Early signs of neurolipidosis-related behavioural alterations in a murine model of metachromatic leukodystrophy

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Behavioural Brain Research 189 (2008) 306–316

Research report

Early signs of neurolipidosis-related behavioural alterations in a murine model of metachromatic leukodystrophy Stijn Stroobants a , Toon Leroy b , Matthias Eckhardt c , Jean-Marie Aerts b , Daniel Berckmans b , Rudi D’Hooge a,∗ a

Laboratory of Biological Psychology, Department of Psychology, University of Leuven, Tiensestraat 102, B-3000 Leuven, Belgium b Measure, Model & Manage Bioresponses (M3-BIORES), Department of Biosystems, University of Leuven, Kasteelpark Arenberg 30, B-3001 Leuven, Belgium c Institute of Physiological Chemistry, Faculty of Medicine, University of Bonn, Nussallee 11, D-53115 Bonn, Germany Received 16 October 2007; received in revised form 10 January 2008; accepted 14 January 2008 Available online 31 January 2008

Abstract Arylsulfatase A (ASA)-deficient mice represent an animal model for the lysosomal storage disorder metachromatic leukodystrophy (MLD). Although the model has been applied in pathophysiological and therapeutic studies, the behavioural phenotype of ASA−/− mice is only partially characterized, and the most decisive outcome measures for therapy evaluation only emerge beyond 1 year of age. Presently, ASA−/− mice and ASA+/− control mice were studied at 6 and 12 months of age on an extensive battery including tests of neuromotor ability, exploratory behaviour, and learning and memory. Overt signs of ataxia were not observed in 6-month-old ASA−/− mice, but quantitative gait analysis during openfield exploration revealed that ASA−/− mice displayed increased hind base width and increased stride lengths for all paws. Their covert motor incoordination was evident in a correlation analysis which unveiled decreased harmonisation of concurrent gait parameters. For example, while ASA+/− controls demonstrated substantial convergence of front and hind base width (r = 0.54), these variables actually diverged in ASA−/− mice (r = −0.37). Furthermore, various behavioural observations indicated emotional alterations in ASA−/− mice. Six-month-old ASA−/− mice also showed decreased response rates in scheduled operant responding. The present findings could provide relevant behavioural outcome measures for further use of this murine MLD model in preclinical studies. © 2008 Elsevier B.V. All rights reserved. Keywords: Gait analysis; Operant conditioning; Behavioural testing; Mouse models; Metachromatic leukodystrophy; Lysosomal storage disorders

1. Introduction Metachromatic leukodystrophy (MLD) is a recessively inherited neurolipidosis caused by deficiency of the lysosomal enzyme arylsulfatase A (ASA). ASA catalyses degradation of sulfatide (a major myelin component) and related 3-Osulfogalactolipids [53]. The inability to degrade these lipids causes sulfolipid storage that affects myelin-producing oligodendrocytes and central neurons, resulting in astrogliosis, activation of microglia and progressive demyelination [20]. In the most frequent late-infantile form of the disease, progressive symptoms usually appear at the approximate age of 2 years.

∗

Corresponding author. Tel.: +32 16 326142; fax: +32 16 326099. E-mail address: [email protected] (R. D’Hooge).

0166-4328/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2008.01.008

These children initially display gait disturbances and ataxia, later develop epileptic seizures, spastic quadriplegia and optic atrophy, and eventually die in a decerebrated state [21]. The juvenile and adult forms of MLD typically run a milder course, but psychiatric symptoms may occur in these types before other signs of deterioration [2]. ASA−/− mice with a targeted disruption of the ASA gene were generated [25], which display an MLD-like pattern of sulfatide storage and brain cholesterol depletion [33], but not the widespread demyelination of the human disease [25,54]. Nevertheless, this model has been instrumental for exploratory therapeutic studies that may eventually lead to effective MLD treatment. ASA−/− mice have been treated by bone marrow stemcell gene therapy, but improvement was predominantly limited to visceral pathology [22,34–36,38]. More promising results were recently obtained with a slightly different stemcell trans-

S. Stroobants et al. / Behavioural Brain Research 189 (2008) 306–316

plantation approach [4,5], enzyme replacement therapy [37], and co-expression of formylglycine-generating enzyme in direct gene therapy [31]. Behavioural characterisation of this murine MLD model is still incomplete, and the most distinct behavioural abnormalities only appear beyond 1 year of age [12,13,25,38]. In the present study, we compared ASA+/− and ASA−/− littermate mice in an effort to detect early signs of lysosomal storagerelated impairment using sensitive tests of neuromotor ability, exploratory behaviour, operant responding and learning and memory. ASA+/− mice were used as controls because earlier studies did not show any pathological alterations in heterozygous mice, and because they are efficiently derived littermates from ASA+/− × ASA−/− pairings. We analysed behavioural elements that were not examined before, and proposed novel clinically relevant outcome measures for future preclinical experiments. In relation to the devastating ataxia in MLD patients, and ataxic signs in ASA-deficient mice [25], we included detailed quantitative gait analysis in ASA−/− mice to detect early signs of altered gait. Previously, neuromotor disability and ataxia were assessed in ASA−/− mice with methods that were relatively insensitive (e.g., rotarod), aspecific (e.g., open field), and/or imprecise (e.g., paper-and-ink gait analysis) [12]. In the present gait analysis procedure, we used a ventrally positioned camera to video-track mice in an open-field arena, and extract specific gait parameters. We also examined genotype-dependent inter- and intratask correlations between these gait variables and other behavioural measures.

