IntroductionHypothalamic neuropeptides orexins, orexin A and orexin B, which are also known as hypocretin 1 and hypocretin 2, respectively (; ), have been shown to be important factors for maintaining sleep/wakefulness and regulating feeding behavior, emotion, the reward system, and energy homeostasis (;;;,;; ). Orexin-producing neurons (orexin neurons) send projections widely throughout the central nervous system (CNS), including the cerebral cortex, limbic system including the amygdala, bed nucleus of the stria terminalis (BST) and hippocampus, hypothalamus such as the arcuate nucleus (ARC) and tuberomammillary nucleus (TMN), and brain stem areas including the central gray, locus coeruleus (LC), and dorsal raphe (DR) (;; ).There are two orexin receptor subtypes, orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R). OX1R exhibits higher affinity to orexin A over orexin B, while OX2R shows similar affinities to both isopeptides. Expression of OX1R is observed in various areas of the brain including the prefrontal and infralimbic cortex, hippocampus, amygdala, BST, paraventricular thalamic nucleus (PVT), anterior hypothalamus, DR, ventral tegmental area (VTA), LC, and laterodorsal tegmental nucleus (LDT)/pedunculopontine nucleus (PPT) (;;;; ).

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Expression of OX2R is observed in the amygdala, BST, PVT, DR, VTA, and LDT/PPT (;; ). Many studies using OX2R −/− mice have suggested important physiological roles of OX2R in the maintenance of wakefulness states (; ) and the regulation of metabolic states and feeding behavior.Recently, studies using selective OX1R antagonists have suggested that OX1R plays important roles in the regulation of feeding behavior, the reward system, emotion, and the autonomic nervous system. On the other hand, only limited information is available thus far regarding the phenotype of OX1R-deficient mice, although a recent study suggested a role of this subtype in formation of emotional memory and emergence of fear-related behaviors. In order to obtain further information about the physiological roles of OX1R, we performed a series of behavioral tests on mice lacking the orexin-1 receptor ( Ox1r −/− mice). We found that Ox1r −/− mice showed altered depression-like behavior, increased anxiety-like behavior, impairment of sensorimotor gating, abnormal social behavior, and decreased locomotor activity compared with the wild-type control mice. Collectively, this study suggests that OX1R might be involved in regulation of mood and anxiety.

Materials and Methods AnimalsAll experimental procedures used in this study were approved by the Animal Experiment and Use Committee of Kanazawa University (AP-111947), and were in accordance with National Institute of Health (NIH) guidelines. Ox1r −/− mice were obtained by mating of heterozygous Ox1r +/− mice. Genotyping of these mice was performed by PCR using DNA samples prepared from the tails. We used the following primers for genotyping; common, 5′-CTCTTTCTCCACAGAGCCCAGGACTC-3′, wild-type, 5′-GCAAGAATGGGTATGAAG GGAAGGGC-3′, and knockout, 5′-TGAGCGAGTAACAACCCGTCGGATTC-3′. The mice were backcrossed to wild-type C57Bl/6J mice at least 10 times. The wild-type littermates of the mutants were used as controls. To minimize a “litter effect,” two to five pups from four litters were used in the experiments.

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Ox1R −/− and wild-type mice were group-housed four per cage (one to two Ox1R −/−s and two to three wild-types in a cage). To generate the Ox1R −/− and wild-type mice, we mated four dams with one male per cage at the time of mating. Pups from the four dams were group-housed in a cage until weaning. Only two to five pups per four dams (from a mating cage) were transferred to a cage at the time of weaning and used in this study. The mice were transferred to Fujita Health University from Kanazawa University at the age of 8–10 weeks. Mice were maintained under a strict 12 h light/dark cycle (lights on at 7:00 at Fujita Health University) in a temperature- and humidity-controlled room.

Food and water were available ad libitum. Two weeks after arrival, mice were subjected to a battery of behavioral tests. All behavioral testing procedures were approved by the Institutional Animal Care and Use Committee of Fujita Health University. All efforts were made to minimize the animals' suffering and discomfort and to reduce the number of animals used. Behavioral ExperimentsAll behavioral experiments were performed during the light phase (9:00–16:00). Only male mice were used.

Mice were group-housed, four mice per cage. Behavioral experiments of this study were performed as previously described. General health screening including measurement of body weight and body temperature were also conducted (, ).

Neuromuscular Strength TestNeuromuscular strength of the mice was examined in the grip strength and wire hang tests. In order to assess forelimb grip strength, we used a grip strength meter (O'Hara & Co., Tokyo, Japan). In this test, we lifted the mice and held them by their tail. As a result mice could grasp a wire grid with their forepaws.

