Behavioural abnormalities of the hyposulphataemic Nas1 knock-out mouse

Behavioural Brain Research 154 (2004) 457–463

Research report

Behavioural abnormalities of the hyposulphataemic Nas1 knock-out mouse Paul Anthony Dawson, Sarah Elizabeth Steane, Daniel Markovich∗ Department of Physiology and Pharmacology, School of Biomedical Sciences, The University of Queensland, St. Lucia, Qld. 4072, Australia Received 20 November 2003; received in revised form 14 March 2004; accepted 15 March 2004 Available online 27 April 2004

Abstract We recently generated a sodium sulphate cotransporter knock-out mouse (Nas1−/−) which has increased urinary sulphate excretion and hyposulphataemia. To examine the consequences of disturbed sulphate homeostasis in the modulation of mouse behavioural characteristics, Nas1−/− mice were compared with Nas1+/− and Nas1+/+ littermates in a series of behavioural tests. The Nas1−/− mice displayed significantly (P < 0.001) decreased marble burying behaviour (4.33 ± 0.82 buried) when compared to Nas1+/+ (7.86 ± 0.44) and Nas1+/− (8.40 ± 0.37) animals, suggesting that Nas1−/− mice may have decreased object-induced anxiety. The Nas1−/− mice also displayed decreased locomotor activity by moving less distance (1.53 ± 0.27 m, P < 0.05) in an open-field test when compared to Nas1+/+ (2.31 ± 0.24 m) and Nas1+/− (2.15 ± 0.19 m) mice. The three genotypes displayed similar spatiotemporal and ethological behaviours in the elevated-plus maze and open-field test, with the exception of a decreased defecation frequency by the Nas1−/− mice (40% reduction, P < 0.01). There were no significant differences between Nas1−/− and Nas1+/+ mice in a rotarod performance test of motor coordination and in the forced swim test assessing (anti-)depressant-like behaviours. This is the first study to demonstrate behavioural abnormalities in the hyposulphataemic Nas1−/− mice. © 2004 Elsevier B.V. All rights reserved. Keywords: NaSi-1; Sulphate; Behaviour; Marble bury; Locomotion; Defecation

1. Introduction Inorganic sulphate (SO4 2− ) is the fourth most abundant anion in mammalian plasma and is essential for numerous physiological functions [22]. Sulphate conjugation is an important step in the biotransformation of xenobiotics [12] and in the activation of endogenous compounds such as heparin and heparan sulphate [24]. In addition, sulphation of structural components such as glycosaminoglycans and cerebroside sulphate is essential for the maintenance of normal structure and function of tissues [25]. Disturbances of sulphate metabolism and transport have been associated with human syndromes and diseases including metachromatic leukodystrophy, Hunter’s syndrome, Morquio’s syndrome, Maroteaux–Lamy syndrome, multiple-sulfohydrolase deficiency, and osteochondrodysplasias [15,35]. Behavioural problems are a common feature in mucopolysaccharide disorders, including Hunter’s disease and Sanfilippo’s syndrome (reviewed in [3]), which are caused ∗

Corresponding author. Tel.: +61-7-3365-1400; fax: +61-7-3365-1766. E-mail address: [email protected] (D. Markovich).

0166-4328/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2004.03.013

by a deficiency of iduronate sulphatase and heparin sulphate metabolizing enzymes, respectively. Animal models of these disorders also display behavioural abnormalities including altered memory, learning and neuromotor function [21,38]. Recently, Han and coworkers [13,14] reported a decreased sulphatide content in brain tissue and cerebroside fluid derived from Alzheimer’s disease (AD) patients. The sulphatide deficiency in these patients was detected at the earliest clinical stage of AD and was proposed to occur prior to the appearance of clinical symptoms [13]. Despite the important roles of sulphate in mammalian physiology, serum SO4 2− levels are rarely measured in a clinical setting and little is known about the physiological consequences of disturbed sulphataemia. In humans, the sodium sulphate cotransporter, NaSi -1, is expressed primarily in the kidney [20] and has been proposed to play a major role in maintaining serum SO4 2− concentrations within the normal physiological range of 0.33–0.47 mmol/l [5,6,17]. We have cloned the mouse and human NaSi -1 genes, designated Nas1 and NAS1, respectively [4,20], and recently generated a Nas1 knock-out mouse that lacks a functional