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Inc., St. Albans, Vermont, USA). Mice were first trained to maintain balance for 2 min at a constant speed of 4 rpm. This training trial was followed by four test trials, during which the rod accelerated from 4 to 40 rpm in 5 min. Consecutive trials were separated by a 10 min intertrial interval. Latency to falling off the rod was recorded up to 5 min.

2.3. Exploratory tests Open-field exploration was tested in a brightly illuminated 50 cm × 50 cm square arena subsequent to 30 min of dark adaptation. Movements in the arena were video-tracked for 10 min (EthoVision: Noldus Bv, Wageningen, The Netherlands). Total path length, percentage of path length in the centre (centre = 30-cm circle), latency to first centre entry, and number of centre and corner entries were assessed. In the social exploration test, the same procedure was applied as in open field, except that a round wire cage with two female mice was placed in the centre of the arena. Anxiety-related exploration was measured in an elevated plus maze, which mice could freely explore for 10 min. The arena consisted of a plus-shaped maze with two arms (5 cm wide) closed by side walls and two arms without walls. Four IR beams recording open and closed arm entries, and one recording the percentage of time per minute spent in the open arms were connected to a computerized activity logger.

2.4. Passive avoidance learning Passive avoidance learning was examined in a cage consisting of a light and a dark compartment containing a grid floor. After 30 min adaptation to the dark, the mouse was placed in the light compartment for a training trial. After 5 s, the sliding door to the dark compartment was opened and step-through latency was manually recorded. When all four paws were placed on the grid floor, a mild electric shock (0.3 mA, 1 s) was delivered, using a constant current shocker (MED Associates Inc., St. Albans, Vermont, USA). Retention was tested 24 h later, and latency to entrance was recorded up to a 300-s cut-off value.

2. Methods 2.1. Subjects Mice with a targeted disruption of the arylsulfatase A gene were generated as described previously [25], using 129/OlaHsd-derived E14-1 ES cells [30]. ES cells were injected into C57BL/6 blastocysts and chimeric male mice were bred with 129/OlaHsd female mice to generate ASA+/− , and subsequently ASA−/− mice with a pure 129/OlaHsd background. For the present experiments, ASA+/− and ASA−/− littermates were efficiently bred from ASA+/− × ASA−/− pairs, whereas in some of the previous studies, knockouts and control mice from independent breeding lines were compared [12,13,35,38]. Alcian blue staining was used to confirm the absence of sulfatide storage in the ASA+/− mice. All animals were housed at standard laboratory conditions (12 h light–dark cycle, constant room temperature and humidity), and all tests were performed during the light phase of their cycle. Food and water were available ad libitum, unless stated otherwise. Knockout and control groups did not differ regarding body weight and size. All procedures were approved by the ethical research committee of the university. We used 36 6month-old ASA+/− mice (12 females, 24 males) and 50 ASA−/− littermates (30 females, 20 males). Surviving mice were partly reexamined at 1 year of age, and used for longitudinal comparison (numbers are indicated in the figures).

2.2. Basic neuromotor tests Cage activity was recorded in 20 cm × 30 cm transparent cages, placed between three IR beams. Total number of beam crossings was recorded during 23 h. Grip strength was measured using a T-shaped bar connected to a digital dynamometer (Ugo Basile, Comerio, Italy). Mice were placed in such a way that they grabbed the bar spontaneously, and were softly pulled backwards by the tail until they released their grip. Ten such readouts were recorded. Motor coordination and equilibrium were tested using an accelerating rotarod (MED Associates

