Behavioural Brain Research 272 (2014) 100–110

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Heterozygous mice deficient in Atp1a3 exhibit motor deficits by chronic restraint stress Hiroki Sugimoto a , Keiko Ikeda a,b , Kiyoshi Kawakami a,∗ a b

Division of Biology, Center for Molecular Medicine, Jichi Medical University, Shimotsuke 329-0498, Tochigi, Japan Biology, Hyogo Medical College, Nishinomiya 663-8501, Hyogo, Japan

h i g h l i g h t s • • • •

Atp1a3+/− exhibited shorter stride length at 4 weeks of age. Shorter stride length was persistently seen in chronically-stressed Atp1a3+/− mice. Shorter hanging time was observed after chronic restraint stress in Atp1a3+/− . Atp1a3+/− is a useful animal model of RDP.

a r t i c l e

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Article history: Received 3 May 2014 Received in revised form 13 June 2014 Accepted 23 June 2014 Available online 29 June 2014 Keywords: Rapid-onset dystonia-parkinsonism (RDP) DYT12 Na,K-ATPase ␣3 subunit Gene-knockout mice Chronic restraint stress Footprint analysis

a b s t r a c t Dystonia is a neurological disorder with involuntary and simultaneous contractions of agonist and antagonist muscles. Rapid-onset dystonia parkinsonism (RDP), one of the heredity forms of dystonia, is caused by mutations of Na,K-ATPase ␣3 subunit gene (ATP1A3). The abrupt onset of bulbar and limb symptoms of RDP are often triggered by physical and/or emotional stress. We reported previously that Atp1a3-deficient heterozygous mice showed higher locomotor activity and developed enhanced dystonia symptoms after kainate injection into the cerebellum, but not spontaneous movement disorder like RDP patients. Here we show that Atp1a3-deficient heterozygous mice exhibited shorter stride length at 4 weeks of age without stress and at later stages under chronic restraint stress loading. Shorter hanging time in the hanging box test was also observed after stress loading. Shorter stride length and hanging time may be relevant to certain phenotypes, such as gait abnormality, observed in RDP patients. Atp1a3 was widely expressed in the brain, including basal ganglia and cerebellum, and spinal cord of young mice, and the expression pattern was compatible with movement abnormalities under lack of one of alleles. Our results demonstrated the usefulness of Atp1a3-deficient heterozygous mice as an animal model of RDP and its potential use to explore the pathophysiology of movement abnormality in this disorder. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Dystonia is a neurological movement disorder characterized by involuntary movement and simultaneous contractions of agonist and antagonist muscles [8,4]. DYT12, known as rapid-onset dystonia-parkinsonism (RDP), is one of the heredity dystonia forms and often triggered by physical and emotional stress. Abrupt onset of bulbar symptoms and limb dystonia is observed between ages of 4 and 55 years, and symptoms stay for life [6]. The causative gene of DYT12 is ATP1A3, which encodes the ␣3 subunit of the Na,K-ATPase [14]. In an autosomal dominant manner, missense

∗ Corresponding author. Tel.: +81 285 58 7311; fax: +81 285 44 5476. E-mail address: [email protected] (K. Kawakami). http://dx.doi.org/10.1016/j.bbr.2014.06.048 0166-4328/© 2014 Elsevier B.V. All rights reserved.

mutations, a deletion, or an insertion in ATP1A3 are linked to DYT12 [12,7,26,3]. Recently, ATP1A3 was also identified as the affected gene in alternating hemiplegia of childhood (AHC) [21,42,25]. AHC is a neurological disorder characterized by transient hemiplegia with other paroxysmal symptoms, and sometimes presents with symptoms common with RDP, i.e., dystonia [42]. Manifestation of symptoms begins before 18 months of age in AHC patients [42]. Both diseases show a phenotypical continuum caused by mutation of ATP1A3 [42], and substitutions of amino acids at different positions or substitution of different amino acids at the same positions in ATP1A3 is thought to affect the manifestations of RDP and AHC [25]. It is possible that AHC is the severe phenotype of RDP [42]. The Na,K-ATPase consists of ␣ (␣1–␣4) and ␤ subunits (␤1–␤3), and maintains the electrochemical gradient of Na+ and K+ across