Next, we gently pulled the mice backward with their tail after maintaining a posture parallel to the table surface until they released the grid. Each mouse was tested three times and the highest value of grip strength (Newton) was used for analysis. In the wire hang test, after placing mice on a wire mesh, it was gently inverted and waved about. Mice gripped the wire to stop themselves falling off.

Latency to fall off the wire mesh was recorded. Rotarod TestThe test was performed to examine motor coordination and balance. This was performed using an accelerating rotarod (UGO Basile Accelerating Rotarod) where a mouse was placed on the rotating drum (3 cm diameter).

The time each animal was able to maintain its balance on the rotating drum was measured. The starting speed of the rotarod was 4 rpm, maximum was 40 rpm, and the test duration was 5 min.

Hot Plate TestWe performed a hot plate test to evaluate sensitivity to a painful stimulus or nociception. Mice were placed on a hot plate (Columbus Instruments, Columbus, OH) with a temperature of 55.0 ± 0.3°C, and latency to the first paw response (sec) was recorded manually (cut-off time: 15 s). The paw response was either a foot shake or a paw lick. Light/dark Transition TestWe used an apparatus consisting of a cage (21 × 42 × 25 cm) which was divided into two equal compartments by a black partition containing a small door (O'Hara & Co., Tokyo). One compartment was brightly illuminated (390 lux) and the other was dark (2 lux). Mice were placed into the dark side and allowed to move freely between the two chambers through the small door for 10 min. The distance traveled, total number of transitions, time spent in the light chamber, and latency to enter the light chamber were recorded automatically using ImageLD software.

Elevated Plus Maze TestWe used an apparatus consisting of two open arms and two closed arms. The open arms (25 × 5 cm) were surrounded by 3-mm high Plexiglas ledges to minimize the likelihood of mice falling down from the apparatus. The closed arms were the same size, with 15 cm high transparent walls (O'Hara & Co, Tokyo). This apparatus was made of white plastic. It was elevated 55 cm above the floor.

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Each mouse was placed in the central area of the maze (5 × 5 cm), facing one of the closed arms. Mouse behavior was recorded during a 10-min test period.

The number of entries into arms, time spent in open arms, and distance traveled (cm) were recorded. Data were analyzed automatically using ImageEP software. Social Interaction Test in a Novel EnvironmentThe social interaction test in a novel environment was performed in two mice of the same genotype, which were previously kept in different cages.

They were placed into a box (40 × 40 × 30 cm) together and allowed to move freely for 10 min. Mouse behavior was analyzed automatically using ImageSI software. The automatic scoring by the ImageSI was validated by comparing it with manual scoring by actual observation of a well-trained experimenter (weight-matched male C57BL/6J pairs, n = 8: for the total duration of contacts, r = 0.978, p.

Normal general health, neurological and motor function and nociception in Ox1r −/− mice. No significant differences were found between genotypes in the body weight (A), body temperature (B) and grip strength (C). A significant reduction in the wire hang latency was observed in Ox1r −/− mice (D). No significant difference in the latency to fall off in the rotarod test was observed in Ox1r −/− and wild-type mice (E).

Hot plate latency was lower in Ox1r −/− mice (F). Asterisk Indicates a significant difference from wild-type mice ( p.

We evaluated anxiety-like behavior in Ox1r −/− mice. In the light/dark transition test, no significant differences were observed in the distance traveled in the light chamber, number of transitions, stay time in the light chamber and latency to enter the light chamber Figures; t (31) = 0.225, p = 0.8237; t (31) = 1.242, p = 0.2234; t (31) = 0.69, p = 0.495; t (31) = 0.235, p = 0.816, respectively, although distance traveled in the dark chamber was significantly lower in the Ox1r −/− mice as compared with wild-type mice Figure; t (31) = 2.228, p = 0.0333.

In the elevated plus maze test, we observed significant reductions in the distance traveled, number of arm entries, percentage of open arm entries and percentage of time spent in open arms in Ox1r −/− mice as compared with controls Figure; t (28) = 4.029, p = 0.0004; t (28) = 3.541, p. Decreased locomotor activity and increased anxiety-like behavior in Ox1r −/− mice.