2.5. Operant conditioning and response suppression In preparation to and throughout the conditional emotional response (CER) test, mice were placed on a quantitative food restriction schedule in such a way that their weight was approximately 80–90% of its initial value. Instrumental learning, and CER acquisition and extinction were tested in eight automated operant chambers (Coulbourn Instruments, Allentown, PA). Mice were first trained during daily 30-min trials to learn to use a nosepoke device to obtain food pellets (Noyes precision pellets; Research Diets, New Brunswick, NJ). Mice received food as a reward during all trials, but the reinforcement schedule was gradually changed during the course of the experiment. Rate of nosepoking during each trial was recorded with Graphic State 3.0 software (Coulbourn Instruments, Allentown, PA). Training started with three continuous reinforcement trials (CFR, every nosepoke was rewarded), followed by five fixed-ratio (FR) trials: one FR3, one FR5 and three FR10 (i.e., 3, 5 and 10 nosepokes for a single reward, respectively). Training ended with two-variable ratio 10 trials (VR10, nosepokes were rewarded on average every 10 nosepokes) and three-variable interval 30 trials (VI30, nosepokes were reinforced on average every 30 s). Mice went through a shorter reinforcement schedule at 1 year of age, that consisted of two CFR, one FR5, two FR10, two VR10 and three VI30 trials. Subsequently, eight CER acquisition trials, each consisting of a 20-s auditory cue followed by a 0.2-mA shock, were presented with a variable 3-min intertrial interval, while nosepoking continued to be reinforced on a VI30 schedule. During the final 21 extinction trails, auditory signals were no longer accompanied by shocks, but nosepoking continued to be reinforced on a VI30 schedule. Rates of nosepoking during the presentation of the auditory cues were compared with nosepoking during intertone intervals, and suppression ratios (SR) were calculated as SR = RR-cue/(RR-cue + RR-int) with RR-cue and RR-int representing mean response rates (per minute) in the presence vs. absence of the auditory cue. An SR of 0.5 thus means that responses were unsuppressed, whereas a value of 0 indicates complete suppression.

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Table 1 Variables defined and included for gait analysis Variable

Description

Total time Total distance Total step cycles Front base Hind base Front/hind distance Stride length

Total time a mouse spent walking Total distance a mouse travelled during a recording Number of step cycles during a recording (one step cycle = one step by all paws, such that a step from one paw is 0.25 cycle) Distance between the contralateral front paw prints Distance between the contralateral hind paw prints Distance between the front paw print and the subsequent hind paw print in a step cycle Distance between unilateral front or hind prints

2.6. Open-field gait analysis

2.7. Alcian blue staining

Mice were placed for 5 min in a transparent 53.0 cm × 34.5 cm × 26.0 cm (length × width × height) plexi-glass open field. Paw placement was recorded with a USB webcam placed 80 cm underneath the plexi-glass cage. To provide sufficient illumination, two fluorescent tubes were placed laterally to the experimental setup at a distance of 23 cm. The webcam captured images with a resolution of 640 pixels × 480 pixels at a frequency of 30 Hz. The position of each of the paws was measured for gait reconstruction. An algorithm was developed in Matlab (version XY; MathWorks Inc., Natick, Massachusetts, USA) for analyzing the acquired images. Several parameters were extracted from these video data (described in Table 1 and illustrated in Fig. 1). First, the number of ambulatory episodes made by each mouse was counted. During these separate sequences, parameters were recorded that could be summed or averaged to summarize the gait characteristics of each tested mouse (several mice that did not display useful ambulatory episodes during the recording had to be excluded from analysis).

Six- and 12-month-old ASA−/− , ASA+/− and ASA+/+ mice were anaesthetized with tribromoethanol and killed by transcardial vascular perfusion with phosphate-buffered saline followed by Bouin’s fixative (diluted 1:4 in phosphatebuffered saline [54]). Brains were removed, postfixed in Bouin’s fixative, and cryoprotected in 20% (w/v) sucrose in phosphate-buffered saline. Cryosections (16 ␮m) were cut in a cryostat and stored at −60 ◦ C. Histochemical staining of sulfolipid storage material was done with minor modifications as described [46]. Slides were equilibrated in 0.025 M sodium acetate (pH 5.7), containing 0.3 M MgCl2 and 2.5% glutaraldehyde and stained in the same buffer containing 0.025% alcian blue 8GX (Sigma, Taufkirchen, Germany). Thereafter, slides were rinsed thoroughly in 0.025 M sodium acetate (pH 5.7), containing 0.3 M MgCl2 and 2.5% glutaraldehyde and mounted with Kaiser’s gelatine (Merck, Darmstadt, Germany). In order to be able to detect low amounts of storage material, sections were not counter-stained.

2.8. Statistics Data are presented as mean and standard error of the mean (S.E.M.). Longitudinal comparisons were carried out using analysis of variance (ANOVA) procedures with Fisher LSD tests for post-hoc analyses. The significance threshold was set at α = 0.05. Possible gender effects were controlled for in view of their potential interaction with behavioural test results. Gender differences were occasionally found, but since they did not influence the conclusions of this paper, they were not reported. A genotype-dependent correlation analysis was performed on the gathered data. Correlations within and between tests were calculated with an extra focus on gait parameters. Differences between correlation coefficients of ASA−/− and ASA+/− mice were evaluated using Fisher’s Z-tests.

