identified as an orphan receptor ligand, and subsequently as an appetite regulator (Sakurai et al., 1998). Orexin exists as two molecular forms derived from the same precursor: a 33-residue peptide known as orexin A, and a 28-residue peptide known as orexin B. Although it was thought originally that orexin had no functional or structural identity with any other known regulatory peptide, orexin has been considered to belong to the incretin gene family of peptides, including members of the secretin–glucagon superfamily such as growth hormone releasing hormone, pituitary adenylate cyclase-activating polypeptide, vasoactive intestinal peptide, and glucagon-like peptides (Alvarez and Sutcliffe, 2002; Tam et al., 2011). Subsequently, orexin receptors have been found to have two forms: orexin receptors 1 and 2 (OX1R and OX2R) (Sakurai et al., 1998; Tam et al., 2011; Wong et al., 2011). In mammals, neuronal cell

⁎ Corresponding author at: Laboratory of Regulatory Biology, Graduate School of Science and Engineering, Graduate School of Innovative Life Science, University of Toyama, 3190-Gofuku, Toyama 930-8555, Japan. Fax: +81 76 445 6549. E-mail address: [email protected] (K. Matsuda). 1 Contributed equally to this work.

http://dx.doi.org/10.1016/j.yhbeh.2014.06.004 0018-506X/© 2014 Elsevier Inc. All rights reserved.

bodies containing orexin-like immunoreactivity are located in the lateral hypothalamus, which is referred to as the “orexigenic center”, and nerve fibers containing orexin-like immunoreactivity are widely distributed in various regions including the cerebral cortex, hippocampus, limbic system and brainstem, suggesting that orexin controls multiple brain functions such as emotional regulation (de Lecea et al., 1998; Li et al., 2013; Sakurai and Mieda, 2011; Thannickal et al., 2000). Indeed, consistent with this observation, orexin has been found to regulate rhythmicity such as the sleep–wakefulness cycle (Kilduff and Peyron, 2000; Kohsaka et al., 2001; Porkka-Heiskanen et al., 2004; Sakurai, 2005; Sakurai et al., 1998). The orexin gene or cDNA has also been characterized in nonmammalian species. The structure of fish orexin appears to be conserved (Kaslin et al., 2004; Tam et al., 2011; Wong et al., 2011). Recent studies indicate that some neuropeptides influence food consumption and related behavior in non-mammalian vertebrates, notably teleost fish (Kang et al., 2011; Lin et al., 2000; Matsuda et al., 2011a). Orexin is widely distributed in the brain of teleosts and amphibians, and seems to exert central and neuroendocrine functions (Huesa et al., 2005; Kaslin et al., 2004; López et al., 2009). In particular, its effect on feeding behavior in teleosts has been well studied. In the goldfish, ICV administration of orexin A stimulates food intake, and this orexigenic