(A) In the light/dark transition test, no significant differences between Ox1r −/− and wild-type mice were found in number of transitions (A 2 ), time spent in the light chamber (A 3 ) and latency to enter the light chamber (A 4 ). The distance traveled in the dark chamber was significantly shorter in Ox1r −/− mice (A 1 ). (B) In the elevated plus maze test, all the behavioral measures, including distance traveled (B 1 ), number of arm entries (B 2 ), percentage of entries into open arms (B 3 ), and percentage of time spent in open arms (B 4 ) were significantly decreased in Ox1r −/− mice compared with wild-type mice. (C) In the open field test, there were no significant differences between Ox1r −/− and wild-type mice in the total distance traveled (C 1 ), vertical activity (C 2 ), time spent in the center (C 3 ) and stereotypic counts (C 4 ). Asterisk Indicates a significant difference from wild-type mice ( p. In the open field test, although there were no significant differences in distance traveled, center time, and stereotypic counts (Figures ) between mutant and control mice, tendency of reduction in the number of vertical activities was observed in mutant mice (Figure ). Decreased locomotor activity was consistently observed in other paradigms including the light/dark transition test (Figure ), elevated plus maze test (Figure ) (number of entries; p = 0.0014, entries into open arms; p = 0.0005, distance traveled; p = 0.0004, time in open arms; p = 0.0001).

Increased Acoustic Startle Response and Decreased Sensorimotor Gating Function in Ox1r −/− MiceOx1r −/− mice displayed significantly greater startle response to the 110 dB and 120 dB stimuli Figure; Genotype effect, F (1, 31) = 23.952, p. Decreased Social Interaction in Ox1r −/− MiceIn the social interaction test, we observed decreased distance traveled in Ox1r −/− mice as compared with wild-type controls although the difference between the genotypes did not reach a significance level Figure; t (9) = 1.874, p = 0.0937. The statistical analysis of the total duration of contact, number of contacts, total duration of active contact, and mean duration per contact did not show any significant differences between genotypes Figure; t (9) = 0.875, p = 0.4042; t (9) = 1.423, p = 0.1884; t (9) = 0.923, p = 0.3801; t (9) = 0.349, p = 0.7351, respectively. Abnormal social behavior in Ox1r −/− mice. Social interaction test in a novel environment (A). Total duration of contact (A 1 ), number of contacts (A 2 ), total duration of active contact (A 3 ), and mean duration of contact (A 4 ) were not significantly different between each genotype.

Reduction of distance traveled was seen in mutant mice (A 5 ). In the sociability test (B), control mice, but not mutant mice, spent significantly longer time in the chamber with a novel conspecific (stranger 1) than in the empty side (B 1 ) and significantly longer time around the cage containing stranger 1 than that around the empty cage (B 2 ). In social novelty test, wild-type control mice tended to spend longer time around the cage containing stranger 2 than that containing stranger 1, but Ox1r −/− mice did not show this tendency (B 3).

Ox1r −/− mice showed significantly shorter time spent around the cage with stranger 2 when compared with control mice (B 4). In a 24-h home cage social interaction test (C), mean number of objects (C 1) and activity level (C 2) were lower in Ox1r −/− mice than control mice. In the sociability and social novelty preference test, we found several differences between Ox1r −/− mice and wild-type control mice. In the sociability test, wild-type mice showed significantly longer time spent in a chamber containing a novel conspecific (stranger 1) in a wire cage as compared with that spent in a chamber with an empty cage Figure; t (18) = 2.882, p = 0.0099 and also significantly longer time spent around the wire cage containing stranger 1 than that spent around the empty cage Figure; t (18) = 3.001, p = 0.0077. However, Ox1r −/− mice showed similar contact times for both cages for time spent in each chamber, t (13) = 0.417, p = 0.6833; for time spent around each cage, t (13) = 0.656, p = 0.5234. Moreover, Ox1r −/− mice exhibited significantly decreased social contact than wild-type mice for time spent in chamber with stranger 1, t (31) = 2.152, p = 0.0393; for time spent around cage with stranger 1, t (31) = 2.47, p = 0.0192. After this session, we introduced another novel conspecific (stranger 2) to the mice (social novelty test).

In this paradigm, wild-type control mice tended to spend longer time around the cage containing stranger 2 than that containing stranger 1 Figure; t (18) = 1.765, p = 0.0946, although there was no difference between time spent in the chamber containing stranger 1 and time spent in the chamber containing stranger 2 in the control mice t (18) = 0.515, p = 0.6127. In contrast, the time spent around each cage and time spent in each chamber were statistically indistinguishable between stranger 1 side and stranger 2 side Figure; t (13) = 0.633, p = 0.5374; t (13) = 0.06, p = 0.9528, respectively. Ox1r −/− mice showed shorter time spent around the cage with stranger 2 when compared with control mice for time spent in chamber with stranger 2, t (31) = 1.174, p = 0.2493; for time spent around cage with stranger 2, t (31) = 2.857, p = 0.0076.In the 24-h home cage social interaction test, mean number of objects was significantly higher in Ox1r −/− mice Figure; Genotype effect, F (1, 9) = 12.312, p = 0.0066; Genotype × Time interaction, F (167, 1503) = 1.555, p.