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NaSi -1 protein [11]. The Nas1−/− mice exhibit increased urinary SO4 2− excretion and hyposulphataemia. Our observations of Nas1−/− mice hiding at the back of their cages, have led us to study their behaviour in tests examining different aspects of stress, anxiety and depression. These experiments include the marble bury test, which is associated with defensive-like behaviour [26], the elevated-plus maze test, which is associated with anxiety-like behaviour [28], the open-field test, which is based on free exploration of an unfamiliar environment [10] and the forced swim test, which can measure (anti-)depressant-like behaviours [2]. We also compared the motor coordination of Nas1−/− and Nas1+/+ mice using a rotarod performance test [29]. The present study is the first to investigate the behaviour of hyposulphataemic mice lacking a functional NaSi -1 gene. 2. Materials and methods 2.1. Experimental animals The Nas1 knock-out strain of mice was recently generated in our laboratory [11]. Same-litter groups of male mice were housed three to five per cage (25 cm × 42 cm × 12 cm) at a constant temperature (23 ± 1 ◦ C) with a 12 h/12 h light/dark cycle (lights on at 06:00 h and off at 18:00 h). To facilitate adaptation, mice were transported to the behavioural studies facility at least 24 h prior to testing. Experiments were conducted between 08:00 h and 13:00 h with the lighting level adjusted to 100 lx. Other than the brief testing periods, mice had access to food (standard rodent chow, no. AIN93G; Glen Forrest Stockfeeders, Glen Forrest, Western Australia) and water ad libitum. One group of mice was tested sequentially in the elevated-plus maze, marble bury test and open-field test at 1-week intervals. A second group of mice was tested sequentially in the rotarod and forced swim tests with a 3-day interval. We have used young (4–7 weeks) mice in this study because older adult Nas1−/− mice develop seizures [11] which could possibly affect their behaviour in our experiments. All experiments conformed to the guidelines of the University of Queensland Animal Ethics Committee. 2.2. Marble bury test Subjects were 21 Nas1+/+, 35 Nas1+/− and 15 Nas1−/− male mice aged between 5 and 6 weeks. The tests were performed as previously described [27] using transparent polycarbonate cages (30 cm × 15 cm × 13 cm high) in which 10 glass marbles (1.5 cm diameter) were spaced evenly on top of a 5-cm deep layer of slightly compacted sawdust. Mice were placed individually in a cage and left undisturbed for 30 min. At the end of the 30-min test period, the number of marbles buried (greater than or equal to 2/3 covered with bedding) were counted. Cages and marbles were cleaned with 70% ethanol and the sawdust replaced between subjects.

2.3. Open-field test Subjects were 18 Nas1+/+, 24 Nas1+/− and 12 Nas1−/−, male mice aged between 6 and 7 weeks. The open-field test was carried out essentially as described elsewhere [10]. The apparatus consisted of a square box (60 cm × 60 cm × 40 cm high). Mice were placed individually into the centre of the box and their behaviour over the 14-min test period was recorded on videotape by a video camera above the apparatus. For the purpose of collecting meaningful spatiotemporal and ethological data, the recorded image of the open-field was divided into 16 squares, which allowed measurements to be taken in the central 4 squares (ce) and the peripheral 12 squares (pe). The spatiotemporal parameters were: ceTIME, time spent in the central region of the open-field (entry defined as all four paws into the region); ceFREQUENCY, frequency of entry into the centre; and total distance travelled (measured using the SMART videotracking software). The ethological parameters consisted of: peREAR, the mouse reared on its hindpaws (periphery only); peGROOM and ceGROOM, licking and/or scratching of fur on body and/or head; peSTRETCH and ceSTRECH, the mouse stretched forward and then retracted to its original position without locomoting forward; and BOLI. The apparatus was cleaned with 70% ethanol followed by wet and dry paper towels, after each test. 2.4. Elevated-plus maze Subjects were 21 Nas1+/+, 31 Nas1+/− and 15 Nas1−/− male mice aged between 4 and 5 weeks. The apparatus and procedure were based on those described previously [28]. The elevated-plus maze consisted of four arms (30 cm × 5 cm) extending from a central platform (5 cm × 5 cm). Two opposing arms were closed-in by a 30-cm wall, while the other two arms were open with a 0.5-cm lip around the perimeter to prevent the mouse from falling from the arm. The entire apparatus was elevated to 60 cm above ground level. Mice were placed individually onto the central platform of the maze and the entire 14-min test period was recorded on videotape by a video camera above the maze. Behavioural indicators comprised both conventional spatiotemporal and ethological measurements [28]. Conventional measurements were the number of entries into (FREQUENCY) and time spent (TIME) on the central platform (ce) and on the open (o) and closed (c) arms (entry defined as all four paws into an arm, or as two paws into the central platform). Ethological measurements were divided into closed (c) and open (o) when referring to the closed arms/central platform and open arms, respectively and consisted of frequency scores for: cREAR, the mouse reared on its hindpaws (protected only); cDIP and oDIP, exploratory movement of the head/shoulders over the sides of the maze; cGROOM and oGROOM, licking and/or scratching of fur on body and/or head; cSTRETCH and oSTRETCH, the mouse stretched forward and then