H. Sugimoto et al. / Behavioural Brain Research 272 (2014) 100–110

the cell membrane using the energy of ATP hydrolysis [31,2]. The ␣3 subunit is predominantly expressed in neurons, including the basal ganglia and cerebellar cortex, which play important roles in motor function in both adult and juvenile mice [11,24]. So far, two lines of Atp1a3-deficient mice, Atp1a3tm1Ling/+ [36] and our Atp1a3+/− [24], and a line of mice with point mutation in Atp1a3 (Myk/+ ) [13] have been reported. Atp1a3tm1Ling/+ mice exhibit lower memory function [36] and motor deficits in rotarod and balanced beam tests after stress loading [15]. Atp1a3+/− mice show higher locomotor activity and enhanced dystonia symptoms following intracerebellar administration of kainate [24]. Myk/+ mice have low body weight, motor deficits including gait abnormality, and cognitive impairment under non-stress conditions. Such phenotypes are also observed in AHC patients [13,28]. However, none of the above mice are reported to show spontaneous dystonia movement. Because stress is known to trigger the expression of dystonia in RDP and the stress loading such as application of chronic stress to mice is a useful method for induction of neurobehavioral signs [33,20,49,27,15,32,51], we performed in the present study various behavioral tests under specific stress loading (restraint) and examined whether Atp1a3+/− of both sexes show phenotypes relevant to the symptoms of RDP, such as gait abnormality, slowness of movement, postural instability, and psychiatric depression-like feature [41]. We also completed comprehensive examination of Atp1a3 mRNA expression in young wild-type mice. 2. Materials and methods 2.1. Animals Atp1a3-deficient mice were established as described previously [24]. The heterozygous mice of Atp1a3 (Atp1a3+/− ) were backcrossed to C57BL6/J for 22–24 generations. Atp1a3+/− and wild-type littermates were used in this study. Mice were housed under a 12 h light/dark cycle (light from 7:00 to 19:00) in a temperaturecontrolled room (22 ± 2 ◦ C). Food and water were provided ad libitum. 2.2. Experimental protocol For each sex and genotype, 4-week mice were divided into two groups (stressed and non-stressed). Footprint analysis, hanging box test, and body-weight measurement were performed at 4, 6, 8, 10, and 12 weeks of age. Stress loading was performed from 4 to 12 weeks of age in the stressed group. After the end of stress loading, mice were tested using a battery of behavioral tests, consisting of open field test, elevated plus maze test, accelerating rotarod test, balanced beam test, grip strength test, and forced swimming test (Table 1). All animal experiments were carried out in a humane manner. The Institutional Animal Experiment Committee of Jichi Medical University approved the study. The study was conducted in accordance with the Institutional Regulation for Animal Experiment and Fundamental Guideline for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions under the jurisdiction of the MEXT of Japan. 2.3. Stress loading To apply the restraint stress, a mouse was placed in a holed 50 mL plastic tube (30 mm diameter × 115 mm length) for 2 h at room temperature. Restraint stress was loaded 6 times in 2 weeks from 4 to 12 weeks of age.

101

Table 1 Experimental schedule. Age (weeks)

Behavioral tests

Stress loading

4

Footprint analysis & hanging box test Footprint analysis & hanging box test Footprint analysis & hanging box test Footprint analysis & hanging box test Footprint analysis & hanging box test Open field test Elevated plus maze test Accelerating rotarod test Balanced beam test Grip strength test Forced swimming test

None

6 8 10 12 13 14 15 16

After hanging box and footprint test in 4-week-old, restraint stress was applied to the stressed group until 12 weeks of age

No stress loading after 12 weeks of age

2.4. Footprint analysis Mice were trained to walk along 40 cm × 10 cm runaway with 15 cm wall on white paper into the escape box. Then, the forelimbs and hindlimbs were painted with red and black ink, respectively. Mice were first given a test trial. If mice did not walk, the test trial was repeated. The footprint patterns were assessed quantitatively by six parameters of strides: left forelimb, right forelimb, left hindlimb, right hindlimb, front base, and hind base. For each walk, three times measurements were taken for each parameter [9]. The mean values were subjected to statistical analysis. To analyze forelimb stride and hindlimb stride, data of the left and right strides were combined. Asymmetry of stride length was expressed as the absolute value of the difference between stride length of left limb and right limb. 2.5. Hanging box test The hanging box apparatus consists of a clear box (25 cm × 25 cm × 40 cm) with a rotatable mesh lid on the top (O’hara & Co., Tokyo, Japan). The mouse was placed on the mesh lid, then the lid was quietly turned upside down. The latency to fall off from the lid was measured. If mice did not fall within 5 min, latency was recorded as 5 min. 2.6. Open field test The open field test was conducted based on the method described previously [24]. The open field apparatus consists of a white square arena (50 cm × 50 cm) with a wall 40 cm in height (O’hara & Co. Ltd.). A mouse was placed in one of corners of the open field and let freely move for 10 min in the field. Total moving distance for 10 min and duration of staying in the central area was analyzed using a video tracking system (Image OF, O’hara & Co.). 2.7. Elevated plus maze test The elevated plus maze test was conducted as described previously [23]. The elevated plus maze apparatus consists of cross platform containing the two open arms without walls (5 cm × 25 cm) and the two closed arms (5 cm × 25 cm) with 15 cm walls (O’hara & Co.). The mouse was placed on the center of a crossshaped pattern and let freely move for 10 min in the plus maze. The time in which the animal stayed in the open arm, closed arm, and center area, were analyzed using a video tracking system (Image EP, O’hara & Co.).

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Fig. 1. Growth curves of Atp1a3+/− and wild type. Body weights of Atp1a3+/− (a3) and wild type (wt) were measured at 4 weeks of age. Then, mice of each genotype were divided into stressed and non-stressed groups, and restraint stress was applied to the stressed group. (A) Body weight of female mice. Body weights of 4-week-old Atp1a3+/− (open circle, n = 20) and wild type (open square, n = 19). Changes in body weight between 6 and 12 weeks of age in non-stressed Atp1a3+/− (open circles, n = 10), non-stressed wild type (open squares, n = 9), stressed Atp1a3+/− (solid circles, n = 10), and stressed wild type (solid squares, n = 10). There was no significant difference in body weight. (B) Body weight of male mice. Body weights of 4-week-old Atp1a3+/− (open circle, n = 19) and wild type (open square, n = 17). Changes in body weight between 6 and 12 weeks of age in non-stressed Atp1a3+/− (open circles, n = 9), non-stressed wild type (open squares, n = 8), stressed Atp1a3+/− (solid circles, n = 10), and stressed wild type (solid squares, n = 9). There was no significant difference in body weight. Data are mean ± SD.