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action is mediated via the orexin receptor, subsequently leading to activation of the neuropeptide Y (NPY) Y1 receptor (Matsuda, 2009; Matsuda et al., 2011a; Nakamachi et al., 2006; Volkoff et al., 1999). In this species, several neuropeptides regulating food intake affect swimming pattern (Maruyama et al., 2008; Matsuda et al., 2005, 2006, 2007, 2011a, 2013; Yahashi et al., 2012), and ICV administration of orexin A also influences locomotor activity (Nakamachi et al., 2006). Behavioral changes induced by orexin A suggest its psychophysiological roles in goldfish. In rodents, orexin A induces anxiety-like behavior when ICV-injected (Avolio et al., 2011; Ito et al., 2008; Lungwitz et al., 2012; Suzuki et al., 2005). This anxiety-like behavior is attenuated by treatment with the OX1R antagonist, SB334867, suggesting that the psychophysiological action of orexin A is mediated by the OX1Rsignaling pathway in mammals (Rodgers et al., 2013; Staples and Cornish, 2014). Information about the orexin receptor in nonmammals, including birds, amphibians and teleost fish, has been insufficient. To date, one receptor for orexin has been identified in chicken, Xenopus and zebrafish (Ohkubo et al., 2003; Tam et al., 2011; Yokogawa et al., 2007), and this receptor corresponds structurally to mammalian OX2R (Wong et al., 2011). However, there is little information about the effect of orexin A on emotional behavior in fish. Therefore, we investigated the roles of orexin A in goldfish by focusing on its locomotor and psychomotor activities in this species. Materials and methods Animals Young goldfish (Carassius auratus, 7–10 g body weight, BW) of both sexes were obtained commercially and kept under controlled light/dark conditions (12 L/12 D) in a temperature-regulated water tank (20–24 °C) for 2 weeks before use in the experiments. The fish were fed a commercially available granular diet (Tetragold, Tetra GmbH, Melle, Germany, containing 32% protein, 5% dietary fat, 2% dietary fiber, 6% minerals, 8% water and 47% other components including 17% carbohydrate) once a day at noon. All animal experiments were conducted in accordance with the University of Toyama guidelines for the care and use of animals. Chemicals Synthetic human orexin A (Peptide Institute Co., Osaka, Japan) was used in this study. Its effectiveness in terms of enhancing the feeding behavior and affecting the swimming activity of goldfish has been confirmed previously (Kojima et al., 2009; Miura et al., 2007; Nakamachi et al., 2006; Yokobori et al., 2011). The orexin receptor 1 antagonist SB334867 (N-(2-methyl-6-benzoxazolyl)-N′-1,5-naphthyridin-4-yl urea, Tocris Cookson Ltd., Bristol, UK), the central-type benzodiazepine receptor (CBR) inverse agonist FG-7142 (an anxiogenic agent, Nmethyl-9H-pyrodo[5,4-b]indole-3carboxamide, Sigma-Aldrich, St. Louis, MO, USA) and the CBR agonist diazepam (7-chloromethyl-5phenyl-3H-1,4-benzodiazepin 2(1H)-one, Sigma-Aldrich) were used. Orexin A was dissolved in a saline solution containing 0.6% NaCl and 0.02% Na2CO3 at a concentration of 1.0 mM and stored at −80 °C before use. SB334867, FG-7142 and diazepam were dissolved in 20% dimethyl sulfoxide at a concentration of 20 mM for storage and diluted with saline before use. 3-Aminobenzoic acid ethyl ester (MS-222) for anesthesia was obtained commercially from Sigma-Aldrich. Effect of ICV administration of orexin A on locomotor activity, and effect of ICV preinjection of SB334867 on orexin A-induced hypermotility Detailed descriptions of the methods used to evaluate locomotor activity in goldfish have been reported elsewhere (Matsuda et al., 2011b, 2012, 2013). Briefly, 2 h prior to starting the experiments, at noon, each fish was supplied with food equivalent to at least 2% of its