3. Results 3.1. Sulfolipid storage in ASA−/− vs. ASA+/− mice

Fig. 1. Different gait parameters were recorded with a ventrally placed camera during free exploration in an open-field arena. A typical footprint pattern in a control mouse illustrates the different measures that could be extracted from such a recording.

All experiments in this study were done with heterozygous ASA+/− mice as controls for efficient littermate breeding. We therefore reexamined whether ASA+/− mice displayed any sulfolipid storage. As reported previously [54], sulfolipid storage was clearly present throughout the brain in 6-month-old ASA−/− mice (not shown), and increased with age. Alcian blue-reactive storage material was prominent in cerebellum of 12-monthold ASA−/− mice, especially in white matter (Fig. 2A), but it was undetectable in ASA+/− cerebellum (Fig. 2B). Alcian bluereactive material was even undetectable in brain of 23-month-old ASA+/− mice (data not shown). As an example, age-dependent increase of storage is illustrated in cerebellum (Fig. 3A and B), and corpus callosum (Fig. 3E and F) of ASA−/− mice.

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the centre (F1,56 = 6.8, p < 0.05), a larger amount of time spent in the centre (F1,56 = 9.3, p < 0.01), a lower latency to the first centre-entry (F1,56 = 5.7, p < 0.05), and a higher number of centre entries (F1,56 = 9.3, p < 0.01). Elevated plus maze recordings (Fig. 6) showed that ASA−/− mice spent relatively more time in the open arms and had relatively more open arm entries (F1,56 = 20, p < 0.001 and F1,56 = 240, p < 0.001, respectively). These effects were not influenced by age. 3.3. Passive avoidance learning, operant conditioning, and operant response suppression During the training phase of the passive avoidance task (Fig. 7A), ASA−/− mice displayed longer step-through latencies than ASA+/− mice (F1,70 = 4.2, p < 0.05). No differences were found in the testing phase. Step-through latency during testing was significantly higher than during training (F1,70 = 179, p < 0.001), indicating successful learning in both genotypes. At the age of 6 months (Fig. 7B), there was a significant interaction between trial and genotype on nosepoke rates during the different phases of the operant conditioning (F13,25 = 2.1, p < 0.05). Nosepoke rates were initially similar in both genotype groups, but rates of ASA−/− mice progressively fell behind those of controls during the VR10 and VI30 schedules. During CER acquisition and extinction, however, no differences were found between genotypes in nosepoke rates or suppression ratios (not shown in figure). At 1 year of age, no genotype differences were found in any phase of the procedure. Fig. 2. Photomicrographs of alcian blue-stained cryosections that illustrate sulfolipid storage in cerebellum of 12-month-old ASA−/− mice (A), but not in ASA+/− mice (B). In ASA−/− mice, alcian blue-stained storage material is prominent and almost exclusively situated in white matter. No alcian blue labeling was detected in sections from ASA+/− mice. Scale bar, 250 ␮m.

Conversely, storage material was undetectable in comparable sections of ASA+/+ and ASA+/− brains (Fig. 3C, D, G and H). 3.2. Basic neuromotor and exploratory tests There was no difference between genotypes in their cage activity level or grip strength (data not shown). In the rotarod test (Fig. 4), there was a significant interaction between age and genotype (F1,56 = 4.2, p < 0.05). ASA+/− mice performed consistently better than ASA−/− mice, but only significantly so at 1 year of age (Fisher LSD, p < 0.01). Older mice were generally more active (F1,56 = 4.7, p < 0.05) and displayed a stronger grip (F1,45 = 4.8, p < 0.05). In the open-field test (Fig. 5), ASA−/− mice made less corner entries (F1,56 = 120, p < 0.01). This effect was not influenced by age, but older mice generally spent a larger percentage of time in the centre (F1,56 = 4.5, p < 0.05). Older mice also displayed an increased total path length (F1,56 = 70, p < 0.05). There was no difference between genotypes regarding social exploration (data not shown). Older mice had a larger total path length (F1,56 = 5.4, p < 0.05), a higher relative path length in

3.4. Open-field gait analysis Gait was analysed during ambulatory episodes that were recorded while the mice were freely exploring an adapted open-field arena. Because 1 year old ASA−/− mice displayed much less ambulatory episodes in this environment compared to ASA+/− mice, only data acquired from 6-month-old mice are reported here (also 5 ASA+/− mice and 13 ASA−/− mice had to be excluded from final analysis because of a lack of useful ambulatory episodes). Velocity, distance and time moved, number of ambulatory episodes, and total number of recorded step cycles were not different between the 6-month-old groups (i.e., no overt signs of ataxia could be observed in these ASA−/− mice). However, detailed inspection showed a distinctive gait pattern in the ASA−/− mice (Fig. 5). Specifically, ASA−/− mice showed a larger maximum hind base (HB) width (F1,56 = 7.8, p < 0.01; Fig. 8A), but no difference was found between genotypes in the distance between the front paws (front base width, FB). ASA−/− mice also displayed decreased mean front/hind distance (FHD; F1,56 = 5. 8, p < 0.05; Fig. 8B), and a trend towards reduced maximum right FHD (F1,56 = 3.5, p = 0.067). On the left side, there was only a trend towards decreased mean FHD (F1,56 = 3.7, p = 0.061). Finally, ASA−/− mice took longer steps with all paws (Fig. 8C). This increased stride length was not observed at 12 months, but the number of ambulatory episodes was too small at this age to allow reliable gait measurements.