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retracted to its original position without locomoting forward; and BOLI. The apparatus was cleaned with 70% ethanol followed by wet and dry paper towels, after each test. 2.5. Rotarod Since difficulties in motor performance can confound behavioural assays of exploration and motivation [29], we analysed the motor coordination of six Nas1+/+ and seven Nas1−/− male mice aged 4–6 weeks, in a rotarod test. The rotarod procedure [29] consisted of three trials separated by a 30-min inter-trial period in the home cage. To begin each trial, mice were placed individually on the stationary drum (62 mm diameter). The rotor was started at a fixed speed of 6 rpm and mice walked on the drum for 120 s. Occasionally a mouse fell from the drum, in which case the timer was stopped and the mouse replaced on the stationary drum, before recommencing timing at 6 rpm. After 120 s the rotor was switched off and restarted at a fixed speed of 12 rpm. The latency for the mouse to fall from the drum was recorded, up to a maximum time of 300 s. The mouse was returned to the home cage for the 30-min inter-trial period. Trials 2 and 3 were carried out in exactly the same way. 2.6. Forced swim test Subjects were six Nas1+/+ and seven Nas1−/− male mice aged 4–6 weeks. The procedure for the forced swim test was previously described [2]. On day 1, each animal was placed in a cylinder (18 cm diameter × 22 cm high) filled to a depth of 15 cm with 30 ◦ C water for a 5-min pre-swim. On day 2, the mouse was monitored during a 5-min swim test in which the time spent immobile was recorded. Immobility was defined as the time spent motionless or using only righting movements to stay afloat. 2.7. Statistical analysis Data were analysed using MINITAB (version 13.31). Distance travelled in the open-field was analysed using one-way ANOVA. All other data were analysed using non-parametric tests. Mann–Whitney was used for the two-sample and Kruskal–Wallis analysis of variance for the n sample case. Differences were considered significant at P < 0.05.

3. Results We noted that Nas1−/− mice hid at the back of their cages during weekly cage transfers (unpublished data). These observations led us to compare the behaviour of the three genotypes using a battery of tests, which revealed three abnormal behavioural traits in the Nas1−/− mice. Firstly, in the marble bury test [26], the Nas1−/− mice buried significantly (P < 0.001) less marbles (4.33 ± 0.82

Fig. 1. Marble burying behaviour of Nas1+/+ (black bars, n = 21), +/− (grey bars, n = 35) and −/− (open bars, n = 15) mice. The Nas1−/− mice buried significantly (∗∗ P < 0.001) fewer glass marbles during the 30-min test. Values represent means ± S.E.