BW (140-200 mg diet), and this was entirely consumed within 1 h. For ICV administration of orexin A, each fish was anesthetized in water containing 2 mM MS-222. The animal was then placed in a stereotaxic apparatus and a small part of the parietal bone was carefully removed using a surgical blade (No. 19, Futaba, Tokyo, Japan). One microliter of test solution was injected into the third ventricle using a 10-μl Hamilton syringe and the gap was then filled with a surgical bonding agent (Aron Alpha, Sankyo, Tokyo, Japan). Injection into the correct site was verified by the appearance in the ventricle of concomitantly injected Evans blue dye. The fish in the experimental groups were injected with orexin A (2 and 4 pmol/g BW). Previous studies had indicated that ICV injection of orexin A at 2–4 pmol/g BW was sufficient to enhance food intake (Kojima et al., 2009; Miura et al., 2007; Nakamachi et al., 2006). The fish in the control group were ICV-injected with the same volume of saline. Each fish was exposed to air during ICV injection of orexin A or saline, and then placed in a small white experimental tank (diameter 24 cm, height 15 cm) filled with 4.0 l of tap water. Recording of locomotor activity was started at 15 min after ICV injection to allow recovery from anesthesia and continued for 30 min. Measurement of locomotor activity was performed with a video-tracking system for automatic recording of goldfish behavior (EthoVision Pro, Noldus Information Technology, Wageningen, Netherlands). The ICV-injected dose of SB334867 (10 pmol/g BW) had been determined in previous studies using goldfish (Kojima et al., 2009; Miura et al., 2007). Two or three minutes after ICV injection of 1 μl of SB334867, 1 μl of orexin A (4 pmol/g BW) was injected ICV. Control fish in each experiment were injected with the same volume of dimethyl sulfoxide diluted with saline, and with saline alone. Each fish was exposed to air during the first and second ICV injections, and then placed individually in the tank. Recording of locomotor activity was started 15 min after ICV injection and continued for 30 min as described above. Preference test with intact fish using a tank with black and white background areas or with upper and lower areas Before the experiment, fish were kept in a tank with a white background for 2 h. Then, another tank with black and white background areas or with upper and lower areas was used to investigate the preference of intact fish. These rectangular experimental tanks (for the black and white preference test, length 50 cm, width 10 cm, depth 5 cm; for the upper and lower preference test, length 15 cm, width 5 cm, depth 35 cm) were filled with 2.0–2.5 l of tap water. Each fish was placed in the black background area or in the bottom of the tank, and the time spent (min) in each background area or in the lower or upper area was recorded for 30 min using a video-tracking system as described above. Effect of ICV administration of orexin A, FG-7142 and diazepam on the time spent in the white background area, and on the time taken to move from the lower to the upper area Each fish received the administration of orexin A, FG-7142 and diazepam as described above. The fish in the experimental groups were injected with orexin A (2 and 4 pmol/g BW), FG-7142 (2 and 4 pmol/g BW) or diazepam (4 and 40 pmol/g BW). The ICV-injected doses of FG-7142 and diazepam had been determined in previous studies using goldfish (Faganello and Mattioli, 2007; Matsuda et al., 2011b; 2013). The fish in the control group were injected with vehicle or saline, as in the experimental group. Each fish was exposed to air during ICV injection. Each fish that had received ICV injection of test solutions, saline or vehicle was placed individually in the black compartment of the tank with areas of black and white background, or in the lower part of the tank. Recording of the time taken to move from the black to the white background area or the time taken to move from the lower to the upper area, was started at 15 min after ICV injection and continued for 30 min, using a video-tracking system.

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saline (29)

Effect of ICV preinjection of SB334867 on the anxiogenic-like action of orexin A

Data analysis

orexin A 2 pmol/g BW (24) orexin A 4 pmol/g BW (18)

(A) 120

Locomotor activity during 30 min (m)

Two or three minutes after ICV injection of 1 μl of SB334867 (10 pmol/g BW), 1 μl of orexin A (4 pmol/g BW) was injected ICV. Control fish in each experiment were injected with the same volume of dimethyl sulfoxide diluted with saline, and with saline alone. Each fish was exposed to air during the first and second ICV injections, and then placed individually in the black background area or in the bottom of the tank. Recording of the time spent in the white background area or the time taken to move from the lower to the upper area, was started at 15 min after ICV injection and continued for 30 min as described above.

319

**

110

**

100 90 80 70 60 50

All the results are expressed as mean ± SEM. Statistical analysis was performed using one- or two-way ANOVA with Bonferroni's method or unpaired Student's t test. Statistical significance was determined at the 5% level.

vehicle + saline (16) vehicle + orexin A 4 pmol/g BW (17) SB334867 10 pmol/g BW + saline (16)

Effect of ICV administration of orexin A on locomotor activity, and effect of SB334867 on hypermotility induced by orexin A Fifteen minutes after ICV injection, the locomotor activity of goldfish treated with orexin A or saline was recorded in the tank during a 30-min observation period. Intracerebroventricular injection of orexin A (2 and 4 pmol/g BW) significantly increased locomotor activity after the start of recording, and continued for 30 min [F(2,68) = 6.80, P = 0.002, η2 = 0.167, Fig. 1A]. Intracerebroventricular injection of SB334867 alone (10 pmol/g BW) had no effect on locomotor activity. In contrast, ICV preinjection of SB334867 completely inhibited the orexin A-enhanced locomotor activity (Fig. 1B). The antagonistic effect of SB334867 on the hypermotility of orexin A was shown to be significant by two-way ANOVA with Bonferroni's method [F(3,69) = 4.37, P = 0.04, η2 = 0.157].