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Fig. 3. Cryosections from mouse brains of the indicated genotypes at 6 and 12 months of age that were stained with alcian blue. Magnifications of equivalent parts of cerebellar white matter (A–D) and corpus callosum (E–H) are shown. Low amounts of storage material were detected in cerebellum (A) and corpus callosum (E) of 6-month-old ASA−/− mice (a few storage bodies are indicated by arrowheads), but storage was more pronounced at 12 months of age (B and F). In contrast, alcian blue-reactive storage material was undetectable in 12-month-old ASA+/+ (C and G) and ASA+/− mice (D and H). Scale bar, 50 ␮m.

Intratask correlation analysis of the gait parameters revealed lower correlations between obviously related parameters in ASA−/− mice compared to ASA+/− mice. A first example of these considerably lower correlations in ASA−/− mice is found in the different FHD measures (Table 2). In contrast to the massive congruence of the mean FHDs in ASA+/− mice (r = 0.88, p < 0.001), they are not even

significantly correlated in ASA−/− mice (r = 0.35, p = 0.10; difference between these correlations: p < 0.01, Fisher’s Z). ASA+/− mice also had a higher correlation between (maximum) right FHD and (mean) left FHD (Z = 2.1, p < 0.05). More modestly, correlations between stride length measures showed a comparable lower correlation in ASA−/− mice (not shown).

Fig. 4. Difference in rotarod performance between 6- and 12-month-old heterozygous ASA+/− (black dots) and ASA−/− (white dots) mice. Accelerated rotarod performance was expressed as the average latency to falling off the rotarod during four trials. There was no significant difference between genotypes at 6 months of age, whereas 12-month-old ASA−/− mice displayed impaired rotarod performance. Data are mean values with S.E.M.s, asterisks indicate significance of differences between heterozygote and knockout values: * p < 0.05, ** p < 0.01 and *** p < 0.001.

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Fig. 5. Difference in open-field performance between 6- and 12-month-old heterozygous ASA+/− (black bars) and ASA−/− (grey bars) mice. While there was no significant difference in total path length during open-field exploration (a), the number of corner crossings was decreased in ASA−/− mice at both ages (b). Data are mean values with S.E.M.s, asterisks indicate significance of differences between heterozygote and knockout values: * p < 0.05, ** p < 0.01 and *** p < 0.001.

Fig. 6. Difference in elevated plus maze performance between 6- and 12-month-old heterozygous ASA+/− (black bars) and ASA−/− (grey bars) mice. Elevated plus maze exploration indicated increased open arm activity in the ASA−/− mice (a and b). At both ages, ASA−/− mice spent more time in the open arms than heterozygotes (a), and showed more activity counts (beam crossings) in the open arms (b). Data are mean values with S.E.M.s, asterisks indicate significance of differences between heterozygote and knockout values: * p < 0.05, ** p < 0.01 and *** p < 0.001.

Table 3 summarizes the genotype differences in the correlation of the distance between the front paws (FB) and the distance between the hind paws (HB). ASA+/− mice showed a positive correlation between mean FB and mean HB (r = 0.54, p < 0.01), whereas this correlation was actually negative in ASA−/− mice (r = −0.37; Z = 3.1, p < 0.01). The print vector lines provide a graphic illustration of this striking discrepancy (Fig. 9). Correlations between mean and maximum base widths followed Table 2 Intercorrelations of different measures of FHD LFHD max

RFHD max LFHD mean RFHD mean

a similar genotype-dependent pattern and differed significantly (all p < 0.05, Fisher’s Z). 3.5. Intertask correlations Intertask correlation showed a negative correlation between total number of runs, total running time and distance, and total number of step cycles in the open-field gait analysis, and the Table 3 Intercorrelations of different measures of base widths

RFHD max

LFHD mean

ASA+/−

ASA−/−

ASA+/−

ASA−/−

ASA+/−

ASA−/−

0.67*** 0.72*** 0.56**

0.61** 0.41* 0.21

– 0.66*** 0.82***

– 0.14 0.58**

– – 0.88***

– – 0.35

Asterisks indicate significance: * p < 0.05, ** p < 0.01 and *** p < 0.001. Abbreviations: LFHD, left front/hind distance; RFHD, right front/hind distance.