buried, n = 15) when compared to Nas1+/+ (7.86 ± 0.44 buried, n = 21) and Nas1+/− (8.40 ± 0.37 buried, n = 35) mice (Fig. 1). There was no significant difference (P = 0.57) in the number of marbles buried between Nas1+/− and Nas1+/+ mice. Secondly, the distance travelled in an open-field was significantly (P < 0.05) reduced by 35% in the Nas1−/− mice (1.53 ± 0.27 m, n = 12) when compared to Nas1+/+ (2.31 ± 0.24 m, n = 18) mice during a test period of 7 min (Table 1). The distance travelled by the Nas1−/− mice was similarly decreased (P < 0.05) for the 7–14- and 0–14-min time periods when compared to the Nas1+/+ mice (data not shown). There was a small but non-significant (P = 0.41) difference (7% reduction) in the distance travelled by the Nas1+/− mice when compared to their Nas1+/+ littermates (Table 1). Thirdly, a significant (P < 0.01) difference was detected in the frequency of defecation (BOLI) between the genotypes in an open-field test (Table 1), with the Nas1−/− mice excreting fewer fecal boli (1.42 ± 0.41, n = 12) when compared to Nas1+/− (4.54 ± 0.49, n = 24) or Nas1+/+ mice (4.17 ± 0.51, n = 18). This result is consistent with our elevated-plus maze data, which revealed a 40% reduction (P < 0.01) in the Table 1 Behaviour of Nas1+/+, Nas1+/− and Nas1−/− mice in the open-field Behaviour

Nas1+/+ (n = 18)

Distance travelled ceTIME ceFREQUENCY peREAR peGROOM ceGROOM peSTRETCH ceSTRETCH BOLI

2.31 41.83 11.44 11.61 1.50 0 4.83 3.44 4.17

± ± ± ± ±

0.24 5.55 2.20 3.15 0.20

± 0.97 ± 0.73 ± 0.51

Nas1+/− (n = 24) 2.15 58.21 13.88 8.00 1.71 0.04 3.75 3.63 4.54

± ± ± ± ± ± ± ± ±

0.19 12.63 2.15 1.84 0.27 0.04 0.83 0.82 0.49

Nas1−/− (n = 12) 1.53 41.42 11.83 8.00 1.91 0.04 5.64 2.64 1.42

± ± ± ± ± ± ± ± ±

0.27∗ 5.37 2.70 2.48 0.37 0.04 1.82 0.97 0.41∗

Data are mean±S.E. over a 7-min test period. pe, periphery of open-field; ce, centre of open-field. ∗ P < 0.05.

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Table 2 Behaviour of Nas1+/+, Nas1+/− and Nas1−/− mice in the elevated-plus maze Behaviour

Nas1+/+ (n = 21)

cTIME cFREQUENCY oTIME oFREQUENCY ceTIME ceFREQUENCY cREAR cGROOM oGROOM cSTRETCH oSTRETCH cDIP oDIP BOLI

272.00 11.62 23.33 2.62 124.67 13.43 4.71 1.71 0 9.00 0.43 4.19 14.48 3.43

± ± ± ± ± ± ± ±

20.65 1.71 5.90 0.79 19.39 1.94 1.40 0.37

± ± ± ± ±

1.22 0.15 1.52 1.91 0.47

Nas1+/− (n = 31) 210.23 10.81 27.35 2.26 182.42 12.45 2.51 1.51 0 9.51 0.69 6.31 18.20 3.69

± ± ± ± ± ± ± ±

16.99 1.01 9.93 0.61 18.34 1.02 0.46 0.22

± ± ± ± ±

0.95 0.20 1.38 1.66 0.39

Nas1−/− (n = 15) 259.00 11.73 19.67 2.13 141.33 12.87 2.60 2.00 0 11.73 0.93 4.20 16.07 1.13

± ± ± ± ± ± ± ±

20.01 1.06 5.31 0.43 20.19 0.98 0.55 0.34

± ± ± ± ±

2.80 0.33 1.19 3.46 0.42∗

Data are mean ± S.E. over a 7-min test period. c, closed arm; o, open arm; ce, centre of maze. ∗ P < 0.05.

number of excreted boli (BOLI) from the Nas1−/− mice when compared to their Nas1+/+ littermates (Table 2). In the open-field (Table 1) and elevated-plus maze (Table 2), no significant (P > 0.25) effect of genotype was detected with respect to the following ethological parameters: rearing, stretching, grooming and head dipping. In addition, Nas1−/− mice and littermate controls exhibited a similar number of entrances into and time spent in the centre of the open-field (Table 1), suggesting that no changes in exploratory- and anxiety-like behaviour existed between the genotype groups for these tests. Furthermore, Nas1−/− mice and their Nas1+/− and Nas1+/+ littermates exhibited similar time spent and the number of entrances into the central platform, and closed and open arms of the maze for a 7-min period (Table 2) and also for the 7–14- and 0–14-min time periods (data not shown), suggesting no changes in anxiety-like behaviour or in general locomotor activity between the three genotypes.