Preference test with intact fish in a tank with black and white background areas, or with upper and lower areas Intact fish that had been transferred to the tank with black and white background areas preferred the black to the white background during the 30 min of observation: the average time spent in the black background area was approximately double that spent in the white background area (Cohen's d = 0.202, P b 0.01, Fig. 2A). Intact fish that had been transferred to the tank also preferred the lower to the upper area during the 30-min observation period: the average time spent in the lower area was approximately 1.5 times that spent in the upper area (Cohen's d = 0.293, P b 0.05, Fig. 2B).

Effect of ICV administration of FG-7142 and diazepam on time spent in the white background area Intracerebroventricular administration of the CBR inverse agonist FG-7142 shortened the time spent in the white background area of the tank, and a significant effect was observed at doses of 2 and 4 pmol/g BW [F(2,52) = 8.90, P = 0.0005, η2 = 0.255, Fig. 2C]. On the other hand, ICV administration of diazepam at 4 and 40 pmol/g BW increased the time spent in the white background area [F(2,57) = 7.48, P = 0.001, η2 = 0.208, Fig. 2E].

SB334867 10 pmol/g BW + orexin A 4 pmol/g BW (22)

(B) 100

Locomotor activity during 30 min (m)

Results

b

90 80

a

a

a

70 60 50 40 30 20

Fig. 1. Effect of ICV injection of orexin A on locomotor activity (swimming distance) in goldfish kept in a tank (A). Effect of ICV preinjection of SB334867 on orexin A-induced action (B). The results are expressed as the mean ± SEM. Numbers in parentheses indicate the number of fish in each group. Statistical significance of differences was evaluated by one-way ANOVA (*P b 0.05, **P b 0.01) (A) or two-way ANOVA (B) with Bonferroni's method. Different superscripts indicate significant differences (P b 0.05).

Effect of ICV administration of FG-7142 and diazepam on time taken to move from the lower to the upper area Intracerebroventricular administration of FG-7142 increased the time taken to move from the lower area to the upper area of the tank, and a significant effect was observed at doses of 2 and 4 pmol/g BW [F(2,52) = 4.70, P = 0.013, η2 = 0.166, Fig. 2D]. On the other hand, ICV administration of diazepam at 4 and 40 pmol/g BW decreased the time taken to move from the lower to the upper area [F(2,55) = 4.84, P = 0.012, η2 = 0.150, Fig. 2F].

Effect of ICV administration of orexin A on time spent in the white background area, and effect of SB334867 on the action of orexin A Intracerebroventricular injection of orexin A (2 and 4 pmol/g BW) significantly shortened the time spent in the white background area [F(2,54) = 3.54, P = 0.036, η2 = 0.116, Fig. 3A]. Intracerebroventricular injection of SB334867 alone had no effect on the time spent in the

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white background area

upper area

black background area

lower area

(B)

30

Time spent in each area during 30 min (min)

Time spent in each area during 30 min (min)

(A) ** 25

(22)

20 15 10 5 0

30

* 25 20 15 10 5 0

vehicle (19)

vehicle (19)

FG-7142 2 pmol/g BW (18)

FG-7142 2 pmol/g BW (18)

FG-7142 4 pmol/g BW (18)

FG-7142 4 pmol/g BW (18)

(D)

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20

12 10 8

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6

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4 2

Time taken to move from the lower to the upper area (min)

Time spent in the white background area (min)

(C)