2.8. Accelerating rotarod test The rotarod apparatus consists of a rotatable rod (33 mm in diameter) flanked by discs at both sides (O’hara & Co.). The mouse was placed on the rotating rod (2 rpm), and the speed was increased by 0.15 rpm/s [24]. The latency to fall from the rod was measured. The latency was scored as 5 min if the mice did not fall within 5 min. The test was performed three times per day for two consecutive days.

solution [4% paraformaldehyde (PFA)/PBS, (pH 7.4)]. The brain was isolated and further fixed at 4 ◦ C in fixation solution for 24 h. Samples were immersed in 18% sucrose/PBS, embedded in optimal cutting temperature (OCT) compound (Sakura Finetek, Torrance, CA), then frozen on dry ice, and cut into 20 ␮m-thick cryosections. In situ hybridization was performed as described previously [24] using isoform-specific digoxigenin-UTP (Roche Diagnostics, Basel, Switzerland)-labeled riboprobes for Atp1a3 at 50 ◦ C [24]. Signals were detected using an anti-digoxigenin antibody conjugated with alkaline phosphatase (Roche) and NBT/BCIP (Roche) for chromogen.

2.9. Balanced beam test 2.13. Statistical analysis The balanced beam apparatus (O’hara & Co.) consists of 1-m long bar (28 or 11 mm in diameter) with an escape box on one end [24]. Over a period of three consecutive days, mice were trained to reach the escape box on a bar 28 mm in diameter from the opposite end. After the last training, the test trial was performed three times using the 11 mm diameter bar. The latency to reach the escape box was measured. If mice did not reach the escape box within 1 min or fell from the bar, latency was recorded as 1 min. The minimum value in the three trials was used for statistic analysis.

All statistic analyses were performed using the R package. Twoway analysis of variance (genotype × stress) was used to examine the main effect and interaction effect in the all behavior data. When data showed significant effects for main and/or interaction (p < 0.05), the pairwise t test was performed with the Holm adjustment method except for the data of body weight, limb stride and base, and hanging time of mice at 4 weeks of age. Student’s t test was performed for these data at 4 weeks of age.

2.10. Grip strength test

3. Results

In this test, the mouse was picked up by the tail and forced to hold the mesh grid of the grip-strength meter in both forelimbs (O’hara & Co.), as described previously in detail [38]. The mouse was subsequently pulled gently backward by the tail. The pulling force applied to the mouse forelimbs was recorded in Newtons (N). The mean value of three trials was used for statistical analysis.

3.1. Restraint-stress loading does not affect body weight of Atp1a3+/−

2.11. Forced swimming test The mouse was floated in a 2.0 L beaker containing 1.5 L water (20–24 ◦ C) for 5 min. The immobility time and latency to freeze were measured. 2.12. RNA probes and in situ hybridization Postnatal day 38 wild-type mice were euthanized by intraperitoneal injection of pentobarbital (140 mg/kg somnopentyl, Kyoritsu Seiyaku co, Tokyo) and transcardially perfused with fixation

Body weight is an index of general health. We measured body weight of mice during stress loading and evaluated whether motor deficits result from differences in body weight. Restraint stress was applied to the stressed group from 4 to 12 weeks of age, and body weight was measured every two weeks during this period. In female and male mice, body weight of Atp1a3+/− was similar to that of the wild type at 4 weeks of age before the restraint stress loading (Fig. 1A and B). During the restraint stress-loading period (6 and 12 weeks of age), body weight gradually increased as the animals grew and there was no significant difference among the non-stressed Atp1a3+/− , non-stressed wild type, stressed Atp1a3+/− , and stressed wild type (Fig. 1A and B). Thus, restraint stress loading did not affect the increment in body weight accompanying growth in both sexes of mice, excluding ill health and/or body size difference as a cause of differences in behavioral analysis (see below).

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Fig. 2. Stress loadings shorten stride length in female Atp1a3+/− compared to wild-type mice. Four-week-old female mice (Atp1a3+/− and wild type) were used for footprint analysis (n = 20 and 19, respectively). Then mice of each genotype were divided into non-stressed and stressed groups, and restraint stress was applied to the stressed group. Footprint test was conducted in 6- to 12-week-old non-stressed Atp1a3+/− , non-stressed wild type, restraint stressed Atp1a3+/− , and restraint stressed wild type were used (open circles, n = 10; open squares, n = 9; solid circles, n = 10; solid squares, n = 10). (A) Forelimb stride length of female mice. (B) Hindlimb stride length of female mice. (C) Asymmetry of forelimb stride. (D) Asymmetry of hindlimb stride. (E) Width between forelimb. (F) Width between hindlimb. Data are mean ± SD. # p < 0.05, for non-stressed Atp1a3+/− and non-stressed wild type. * p < 0.05, for stressed Atp1a3+/− and stressed wild type.