FB max

HB max FB mean HB mean

HB max

FB mean

ASA+/−

ASA−/−

ASA+/−

ASA−/−

ASA+/−

ASA−/−

0.56** 0.49* 0.20

0.05 0.72*** −0.45*

– 0.60** 0.81***

– −0.17 0.75***

– – 0.54**

– – −0.37

Asterisks indicate significance: * p < 0.05, ** p < 0.01 and *** p < 0.001. Abbreviations: FB, front base; HB, hind base.

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Fig. 7. Aversive and appetitive conditioning in 6-month-old ASA−/− and heterozygous ASA+/− mice (black and grey bars, respectively). Step-through latencies were recorded during training and testing trials of the passive avoidance test (a). ASA−/− mice had longer step-through latencies than heterozygotes during training, but no significant difference occurred during testing. Nosepoke rates were measured during different reinforcement schedules in appetitive operant conditioning (b). ASA−/− mice displayed progressively less nosepokes than heterozygotes during the different phases of the procedure (significant interaction between trial and genotype on nosepoke rates during the different phases: p < 0.05). Data are means with S.E.M.s. Abbreviations: CRF, continuous reinforcement; FR, fixed-ratio reinforcement; VR10, variable ratio reinforcement; VI30, variable interval 30 s.

latency to enter the centre in the open-field test in both genotypes. There was especially much congruence between open-field gait analysis and social exploration measures. The total number of counts in the elevated plus maze was positively correlated with each of the gait parameters as well. Several intertask correlations were different between the genotypes. Cage activity was correlated with front and hind base measures in ASA+/− mice, but not in ASA−/− mice (Table 4). Also, larger FHD correlated with worse rotarod performance in ASA+/− mice, but not in ASA−/− mice. Mean FB was positively correlated with most open-field measures in ASA+/− mice, but none of these correlations was significant in ASA−/− mice (even oppositely directed; all p < 0.05, Fisher’s Z). HB was positively correlated with elevated plus maze measures in ASA+/− mice, but not in ASA−/− mice (p < 0.01, Fisher’s Z). Correlation analysis in 6-month-old mice revealed highly significant associations between open-field measures, both in ASA−/− and ASA+/− mice. Open-field path length was cor-

related negatively with rotarod performance in ASA−/− mice (r = −0.41; p < 0.05), but not in ASA+/− mice. Conversely, rotarod performance correlated positively with the number of corner entries in ASA+/− mice (r = 0.60), but not in ASA−/− mice (Z = 2.3, p < 0.05). ASA−/− mice with a higher cage activity level ambulated more in the open field (r = 0.37), whereas active ASA+/− mice ambulated less (r = −0.33; Z = −2.3, p < 0.05). At 6 months, percentage of time spent and relative number of counts in the open arms of the elevated plus maze correlated negatively with rotarod performance (r = −0.37, p < 0.05 and r = −0.32, p < 0.05) Step-through latencies during passive avoidance training and testing did not intercorrelate significantly. In ASA+/− mice, step-through latency during training correlated positively with the percentage of time spent in the open arms of the elevated plus maze (r = 0.49, p < 0.05), and with ambulatory measures in the open-field gait analysis (all p < 0.05). Besides some significant intratask correlations, conditioning and CER variables were

Table 4 Correlations of different neuromotor tasks and gait parameters FB max

Activity Grip

HB max

HB mean

ASA−/−

ASA+/−

ASA−/−

ASA+/−

ASA−/−

ASA+/−

ASA−/−

0.54** −0.18

0.16 −0.25

0.67*** −0.15

0.07 0.35

0.45* −0.15

0.05 −0.50*

0.41 −0.07

0.07 0.36

LFHD max

Rotarod

FB mean

ASA+/−

RFHD max

LFHD mean

RFHD mean

ASA+/−

ASA−/−

ASA+/−

ASA−/−

ASA+/−

ASA−/−

ASA+/−

ASA−/−

−0.42

−0.15

−0.21

−0.19

−0.54*

−0.31

−0.42

−0.23

Asterisks indicate significance: * p < 0.05, ** p < 0.01 and *** p < 0.001. Abbreviations: FB, front base; HB, hind base; LFHD, left front/hind distance; RFHD, right front/hind distance.