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16 14 12 10 8 6 4 2

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vehicle (20)

vehicle (18)

diazepam 4 pmol/g BW (20)

diazepam 4 pmol/g BW (17) diazepam 40 pmol/g BW (23)

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10 8 6 4 2 0

(F) 14 Time taken to move from the lower to the upper area (min)

(E) 12

*

18

diazepam 40 pmol/g BW (20) Time spent in the white background area (min)

(18)

12 10 8

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6

** 4 2 0

Fig. 2. Black and white background area preference (A) and upper and lower area preference (D) of intact goldfish. The results are expressed as the mean ± SEM. Numbers in parentheses indicate the number of fish in each preference test. Statistical significance of differences between times spent in the white and black background areas or in the upper and lower areas was evaluated by Student's t test (*P b 0.05, **P b 0.01). Effect of ICV injection of FG-7142 (C, D) and diazepam (E, F) on the time spent by a fish in the white background area or the time taken to move from the lower to the upper area of the tank. Statistical significance of differences was evaluated by one-way ANOVA (*P b 0.05, **P b 0.01) with Bonferroni's method.

white background area. In contrast, ICV preinjection of SB334867 completely inhibited the effect of orexin A (Fig. 3B). The antagonistic effect of SB334867 on the action of orexin A was shown to be significant by two-way ANOVA with Bonferroni's method [F(3,73) = 4.78, P = 0.032, η2 = 0.202].

Effect of ICV administration of orexin A on the time taken to move from the lower to the upper area, and effect of SB334867 on the action of orexin A Intracerebroventricular injection of orexin A (4 pmol/g BW) significantly increased the time taken for the fish to move from the lower area

T. Nakamachi et al. / Hormones and Behavior 66 (2014) 317–323

saline (18)

saline (18)

orexin A 2 pmol/g BW (19)

orexin A 2 pmol/g BW (18)

orexin A 4 pmol/g BW (20)

orexin A 4 pmol/g BW (18)

(A)

14

14

12

12

Time taken to move from the lower to the upper area (min)

Time spent in the white background area (min)

(A)

10 * 8

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6 4 2

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10 8 6 4 2 0

0 vehicle + saline (19)

vehicle + saline (18)

vehicle + orexin A 4 pmol/g BW (22)

vehicle + orexin A 4 pmol/g BW (18)

SB334867 10 pmol/g BW + saline (19)

SB334867 10 pmol/g BW + saline (21) SB334867 10 pmol/g BW + orexin A 4 pmol/g BW (18)

SB334867 10 pmol/g BW + orexin A 4 pmol/g BW (17)

(B)

(B) 12

a a

a

10 8 6

b

14

14

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4 2 0

Fig. 3. Effect of ICV injection of orexin A (A) on the time spent by a fish in the white background area of the tank. The results are expressed as the mean ± SEM. Numbers in parentheses indicate the number of fish in each group. Statistical significance of differences was evaluated by one-way ANOVA (*P b 0.05, **P b 0.01) with Bonferroni's method. Effect of ICV preinjection of SB334867 on the action of orexin A in terms of the time spent in the white background area (B). Statistical significance of differences was evaluated by two-way ANOVA with Bonferroni's method. Different superscripts indicate significant differences (P b 0.05).

to the upper area [F(2,51) = 4.76, P = 0.013, η2 = 0.157, Fig. 4A]. Intracerebroventricular injection of SB334867 alone had no effect on the time taken for fish to move from the lower to the upper area of the tank. On the other hand, ICV preinjection of SB334867 completely inhibited the effect of orexin A (Fig. 4B). The antagonistic effect of SB334867 on the action of orexin A was shown to be significant by two-way ANOVA with the Bonferroni's method [F(3,71) = 6.87, P = 0.011, η2 = 0.150]. Discussion In goldfish, the orexigenic response to orexin A is modulated not only by NPY and ghrelin, but also by cocaine- and amphetamineregulated transcript peptides and leptin (Abbott and Volkoff, 2011; Kojima et al., 2009; Miura et al., 2007; Volkoff et al., 1999). Intracerebroventricular injection of orexin A also affects locomotor activity in this species (Matsuda, 2009; Nakamachi et al., 2006). In zebrafish, ICV injection of orexin A induces hypermotility in addition to exerting an

