Elsevier

Behavioural Brain Research

Volume 154, Issue 2, 5 October 2004, Pages 457-463
Behavioural Brain Research

Research report
Behavioural abnormalities of the hyposulphataemic Nas1 knock-out mouse

https://doi.org/10.1016/j.bbr.2004.03.013Get rights and content

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.

Introduction

Inorganic sulphate (SO42−) 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 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 SO42− 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 SO42− 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 NaSi-1 protein [11]. The Nas1−/− mice exhibit increased urinary SO42− 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.

Section snippets

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 (25cm×42cm×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

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 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

Discussion

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

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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