3.2. Restraint-stress loading alters footprint pattern of Atp1a3+/− Gait disturbance due to limb dystonia [45], unsteady gait [5], and tip-toe gait [30] are symptoms of RDP, and physical and/or emotional stress often triggers their expression [6]. In female mice, stride lengths of forelimb and hindlimb of Atp1a3+/− were significantly shorter than those of wild type at 4 weeks of age (Fig. 2A and B, open circle and square). There were no differences in forelimb and hindlimb stride lengths in the non-stressed groups between 6 and 12 weeks of age between Atp1a3+/− and wild type (Fig. 2A and B, open circles and squares). However, the stride length of the forelimb was significantly shorter at 6, 8, and 10 weeks of age in stressed Atp1a3+/− mice compared with the wild-type counterpart (Fig. 2A, closed circles and squares). Furthermore, the stride length of hindlimb of 8- and 10-week-old Atp1a3+/− was also significantly shorter than that of the wild type of similar age in the stressed group (Fig. 2B, closed circles and squares). On the other hand, stride lengths of forelimb and hindlimb were not significantly different in 12-week-old mice of the different groups (Fig. 2A and B). There were no differences in asymmetry of stride length (representing the

difference between stride length of the left limb and right limb) of both forelimb and hindlimb at all ages in all groups (Fig. 2C and D). Moreover, the front and hind bases were not significantly different at all ages in all groups (Fig. 2E and F). In male mice, stride lengths of forelimb and hindlimb were significantly shorter in 4-week-old Atp1a3+/− than wild type (Fig. 3A and B, open circle and square). Furthermore, there was no significant difference in stride length between non-stressed Atp1a3+/− and non-stressed wild type (Fig. 3A and B, open circles and squares). In addition, there was no significant difference in stride length between stressed Atp1a3+/− and stressed wild type of other ages (Fig. 3A and B, closed circles and squares). However, the stride lengths of both limbs were significantly shorter in 8-week-old stressed Atp1a3+/− than non-stressed Atp1a3+/− (Fig. 3A and B, closed and open circle). Further analysis showed no significant differences in asymmetry of stride length of forelimb and hindlimb in all groups of mice at all ages (Fig. 3C and D). The front base was also similar in all groups (Fig. 3E). Only the hind base of 10-week-old stressed Atp1a3+/− was significantly wider than that of the stressed wild-type counterpart (Fig. 3F, closed circle and square).

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Fig. 3. Stress loadings alter footprints of male mice. Four-week-old male mice (Atp1a3+/− and wild type) were used for footprint analysis (n = 19 and 17, respectively). Then mice of each genotype were divided into stressed and non-stressed groups, and restraint stress was applied to the stressed group. Footprint test was conducted in 6–12week-old non-stressed Atp1a3+/− , non-stressed wild type, restraint stressed Atp1a3+/− , and restraint stressed wild type (open circles, n = 9; open squares, n = 8; solid circles, n = 10; solid squares, n = 9). (A) Forelimb stride length of male mice. (B) Hindlimb stride length of male mice. The length of both forelimb and hindlimb strides were shorter in 8-week-old stressed Atp1a3+/− than non-stressed Atp1a3+/− . (C) Asymmetry of forelimb stride. (D) Asymmetry of hindlimb stride. (E) Width between forelimb. (F) Width between hindlimb. Ten-week-old stressed Atp1a3+/− showed wider base than stressed wild type. Data are mean ± SD. # p < 0.05, for non-stressed Atp1a3+/− and non-stressed wild type. * p < 0.05, for stressed Atp1a3+/− and stressed wild type. + p < 0.05, for non-stressed and stressed Atp1a3+/− .

Table 2 summarizes the effects of restraint-stress loading on footprint patterns. Before stress loading, the stride length was shorter in both female and male 4-week-old Atp1a3+/− mice compared with the wild type. Only in females was the stride length shorter in stressed Atp1a3+/− than stressed wild type. In males, the stride length was shorter in stressed Atp1a3+/− than non-stressed Atp1a3+/− and the hind base was wider in stressed Atp1a3+/− than stressed wild type. Thus, restraint-stress loading caused changes in footprint patterns of both male and female Atp1a3+/− , and gender influenced their expression. 3.3. Restraint-stress loading induces motor deficit in male Atp1a3+/− mice in hanging box test The hanging box test was performed on the same day of footprint analysis to evaluate the motor ability (balance and grip strength). In 4-week-old female mice, the hanging time was not significantly different between Atp1a3+/− and the wild-type mice (Fig. 4A, open circle and square). During the period of stress