Time taken to move from the lower to the upper area (min)

Time spent in the white background area (min)

321

12 10 a 8 a

a

6 4 2 0

Fig. 4. Effect of ICV injection of orexin A (A) on the time taken for a fish to move from the lower to the upper area of the tank. The results are expressed as the mean ± SEM. Numbers in parentheses indicate the number of fish in each group. Statistical significance of differences was evaluated by one-way ANOVA (*P b 0.05, **P b 0.01) with Bonferroni's method. Effect of ICV preinjection of SB334867 on the action of orexin A in terms of the time taken to move from the lower to the upper area (B). Statistical significance of differences was evaluated by two-way ANOVA with Bonferroni's method. Different superscripts indicate significant differences (P b 0.05).

orexigenic effect (Yokobori et al., 2011; Yokogawa et al., 2007), and overexpression of orexin promotes wakefulness and inhibits rest (Prober et al., 2006). Taken together, these observations indicate that, in Cypriniformes (including the goldfish and zebrafish), orexin A not only regulates energy homeostasis by stimulating food consumption, but is also involved in the regulation of psychophysiology by affecting locomotor activity, suggesting its involvement in multiple brain functions in teleosts (Appelbaum et al., 2009; Rihel et al., 2010). However, there is no information about the involvement of orexin in emotional behavior in teleost fish, and the behavioral function of the orexin system in fish has not well been studied. Therefore, this study was conducted to clarify the involvement of orexin A and its receptor system in psychophysiological control in the goldfish. Although the synthetic orexin A that we used was of heterologous origin, our previous studies had demonstrated that ICV administration of orexin A enhanced food intake in goldfish and zebrafish, as described above. The primary structure of orexin precursors has been conserved during evolution among vertebrates (Wong et al., 2011). However, the amino acid sequence of zebrafish orexin A shows 32% identity with that of human orexin A