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Fig. 8. Altered gait parameters in 6-month-old ASA−/− mice (grey bars) compared to heterozygous ASA+/− mice (black bars). Measurements of the distance between front paws and hind paws during locomotion indicated decreased maximum hind base in ASA−/− mice (a). Distances between the paw prints on the left and right side, respectively, demonstrated a smaller mean front/hind distance between the right prints (b). However, gait alteration was most obvious in mean and maximum stride length measurements (c). ASA−/− mice showed consistently higher mean and maximum stride lengths compared to heterozygous mice. Data are means with S.E.M., asterisks indicate significance of differences between heterozygote and knockout values: * p < 0.05, ** p < 0.01 and *** p < 0.001. Abbreviations: mn, mean; mx, maximum; FB, front base; HB, hind base; LFHD, left front/hind distance; RFHD, right front/hind distance; LFS, left front stride; RFS, right front stride; LHS, left hind stride; RHS, right hind stride.

hardly associated with other measures. For operant responding variables, this was partially due to the low number of subjects in each of the genotype groups. Total group correlations revealed a strong tendency towards a negative correlation between rotarod performance and fixed-ratio nosepoke rate (r = −0.43; p = 0.05) and a positive correlation between total elevated plus maze activity and VI30 nosepoke rate (r = 0.54; p < 0.05). 4. Discussion Like for several other lysosomal storage disorders [51], a murine MLD model was generated using gene targeting techniques [25]. Although ASA-deficient mice have been used in pathophysiological and therapeutic experiments, additional evaluation criteria would be of great importance, especially in view of recent advances in MLD therapy research [4,31,37]. ASA−/− mice show an age-dependent increase in sulfolipid

storage, which confirms previous reports using histochemical staining with alcian blue [54], and thin layer chromatography [56]. In contrast, storage material was undetectable in ASA+/− mice at 6, 12 and even 23 months of age. We therefore conclude that ASA+/− mice did not accumulate sulfatide in the brain, and can be used as controls to study the effects of sulfolipid storage. Notably, this is in agreement with the earlier suggestion that a 20–50% reduction in lysosomal enzyme activity, which is typically found in heterozygotes, does not influence substrate turnover rate [29]. The present study proposes a combination of behavioural measures that are sensitive to the early effects of sulfolipid storage in ASA−/− mice in comparison to ASA+/− littermates. It confirms and extends our previous observations of MLD-like changes in independent breeding lines that mostly occurred beyond the first year of age [12,13,35,38]. In contrast to previous findings [13], we did not find evidence of hyperactivity in the

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Fig. 9. Unilateral print vectors show differences in the relative positions of front and hind prints in ASA−/− mice (right, n = 8) and ASA+/− heterozygotes (left, n = 8). A few typical examples were shown to illustrate these differences more clearly. Vector lines connect front and hind paw prints. Dotted lines represent the group means, whereas solid lines correspond to individual recordings. The length of the lines indicates the total mean stride length (clearly longer in ASA−/− mice). Notably, the front base obviously correlates strongly with the hind base in ASA+/− heterozygotes (r = 0.54), resulting in an approximately parallel set of print vectors, whereas in knockouts, there was actually a reversed relationship (r = −0.37).

present ASA−/− groups. This could be attributed to differences in genetic background of the tested mice and/or the controlled use of littermates in the present study. Like many other forms of behaviour, home cage activity has proven to be (sub)strain dependent [15,52], and different strains may express a genetic defect in a different way [49]. Progressive neuromotor disabilities in ASA−/− mice are reminiscent of the pathognomonic gait disturbances and ataxia in clinical MLD [12]. To date, ataxia in ASA-deficient mice has been mainly assessed with the rotarod test [13]. Although this is a standard mouse test for motor performance evaluation [11], the present study also shows that it lacks the ability to detect early signs of motor dysfunction. Presently, we report direct parameters of uncoordinated movement in the MLD model. Since detailed gait analysis proved its usefulness in the early detection of motor changes in an amyotrophic lateral sclerosis (ALS) model [55], we employed a novel method for sensitive gait analysis in the open field using ventrally captured images. Previous gait analysis in these mice, using the classic paper-andink runway test [8], did show decreased stride length in MLD mice beyond the first year of age [12,38]. We observed decreased ambulation in 1 year old mutants as well, but the present method revealed differences in several static gait parameters at a much earlier age. Stride length was actually increased in 6-monthold ASA-deficient mice, but it should be noted that decreased [48] as well as increased [23] stride lengths have been reported in ataxic mice. Our present observations seem to indicate that increased stride length might be an early sign of impairment in these ASA-deficient mice, which is overshadowed by profound deterioration at a later age.