loading, the hanging time tended to be longer in the stressed groups compared with the non-stressed groups at 6- to 12-weeks of age, although the difference was not significant (Fig. 4A, closed circles and squares). In the non-stressed groups of Atp1a3+/− and wild-type mice, only a few mice aged 6- to 12-weeks old, hang on the mesh lid for more than 50 s (0 out of 19 in 6-week-old, 1 out of 19 in 8week-old, 2 out of 19 in 10-week-old, 1 out of 19 in 12-week-old). In contrast, in the stressed groups, hanging more than 50 s was noted in 3 out of 20 in 6-week-old, 6 out of 20 in 8-week-old, 6 out of 20 in 10-week-old, and 4 out of 20 in 12-week-old, although more than 50% of mice (42/80) fell within 10 s even in the stressed group. There was no significant difference in the hanging time between 4-week-old male Atp1a3+/− and wild-type mice (Fig. 4B, open circle and square). No 6- to 10-week-old mice hang on the mesh lid for more than 50 s. Furthermore, there was no significant difference in hanging time among mice aged 6- to 10-weeks (Fig. 4B). However, at 12 weeks of age, the hanging time was significantly longer in stressed wild-type mice compared with the other groups (Fig. 4B, closed square). None of the 12-week old mice in the other

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Fig. 4. Prolonged hanging time in male stressed wild-type mice. Hanging time was measured in 4-week-old Atp1a3+/− and wild-type mice. Then restraint stress was applied to the stressed group. (A) Hanging time of female mice plotted by logarithmic scale. Hanging time of 4-week-old Atp1a3+/− (open circle, n = 20) and wild type (open square, n = 19) mice. Hanging time of 6–12-week-old non-stressed Atp1a3+/− (open circles, n = 10), non-stressed wild type (open squares, n = 9), stressed Atp1a3+/− (solid circles, n = 10), and stressed wild type (solid squares, n = 10) mice. Gray dots represent individual mice for each group. (B) Hanging time of male mice plotted by logarithmic scale. Hanging time of 4-week-old Atp1a3+/− (open circle, n = 19) and wild type (open square, n = 17). Hanging time of 6–12-week-old non-stressed Atp1a3+/− (open circles, n = 9), non-stressed wild type (open squares, n = 8), stressed Atp1a3+/− (solid circles, n = 10), and stressed wild type (solid squares, n = 9). Data are mean ± SD. * p < 0.05, for stressed Atp1a3+/− and stressed wild type. $ p < 0.05, for non-stressed and stressed wild type. + p < 0.05, for non-stressed Atp1a3+/− and stressed wild type.

Fig. 5. Results of behavior tests in female mice. (A) and (B) Open field test. (A) Total moving distance in the open field. (B) Percentage of stay in center area of the open field. (C) Time spent in each arm of the elevated plus maze. (D) and (E) Accelerating rotarod test. (D) Latency to fall from the rotating rod (33 mm in diameter). (E) Learning rate calculated by latency of day 2/latency of day 1. (F) Balanced beam test. Time to reach the goal box on the narrow rod (11 mm in diameter). (G) Grip strength test. (H) and (I) Forced swimming test. (H) Immobility time in water. (I) Latency to freeze. Stripped open bars: non-stressed Atp1a3+/− (n = 10), open bars: non-stressed wild type (n = 9), stripped solid bars: stressed Atp1a3+/− (n = 10), solid bars: stressed wild type (n = 10). Data are mean ± SD.

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Fig. 6. Results of behavior tests in male mice. (A) and (B) Open field test. (A) Total moving distance in the open field. (B) Percentage of stay in center area of the open field. (C) Time spent in each arm of the elevated plus maze. (D) and (E) Accelerating rotarod test. (D) Latency to fall from the rotating rod (33 mm in diameter). (E) Learning rate calculated by latency of day 2/latency of day 1. (F) Balanced beam test. Time to reach the goal box on the narrow rod (11 mm in diameter). (G) Grip strength test. (H) and (I) Forced swimming test. (H) Immobility time in water. (I) Latency to freeze. Stripped open bars: non-stressed Atp1a3+/− (n = 9), open bars: non-stressed wild type (n = 8), stripped solid bars: stressed Atp1a3+/− (n = 10), solid bars: stressed wild type (n = 9). Data are mean ± SD.

groups hang on the mesh lid for more than 10 s. On the other hand, the hanging time was longer than 10 s in 6 out of 9 (67%) in 12-week-old stressed wild-type mice. Restraint-stress loading prolonged hanging time in male wild-type only but not in Atp1a3+/− . Thus, restraint-stress loading affected motor strength of Atp1a3+/− . 3.4. Restraint-stress loading does not affect anxiety-like, depression-like behavior, and motor coordination To evaluate the differential effects of stress loading on other behaviors (anxiety-like and depression-like behavior and locomotion activity) in Atp1a3+/− and wild type, we performed a comprehensive behavioral test battery including open field test, elevated plus maze test, accelerating rotarod test, balanced beam Table 2 Summary of significant differences by footprint analysis.