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(Wong et al., 2011). Our data support some previous studies demonstrating that synthetic human orexin A is able to affect physiological activity in goldfish and zebrafish (Nakamachi et al., 2006; Volkoff and Peter, 2000, 2001; Volkoff et al., 1999, 2003; Yokobori et al., 2011; Yokogawa et al., 2007). In mammals, there is clear evidence that various neuropeptides implicated in the control of appetite and satiety also exert psychophysiological effects (do Rego et al., 2006; Roy et al., 2006; Refojo and Holsboer, 2009; Vaudry et al., 2009; Rotzinger et al., 2010). More recent findings suggest that orexin A is involved in emotional behavior, particularly anxiety, in rodents (Johnson et al., 2012; Lungwitz et al., 2012; Rodgers et al., 2013; Staples and Cornish, 2014). Recent reports have sufficiently confirmed that the swimming pattern of a fish in a tank can be used for psychophysiological evaluation, including that of anxiety-like behavior (Backström et al., 2011; Blaser and Rosemberg, 2012; Cachat et al., 2010; Faganello and Mattioli, 2007; Grossman et al., 2010; Khor et al., 2012; Maaswinkel et al., 2012; Matsuda et al., 2011b, 2012, 2013; Maximino et al., 2010a, 2010b). Our previous studies have revealed that neuropeptides such as endozepines, including octadecaneuropeptide (ODN), which is derived from the diazepambinding inhibitor, and corticotropin-releasing hormone (CRH) are involved in the control of not only satiety but also locomotor activity (Matsuda et al., 2011b; 2013). Using the black/white preference test or lower/upper preference test in fish tanks, it has been shown that ICV administration of ODN and CRH induces anxiety-like behavior (Matsuda et al., 2011b; 2013). The available data suggest that neuropeptides related to feeding regulation act as psychophysiological factors (Matsuda, 2013; Matsuda et al., 2011a). We have therefore analyzed swimming patterns to evaluate the effect of orexin A on psychomotor activity in a goldfish model using two preference tests. In these tests, the CBR inverse agonist FG-7142 shortened the time spent in the white background area, and prolonged the time taken to move from the lower area to the upper area of the tank, whereas the CBR agonist diazepam appeared to exert effects opposite to those of FG-7142, indicating that FG-7142 exerts an anxiogenic-like action, whereas diazepam exerts an anxiolytic-like action. Surprisingly, ICV administration of orexin A reduced the time spent in the white background area, and increased the time taken to move from the lower to the upper area of the tank. Since the behavioral action of ICV-injected orexin A mimicked that of ICV-injected FG-7142, but not that of diazepam, our data suggest that systemic orexin A induces anxiety-like behavior in goldfish. In support of this notion, the fact that orexin A increased the locomotor activity of goldfish placed in a tank can be interpreted in terms of escape or exploratory behavior induced by the anxiogenic-like action of orexin A. This study has thus confirmed that orexin A also affects locomotor and psychomotor activities in a goldfish model, providing an example of a neuropeptide that regulates both food intake and psychophysiological activity in fish. In the goldfish, orexigenic action of orexin A is mediated via the orexin receptor, subsequently leading to activation of the NPY Y1 receptor (Matsuda, 2009; Matsuda et al., 2011a; Nakamachi et al., 2006; Volkoff et al., 1999). On the other hand, ICV administration of NPY also induces anxiolytic-, but not anxiogenic-, action perhaps via the NPY Y4 receptor (Matsuda et al., 2011a). Since orexin A and NPY seem to exert opposite psychophysiological actions, there is little possibility of mutual interaction with orexin A and NPY in this case. We then used an antagonist to characterize the orexin receptor responsible for mediating the behavioral changes induced by orexin A. Preinjection of SB334867 suppressed the action of orexin A, indicating that in goldfish, orexin A acts pharmacologically as an agonist of the OX1R to exert its anxiogenic-like effect. ICV injection of SB334867 alone had no effect on locomotor and psychomotor activities. Prior to starting the experiments, each fish was supplied with food. This suggests that the action of endogenous orexin A seems to decrease in the experimental situation. The orexigenic action of orexin A in goldfish is also blocked by treatment with SB334867, suggesting that this action is mediated through an orexin receptor (Kojima et al., 2009; Miura et al.,