Fig. 3. Immobility response time (s) of Nas1+/+ (filled bars, n = 6) and Nas1−/− (open bars, n = 7) mice in a forced swim test. Values represent means ± S.E.

In the rotarod test, Nas1−/− mice (n = 7) and their Nas1+/+ littermates (n = 6) showed no significant differences (P > 0.36) in the latency to fall from the rotarod in three consecutive trials (Fig. 2), indicating no changes to motor coordination. The forced swim test revealed no significant differences (P = 0.85) in the immobility response time between Nas1−/− (65 ± 16 s, n = 7) and Nas1+/+ (65 ± 12 s, n = 6) mice (Fig. 3).

4. Discussion

Fig. 2. Rotarod performance of Nas1+/+ (filled bars, n = 6) and Nas1−/− (open bars, n = 7) mice in three consecutive trials. Values represent means ± S.E. trial 1, Nas1−/− (81.3 ± 42.1), Nas1+/+ (43.8 ± 16.8); trial 2, Nas1−/− (77.3 ± 32.8), Nas1+/+ (40.5 ± 16.8); and trial 3, Nas1−/− (60.86 ± 37.1), Nas1+/+ (97.3 ± 47.0).

To our knowledge, the present study is the first to investigate behavioural characteristics in an animal model of hyposulphataemia. Our investigation of possible behavioural changes in Nas1 knock-out mice was motivated by observations of the Nas1−/− mice hiding at the back of their cage during their weekly transfer to fresh cages. We demonstrated that the Nas1-deficient mice exhibited a decreased marble burying behaviour, together with a decreased locomoter activity in an open-field test and reduced defecation frequency. These behavioural features were associated with

P.A. Dawson et al. / Behavioural Brain Research 154 (2004) 457–463

normal motor coordination in a rotarod test, and no changes in a forced swim test. The observed behavioural differences between Nas1−/− and Nas1+/+ mice are most likely the consequence of disturbed sulphate homeostasis in the Nas1−/− mice. This conclusion is based on our previous study, demonstrating that Nas1−/− mice have very low (>75% reduction) serum SO4 2− levels [11]. We have also shown that Nas1 is primarily expressed in the kidney and ileum but not in the brain [4], suggesting that Nas1-deficiency mediates an indirect effect on behaviour through disturbed sulphataemia. To date, little is known about the consequences of disturbed sulphate homeostasis on human and animal behaviour. Our knowledge is limited to the findings of low plasma SO4 2− levels (60–82% reduction) in Motor Neurone disease (MND), Parkinson’s disease (PD), Alzheimer’s disease (AD) and autistic disorder patients, when compared to age-matched controls [16,36]. The low SO4 2− levels in these patients could possibly be a limiting factor in the formation of SO4 2− conjugates. Indeed, PD, MND, AD and autistic patients have a reduced sulphation capacity, which was proposed to lead to neuronal toxicity and exacerbate the behaviour of these individuals [1,30–32]. The low sulphataemia in MND, AD, PD and autistic individuals, together with the findings of the present study, leads us to speculate that SO4 2− may be an important factor in the modulation of behaviour. Sulphation is an important process in the metabolism of certain neurotransmitters, including dopamine and norepinephrine (reviewed in [37]). Strobel et al. [33] showed that catecholamine sulphates exhibit a plasma half-life of approximately 3 h, which is in contrast to free catecholamines with a half-life of <3 min. Mice lacking the ␣2A -adrenergic receptor, which is important for the release of norepinephrine, showed abnormal behavioural traits including decreased marble burying behaviour [18]. In addition, the serotonin metabolite, serotonin-O-sulphate, has been proposed to contribute a functional role in the serotonergic system beyond serotonin inactivation [34]. Disturbances of the serotonergic pathway have been described in a variety of human neurological illnesses, including mood disorders [40], Parkinson’s disease [23] and autism [19]. Recent studies have shown that reduced 5-HT1A receptor mRNA levels [39] and serotonin uptake inhibitors [27], are correlated with a decreased marble burying behaviour in mice. Taken together, these studies suggest an important, yet poorly understood role of sulphate in neurotransmitter function. One of the most striking behavioural phenotypes of the Nas1−/− mice was their reduced marble bury behaviour, which may reflect an anxiolytic or anti-depressant effect of hyposulphataemia. This interpretation is based on previous studies which reported a decreased marble bury behaviour in mice treated with anxiolytic and anti-depressant agents [8,9,27]. However, our open-field and elevated-plus maze experiments for assessing anxiety, and the forced swim test, which assesses (anti-)depressant related behaviours,