Female

Male

Age (weeks)

4

Non-stressed a3 vs wt Stressed a3 vs wt Non-stressed a3 vs stressed Non-stressed wt vs stressed Non-stressed a3 vs wt Stressed a3 vs wt Non-stressed a3 vs stressed Non-stressed wt vs stressed

*1

6

8

10

*2

*3

*3

*1 *5

12

test, grip strength test, and forced swimming test. After stress loading during 6- to 12-weeks of age, the behavioral test battery was performed at 13- to 16-weeks of age. In the open field test, total distance (Figs. 5A and 6A) and percentage of time staying in the center (Figs. 5B and 6B) were not significantly different among the groups. In addition to the open field test, the elevated plus maze was used to assess anxiety-like behavior. The percentage of time spent in the open arm was not significantly different among the groups (Figs. 5C and 6C). Accelerating rotarod test, balanced beam test, and grip strength test were performed to assess motor function (coordination, balance, and grip strength). In the accelerating rotarod test, no significant differences were observed in fall latency (Figs. 5D and 6D) and learning rate of motor (latency of Day1/Day 2) (Figs. 5E and 6E). In the balanced beam test, the time to reach the goal box (Fig. 5F and 6F) and number of slippage (data not shown) were also not different. Furthermore, the grip strength was similar in all mice (Figs. 5G and 6G). The forced swimming test, which evaluates the tendency for depression-like behavior, showed similar immobility time (Figs. 5H and 6H) and latency to freeze (Figs. 5I and 6I). Thus, stress loading did not any effect on anxiety-like and depression-like behavior and locomotion activity, based on the results of various behavioral tests. 3.5. Expression of Atp1a3 in various brain regions at postnatal day 38

*4

a3—Atp1a3+/− ; wt—wild type. *1 p < 0.05, compared with stride lengths of forelimb and hindlimb. *2 p < 0.05, compared with stride lengths of forelimb. *3 p < 0.05, compared with stride lengths of forelimb and hindlimb. *4 p < 0.05, compared with stride lengths of forelimb and hindlimb. *5 p < 0.05, compared with hind base.

Previous studies have demonstrated the expression of ␣3 subunit in restricted regions of the brain in rats and mice [34,40]. Bøttger et al. also reported the distribution of Atp1a3 protein [11]. The mRNA expression of Atp1a3 in the “rat” brain was also reported previously [44,22]. Here we examined the expression of Atp1a3 in the whole brain of juvenile mice. Atp1a3 was expressed in almost all brain regions of 38-dayold wild-type mice; the cerebral cortex (Cx), limbic system, basal

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ganglia, diencephalon, midbrain, pons, cerebellum, and medulla oblongata, as well as in the spinal cord. In the cerebral cortex, it was expressed in all layers (Fig. 7H–P). As reported previously in rats [22], stronger expression in the deeper layer than superficial layer was observed. In the olfactory and limbic system, Atp1a3 was expressed in olfactory bulbs (Fig. 7A–C), piriform cortex (Fig. 7D–N), septal nucleus (Fig. 7E–H), hippocampal formation (Fig. 7J–P), nucleus accumbens (Fig. 7E), nucleus of the diagonal band (Fig. 7F–H), amygdala (Fig. 7I–K), bed nucleus of stria terminalis (Fig. 7I), and mammillary body (Fig. 7M and N). Furthermore, Atp1a3 was highly expressed in the piriform cortex and hippocampus/dentate gyrus. In the basal ganglia, it was expressed in caudate/putamen and globus pallidus (Fig. 7E–J). The dienchephalon, epithalamus, thalamus, subthalamus, and hypothalamus were also positive, with high intensity in the habenular nucleus of epithalamus (Fig. 7J–L) and laterodorsal thalamic nuclei of thalamus (black asterisk in Fig. 7J), and with moderately high intensity in zona inceruta and subthalamic nucleus of subthalamus (Fig. 7K and L). In the midbrain, the superior colliculus, inferior colliculus, periaqueductal gray, mesencephalic nucleus, substantia nigra (pars compacta), and red nucleus showed positive hybridization signals (Fig. 7M–T). The pons, pontine nuclei and motor nuclei of V (trigeminal) also showed signals (Fig. 7Q–W). The vestibular and cochlear nuclei of VIII were positive (Fig. 7U–W). Positive hybridization signals were also observed in the cerebellum, Purkinje cells, molecular-layer interneurons, and Golgi cells, as described previously [24]. The cerebellar nuclei were also highly positive (Fig. 7V). In the medulla oblongata, various nuclei, including gracilis, and cuneate nuclei were positive (Fig. 7X). Examination of 4-week wild-type mice showed essentially similar expression patterns for Atp1a3 (data not shown). 4. Discussion 4.1. Chronic restraint stress induces motor deficits in Atp1a3+/− To investigate whether stress-load Atp1a3+/− mice show motor deficits and other abnormal behaviors that are observed in RDP patients, comprehensive behavioral analyses were performed. Footprint analysis, which evaluates motor coordination and synchrony [10], is a behavioral test suitable for validation of the usefulness of Atp1a3+/− as an animal model of RDP, because patients with RDP often show gait abnormalities [5,30,45]. Patients with RDP and other parkinsonism or Parkinson’s disease present a wide range of gait abnormalities including a shortened stride length together with several symptoms [35,29,16]. Mice showing Parkinson’s disease/parkinsonism like neurodegeneration by gene mutation or by injection of drugs (e.g., MPTP, nitropropionic acid) are known to show reduced stride length [17,47,1,48]. In our present study, both female and male Atp1a3+/− mice showed shorter stride length compared with wild type at 4 weeks of age under non-stress conditions. Before the onset of RDP, several patients show vague dystonia limited to the distal arm or leg [6]. AHC patients also show unsteady gait [39]. The shorter stride of Atp1a3+/− before stress loading probably reflects antecedent symptoms of RDP and symptoms of AHC. Although we did not observe limb dystonia, bradykinesia, and impairment of postural reflex, Atp1a3+/− exhibited gait abnormalities from 6- to 10-weeks of age under stress loading, like RDP patients. Myk/+ mice are reported to show unsteady and tremorous gait at 4 weeks of age and shorter stride length and wider hind base from 8- to 12-weeks of age in the absence of experimental stress [28]. In contrast, 4-week-old Atp1a3+/− showed mild gait abnormality with shorter stride and no gait abnormality at 6–12 weeks in the absence of stress, but showed stress-dependent gait abnormality at 6–12 week-old. These observations suggest that the phenotype of Myk/+ is more similar to AHC