ASA−/− mice also exhibited decreased front/hind distance values, which were not found in other motor impaired mice [7,14,32], but these measures might be less reliable in this exploration-based procedure that does not limit swerving. More significantly, ASA-deficient mice showed increased maximum distance between the hind paws (hind base width), which is an established characteristic of ataxia in mice [23,28,40]. Most notably, correlation analysis revealed less harmonious gait dynamics in ASA−/− mice compared to ASA+/− mice. Little congruity was found in ASA−/− mice between front/hind distances and individual stride lengths, while these variables are expected to be closely related. A striking mismatch also occurred between the placement of front and hind paws. In ASA+/− mice, the front interpaw distance clearly accorded with the hind interpaw distance, whereas the inverted relationship between these measures in ASA−/− mice may signal early motor incoordination in these animals. Surprisingly, phenotypic gait correlations have not been previously used to distinguish between murine genotypes. Metten et al. [39] did calculate correlations between ataxia rating scales, but did not examine the possibility of genetic differences in correlation coefficients. The present tests did not suggest any neurocognitive defects in 6-month-old ASA−/− mice in passive avoidance learning, or CER acquisition or extinction. Also in a previous study [13], we did not observe any deficit in passive avoidance at 6 months of age, and only a slight alteration at the beginning of water maze acquisition (not in the probe trial), which could have been due to neuromotor defects. The present data confirm the lack of pronounced cognitive deficits in ASA−/− mice below the age of 1 year. On the other hand, we have reported reduced performance in passive avoidance and water maze tasks in 1 year old ASA−/− mice [13]. However, the present study failed to demonstrate differences between ASA+/− and ASA−/− mice in CER acquisition or extinction at 1 year of age. Several plausible explanations could be given for the lack of any deficit in this particular form of learning in the present study, but it is not unlikely that different forms of learning are differently influenced by brain sulfatide accumulation. Further study may provide more detail about which cognitive competences are preserved and which are affected in ASA−/− mice, and about the time course of the changes. However, several behavioural alterations indicated that ASA deficiency leads to emotional dulling manifested as reduced proactive anxiety and anhedonia. Indeed, ASA−/− mice spent more time in the open arms of the elevated plus maze, made less corner entries in the open-field test (note, however, that this alteration correlated exclusively with rotarod performance, indicating that it might be related to their neuromotor defect), and displayed longer step-through latencies during the training phase of the passive avoidance test. Additionally, 6-monthold ASA−/− mice showed lower response rates in scheduled appetitive conditioning that could not be reduced to neuromotor performance as response rates and neuromotor performance (e.g., rotarod, grip strength) were not positively correlated. On the contrary, mice with pronounced rotarod defects even tended to poke more during fixed-ratio schedules. As well, we observed a dissociation of cage and open-field activity in ASA+/− mice,

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whereas these activities were related in ASA−/− mice. Again, this might reflect the emotional indifference of ASA-deficient mice towards contextual stimuli. Progressive-ratio schedules, where the number of responses needed to retrieve a reinforcer gradually increases with the number of reinforcers already obtained, were demonstrated to be especially sensitive to changes in reinforcement efficacy [1,50]. Rats with lesions of the dorsal noradrenergic bundle display lower bar-pressing rates on a VI30 schedule, but show similar response rates to controls on continuously reinforced and fixed-ratio schedules [41]. Furthermore, dopamine depletion in nucleus accumbens causes disruption of operant responding in high response schedules, while leaving operant behaviour in other schedules relatively intact [43]. In the present study, variable reinforcement schedules might have amplified genotype-dependent differences in the reaction to reward unpredictability. The observation that ASA−/− mice progressively fell behind ASA+/− mice in scheduled operant responding might be attributed to emotional dullness or inattentiveness, rather than to neuromotor defects. This is confirmed by the observation that mice that showed intense exploration in the elevated plus maze did display higher VI30 nosepoke rates. Also, similar alterations of operant responding appear after defects in brain systems that control novelty responses [26], and behavioural activation [10]. Human MLD patients display psychiatric symptoms that are especially pronounced in the juvenile/adult forms [17], and human MLD has been proposed as a naturally occurring model of psychosis [27]. However, we have to be cautious in relating the currently observed deficits in ASA−/− mice to the negative symptoms of schizophrenia. Thalamic changes are associated with neuropsychiatric symptoms [3,44,47], but, although ASA−/− mice do display sulfatide accumulation in laterodorsal and reticular thalamic nuclei [54], thalamic storage is not as prominent in mice as in human MLD patients. Purkinje cell degeneration is not detectable in 6-month-old ASA−/− mice [16], but storage is certainly prominent in the cerebellum and might underlie the emotional changes in ASA−/− mice. Cerebellum is usually implemented in motor coordination, but many studies have demonstrated additional roles in cognition [18,19] and emotion [42,45]. Decreased anxiety was found in staggerer mice that display massive cerebellar degeneration [24], and rats with early midline cerebellar lesions showed anxiolysis in elevated plus maze and insensitivity to environmental distractors during object exploration [6]. Bob´ee et al. [6] concluded that cerebellar alterations are associated with disinhibition of emotional behaviour. They linked the observed phenomena to autistic symptoms, which can be part of the human MLD phenotype as well [9]. In conclusion, we were able to detect phenotypic differences between 6-month-old ASA+/− and ASA−/− mice, whereas most previous studies reported robust differences at more advanced ages. Importantly, we uncovered gait alterations in ASA−/− mice using quantitative analysis. Additionally, we found a first indication of possible emotional changes in ASA−/− mice. The observed behavioural discrepancies will be useful for preclinical development and evaluation of therapeutic interventions in ASA-deficient mice.

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