2007). To date, one receptor for orexin has been identified in non-mammalian vertebrates including zebrafish, and this receptor corresponds structurally to the mammalian OX2R (Wong et al., 2011). However, recent studies have failed to identify the gene encoding the OX1R in chicken, Xenopus, zebrafish and goldfish (Panula, 2010; Wong et al., 2011). There has been no molecular information about the fish OX1R. From an evolutionary viewpoint, the OX2R is likely to be a more ancient receptor form in vertebrates. In this study, surprisingly, a mammalian OX1R receptor antagonist, SB334867, blocked the action of orexin A. Further investigations to clarify the relationship between mammalian and non-mammalian orexin receptors, especially the fish receptor, would be warranted. If it was possible to examine whether the fish orexin receptor (OX2R) mediates the anxiogenic-like and orexigenic actions of orexin A, and to clarify the receptor mediating the action of orexin A in non-mammalian vertebrates, then the zebrafish, as well as the goldfish, would become excellent animal models for examining the physiological significance of the orexin system in fish from the viewpoint of comparative neuroendocrinology. Further study, particularly with the use of goldfish orexin A and its receptor, will be required to clarify the molecular mechanisms underlying the action of orexin A in goldfish. In conclusion, this study has shown that orexin A has the potential to affect both locomotor and psychomotor activities via the orexin receptor-signaling pathway in goldfish. These data provide the first evidence for a role of orexin A in the control of emotional behavior in addition to the regulation of food intake in fish. Acknowledgments This work was supported by a Grant-in-Aid from the Japan Society for the Promotion of Science (25650107 to K.M.), and by a research grant from the University of Toyama (K.M.). References Abbott, M., Volkoff, H., 2011. Thyrotropin Releasing Hormone (TRH) in goldfish (Carassius auratus): role in the regulation of feeding and locomotor behaviors and interactions with the orexin system and cocaine- and amphetamine regulated transcript (CART). Horm. Behav. 59, 236–245. Alvarez, C.E., Sutcliffe, J.G., 2002. Hypocretin is an early member of the incretin gene family. Neurosci. Lett. 324, 169–172. Appelbaum, L., Wang, G.X., Maro, G.S., Mori, R., Tovin, A., Marin, W., Yokogawa, T., Kawakami, K., Smith, S.J., Gothilf, Y., Mignot, E., Mourrain, P., 2009. Sleep-wake regulation and hypocretin-melatonin interaction in zebrafish. Proc. Natl. Acad. Sci. U. S. A. 106, 21942–21947. Avolio, E., Alò, R., Carelli, A., Canonaco, M., 2011. Amygdalar orexinergic-GABAergic interactions regulate anxiety behaviors of the Syrian golden hamster. Behav. Brain Res. 218, 288–295. Backström, T., Pettersson, A., Johansson, V., Winberg, S., 2011. CRF and urotensin I effects on aggression and anxiety-like behavior in rainbow trout. J. Exp. Biol. 214, 907–914. Blaser, R.E., Rosemberg, D.B., 2012. Measures of anxiety in zebrafish (Danio rerio): dissociation of black/white preference and novel tank test. PLoS One 7, e36931. Cachat, J., Stewart, A., Grossman, L., Gaikwad, S., Kadri, F., Chung, K.M., Wu, N., Wong, K., Roy, S., Suciu, C., Goodspeed, J., Elegante, M., Bartels, B., Elkhayat, S., Tien, D., Tan, J., Denmark, A., Gilder, T., Kyzar, E., Dileo, J., Frank, K., Chang, K., Utterback, E., Hart, P., Kalueff, A.V., 2010. Measuring behavioral and endocrine responses to novelty stress in adult zebrafish. Nat. Protoc. 5, 1786–1799. de Lecea, L., Kilduff, T.S., Peyron, C., Gao, X., Foye, P.E., Danielson, P.E., Fukuhara, C., Battenberg, E.L., Gautvik, V.T., Barlett, F.S., Frankel, W.N., van den Pol, A.N., Bloom, F. E., Gautvik, K.M., Sutcliffe, J.G., 1998. The hypocretins: hypothalamus-specific peptides with neuroexcitatory activity. Proc. Natl. Acad. Sci. U. S. A. 95, 322–327. do Rego, J.C., Leprince, J., Chartrel, N., Vaudry, H., Costentin, J., 2006. Behavioral effects of 26RFamide and related peptides. 27, 2715–2721. Faganello, F.R., Mattioli, R., 2007. Anxiolytic-like effect of chlorpheniramine in inhibitory avoidance in goldfish submitted to telencephalic ablation. Prog. Neuropsychopharmacol. Biol. Psychiatry 31, 269–274. Grossman, L., Utterback, E., Stewart, A., Gaikwad, S., Chung, K.M., Suciu, C., Wong, K., Elegante, M., Elkhayat, S., Tan, J., Gilder, T., Wu, N., Dileo, J., Cachat, J., Kalueff, A.V., 2010. Characterization of behavioral and endocrine effects of LSD on zebrafish. Behav. Brain Res. 214, 277–284. Huesa, G., van den Pol, A.N., Finger, T.E., 2005. Differential distribution of hypocretin (orexin) and melanin-concentrating hormone in the goldfish brain. J. Comp. Neurol. 488, 476–491. Ito, N., Yabe, T., Gamo, Y., Nagai, T., Oikawa, T., Yamada, H., Hanawa, T., 2008. I.c.v. administration of orexin-A induces an antidepressive-like effect through hippocampal cell proliferation. Neuroscience 157, 720–732.

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