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than RDP [28], while the phenotype of Atp1a3+/− is more similar to RDP than AHC. Stressed male wild-type mice showed increased hanging time in the hanging box test which evaluates motor ability, but this was noted only in 12-week-old mice. It is reported that the struggling of mice during restraint provided increased physical activity [50]. It is possible that chronic restraint stress may result in increased physical activity judged by the longer hanging time in stressed male wild-type mice, when compared with non-stressed male mice. The observation that the stressed male Atp1a3+/− did not show increased hanging time might indicate their less physical activity. This motor deficit could also reflect gait abnormality in human patients. However, no deficits were observed in different behavioral tests including the balanced beam and grip strength test even after stress loading (in 15–16-week-old mice). The fact that the effect of hanging box was only observed at the last period of stress loading (12-week-old) suggest that the effect of stress loading might reach threshold for the expression of motor deficit cumulatively at 12 weeks of age, but the effect disappeared after the period of stress loading. In this context, it is reported that chronic restraint stress decreased locomotor activity of mice in the open field test at 1 day after stress loading, but no decrease in activity in the open field test was observed at 19 days after stress loading [51]. 4.2. Lack of effect of stress loading on anxiety- and depression-like behaviors and motor functions In contrast to the motor deficits observed in footprint and hanging box tests, no anxiety-like and depression-like behaviors were noted. Generally, chronic stress can increase depression-like [49] and anxiety-like behaviors in mice [33,20,32]. However, many studies on chronic stress have failed to demonstrate a consistent behavior phenotype [18,19,43]. For example, housing condition can alter the effects of chronic stress [32]. Chronic restraint stress induced increased anxiety-like behavior in elevated plus maze and forced swimming tests in single-housed mice, but not in group-housed mice [32]. In our study, stressed wild type did not show increased anxiety- and depression-like behaviors compared with non-stressed wild type. This result may be related to our housing condition (group housing of each group of mice) or to non-sustainable effect of stress after the period of stress loading as mentioned above. The present study showed no significant difference between Atp1a3+/− and wild type in rotarod and balanced beam even during non-stress. In contrast, our previous study showed better performance in the accelerating rotarod test in 3-week-old and in balance beam in 11–14-week-old Atp1a3+/− compared to the wild type [24]. The observed differences in rotarod performance may be explained by the different age of mice used in the present and previous studies (13–16-week-old vs 3-week-old, respectively). The better performance in rotarod may be specific to the adolescence period. Moreover, the difference in balance beam performance in adult mice may be explained by housing conditions. All mice in the present study were housed in a disposable individually ventilated cage (IVC) consisting of clear polyethylene terephthalate, thus mice cannot hang from the wire-roof, while in previous study, mice were housed in a conventional wire-roof home cage [24]. IVC housing may affect motor function through differences in exercise in each cage (e.g., wire hanging), although there are no studies that have compared motor function in mice in IVC housing and mice housed in other cage systems. 4.3. Sex difference in behavior phenotype In the present study, the stride length was shorter in both female and male Atp1a3+/− mice under non-stressed and stressed

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Fig. 7. Analysis of mRNA expression in various brain regions of 38-day-old male Atp1a3 wild-type mice. Scale bar = 1 mm. GrA, granule cell layer of accessory olfactory bulb; Gl, glomerular layer of olfactory bulb; GrO, granule cell layer of olfactory bulb; Mi, mitral cell layer of olfactory bulb; AOB, accessory olfactory bulb; AOL, anterior olfactory nucleus, lateral; AOE, anterior olfactory nucleus, external; OCx, orbital cortex; AOM, anterior olfactory nucleus, medial; Pir, piriform cortex; Cx, cerebral cortex; fmi, forceps minor corpus callosum; ac, anterior commissure; Acb, nucleus accumbens; CPu, caudate putamen (striatum); En, endopiriform nucleus; S, septal nucleus; IG, indusium griseum; cc, corpus callosum; LV, lateral ventricle; VDB, nucleus of vertical limb diagonal band; 3V, 3rd ventricle; VP, ventral pallidum; HDB, nucleus of horizontal limb diagonal band; ox, optic chiasm; Tu, olfactory tubercle; 2n, optic nerve; POA, preoptic area; D3V, dorsal 3rd ventricle; TS, triangular septal nucleus; LSD, lateral septal nucleus, dorsal part; GP, globus pallidus; st, stria terminalis; BST, bed nucleus stria terminalis; ic, internal capsule; thal nu, thalamic nucleus; * , laterodorsal thalamic nuclei of thalamus; Amy, amygdala; Hb, habenular nucleus; GrDG, granular layer of dentate gyrus; H, hipoccampal formation; ZI, zona inceruta; hypothal nu, hypothalamic nucleus; fr, fasciculus retroflexus; ml, medial lemniscus; STh, subthalamic nucleus; PMD, premammillary nucleus dorsal; PMV, premammillary nucleus ventral; mt, mammillothalamic tract; PAG, periaqueductal gray; 3N, oculomotor neucleus; MG, medial geniculate nucleus; Mn, mammillary body; SNC, substantia nigra, compact part; SNR, substantia nigra, reticular part; SuG, superficial gray layer of superior colliculus; RMC, red nucleus, magnocellular; Me, mesencephalic nucleus; MnR, median raphe nucleus; Pn,