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argue against this interpretation. Some investigators have described marble burying as a defensive behaviour which reflects certain types of anxiety that are distinct from those measured by the elevated-plus maze or open-field test, in which no obvious threat is presented [39]. Alternatively, it has been suggested that the burying behaviour may be rewarding or that it is a compulsive-like behaviour [8]. Nonetheless, the markedly reduced number of marbles buried by Nas1−/− mice highlights a possible effect of hyposulphataemia on mouse behaviour, which we interpret as a decreased object-induced anxiety. We recently showed a general growth reduction in the Nas1−/− mice up to at least 5 months of age, when compared with their Nas1+/− and Nas1+/+ littermates [11]. While the low body weight phenotype of the Nas1−/− mice is not restricted to the age-group (4–7 weeks) of mice used in the present study, we need to consider the possibility of maturational defects in the Nas1−/− mice that could affect their behaviour. However, we have shown that Nas1−/− mice become sexually mature at the same age as wild-type mice [11]. Furthermore, the vast majority of behavioural phenotypes examined in our experiments, were similar between Nas1−/− and Nas1+/+ mice. Taken together, these findings suggest that the maturation of Nas1−/− mice may be similar to their wild-type littermates. Despite the similarity between Nas1−/− and Nas1+/+ mice in most open-field and elevated-plus maze measurements, Nas1−/− mice consistently displayed a decreased defecation frequency (40% reduction) in both tests, together with a reduced distance of ambulation in an open-field test (35% reduction). While open-field defecation and activity are traits that have been used to assess the emotional reactivity or fearfulness of rodents (reviewed in [7]), we need to consider the possibility that the reduced locomotor activity and defecation frequency of Nas1−/− mice, respectively, could be explained by a general decrease in activity and by a role of the NaSi -1 transporter in the intestinal or renal system. However, our elevated-plus maze experiment revealed no differences between the three genotypes in the number of entries and time spent on the open and closed arms, suggesting that the Nas1−/− mice may have normal levels of general activity. The physiological role of NaSi -1 on fecal boli excretion, is yet unknown, but will come from future studies that aim to measure food intake and fecal excretion of Nas1−/− and Nas1+/+ mice in their home cages. Interestingly, there were no significant differences in behaviour between Nas1+/− and Nas1+/+ mice, suggesting that the intermediate level of sulphataemia in the heterozygotes [11] is not sufficient to cause behavioural changes. In conclusion, the present study provides primary behavioural characteristics of Nas1 knock-out mice and suggests a possible role of sulphate in modulating certain types of behaviour. The results emphasize the consequences of inactivating NaSi -1 on behaviour, expressed here as a decrease in marble bury behaviour, a reduced locomoter response to a novel environment, and a reduction in defecation frequency.

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Future studies of the Nas1-deficient mice will provide necessary information about the neurochemical basis relating hyposulphataemia and behavioural changes.

Acknowledgements The authors thank Drs. Axel Becker (Institute of Pharmacology and Toxicology, University of Magdeburg) and Thomas Burne (School of Biomolecular and Biomedical Science, Griffith University) for valuable discussions. This work was funded in part by the Australian Research Council and the National Health and Medical Research Council (D.M. and P.A.D.).

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