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conditions. Furthermore, only male Atp1a3+/− showed wider hind base at 10 weeks and shorter hanging time at 12 weeks of age. Thus, both female and male mice showed motor deficits, in different behavior tests or parameters. In contrast, Atp1a3tm1Ling/+ showed motor deficits only in females but not in males in rotarod and balanced beam test by restraint-stress loading, and the difference was explained as sex-dependent effect of stress loading [15]. The different effects of stress loading on behavior phenotype between the present and the above study [15] may be due to the duration of stress loading and different gene targeting strategies. Our study applied restraint stress from 4- to 12-weeks of age, while the previous study applied restraint stress for 5 consecutive days [15]. With regard to gene targeting strategy, our Atp1a3-deficient mice were generated from gene targeting that deletes exon 2–6 in Atp1a3 [24], while the other mice line was generated from gene targeting that introduced a point mutation in intron 4 of Atp1a3 adjacent to splice site [36]. As we already mentioned, induction of motor deficits may require longer period of stress loading. Moreover, mice generated by different gene targeting strategies are not completely consistent with phenotype for each other. Thus, the deficits described in our mice may be due to the longer period of stress loading and different gene targeting strategy. Our Atp1a3+/− could be regarded as a complementary model of RDP. Although penetrance and symptoms of RDP in human do not show sex differences, studying the sex difference in motor deficits in mice could shed new light on sex-dependent pathology like Dopa-responsive dystonia [37].

4.4. Expression of Atp1a3 in the brain Gait disturbance is a characteristic feature of patients with Parkinsonism and spinocerebellar degeneration. In mammals, gait control involves several brain regions; e.g., the cerebral cortex, basal ganglia, limbic system, thalamus, brainstem, cerebellum, and spinal cord [46]. In this study, we examined the expression of Atp1a3 at postnatal age of 38 days by in situ hybridization. In situ hybridization studies are powerful for detection the precise distribution of mRNA at single cell level. These studies confirmed the expression of Atp1a3 in almost all brain regions and major neuronal cells. Although it is difficult to identify the main brain region(s) for gait disturbance in our mice as well as patients, the observation of broad expression of Atp1a3 is compatible with the manifestation of gait abnormalities.

4.5. Is the Atp1a3+/− mouse a suitable RDP model? We identified a new motor deficit related to gait abnormality, one of the symptoms of RDP and AHC, in Atp1a3+/− mice. Four-week-old Atp1a3+/− mice showed shorter stride length than wild type before stress loading. This motor deficit disappeared later under non-stress conditions (6- to 12-week-old), but was persistently noted in 6–10-week-old females under stress condition. The abnormal footprint induced by stress loading in mice mimics the stress-triggered expression of RDP symptoms in human. So far, stress loading-induced RDP symptoms like gait disturbance have not been reproduced in any mouse line harboring mutation of Atp1a3 [27,15]. Thus, our Atp1a3+/− mice with gait abnormality similar to that observed in RDP patients, is a suitable model of RDP and can be used to examine the pathology of the RDP

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by chronic restraint-stress loading in addition to other Atp1a3+/− mice described previously [36]. On the other hand, Myk/+ with more severe phenotype than Atp1a3-deficient mice, is considered a suitable model of AHC [28]. The differences between Atp1a3-deficient mice and Myk/+ are probably related to the dominant-negative effect of point mutation in Myk/+ . The expression of Atp1a3 is decreased in heterozygous deficient mice [36,24], but not in Myk/+ , whereas the latter exhibit reduced Na,K-ATPase activity in brain homogenates [13]. Indeed, AHC-causing mutations do not affect the protein expression level compared with the wild type, but show reduced Na,K-ATPase activity in COS-7 cells expressing wild type or mutated ATP1A3 [21]. However, in the present study, Atp1a3+/− mimicked parts of the RDP symptoms, and the phenotype was independent of dominantnegative effect. Thus, in addition to mice with point mutation, Atp1a3-deficient mice are also useful for understanding the disease pathology in ATP1A3.

Acknowledgments We thank all members of the Division of Biology for the helpful discussion and technical assistance. This work was supported by The Science Research Promotion Fund of the Promotion and Mutual Aid Corporation for Private Schools of Japan (to H.S.), and Jichi Medical University Young Investigator Award (to H.S.).

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