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Increased gait variability in mice with small cerebellar cortex lesions and normal rotarod performance
Behavioural Brain Research 241 (2013) 32–37
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Research report
Increased gait variability in mice with small cerebellar cortex lesions and normal rotarod performance Stijn Stroobants ∗ , Ilse Gantois, Tine Pooters, Rudi D’Hooge Laboratory of Biological Psychology, University of Leuven, Belgium
h i g h l i g h t s
Bilateral lesions of the cerebellar cortex were electrolytically produced in mice. Cerebellar lesioned mice showed no overt signs of ataxia or abnormal ambulation. Accelerating rotarod did not distinguish cerebellar lesioned mice from sham controls. Treadmill gait analysis distinguished cerebellar lesioned mice from sham controls. Cerebellar lesions evoked an increase of gait variability in mice.
a r t i c l e
i n f o
Article history: Received 18 October 2012 Received in revised form 22 November 2012 Accepted 24 November 2012 Available online 3 December 2012 Keywords: Cerebellar-lesioned mice Treadmill gait analysis Rotarod Ataxia Behavioural testing
a b s t r a c t The physiological and pathophysiological role of the cerebellum in neuromotor performance and gait is a prominent research topic in contemporary brain research. However, it has proven difficult to measure subtle neuromotor changes and cerebellar dysfunction in laboratory rodents with some of the common behavioural assays. Rotarod assays and gait analyses have been used extensively as indicators of neuromotor performance, and more specifically, cerebellar function. Standard rotarod procedures fail to reveal subtle motor alterations, whereas automated gait analysis could be more sensitive in this respect. In the present study, we compared detailed treadmill gait analysis to the standard accelerating rotarod assay in its ability to reveal neuromotor alterations in mice with small bilateral lesions in the cerebellar cortex. This small lesion model showed no readily observable signs of ataxia or abnormal activity. In the rotarod test, cerebellar-lesioned mice performed at the level of control animals, and basic gait parameters were not altered. However, cerebellar-lesioned mice did show increased front base-width and hind stride length variability, as well as increased stride length incongruity between different paws. We conclude that small cerebellar lesions lead to increased gait variability as it does in humans with cerebellar dysfunction. Treadmill gait analysis is better suited than accelerating rotarod assays to measure such subtle neuromotor defects. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Different behavioural assays have been described to evaluate neuromotor performance in rodents, most of which involve the ability to balance on a rotating cylinder [1]. The rotarod test has been used for more than 50 years as an easy way to test effects of drugs, brain damage or disease states [2]. Most protocols not only assess motor coordination proper, but various other aspects of neuromotor functioning as well, such as motor learning manifested by improved performance over sequential trials (e.g., [3–5]).
∗ Corresponding author at: Laboratory of Biological Psychology, University of Leuven, Tiensestraat 102, B-3000 Leuven, Belgium. Tel.: +32 16 325639; fax: +32 16 326099. E-mail address: [email protected] (S. Stroobants). 0166-4328/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbr.2012.11.034
However, several authors have questioned the sensitivity of rotarod procedures to detect subtle alterations in neuromotor function, especially in the early stages of progressive disease (e.g., [6,7]). Footprint gait analysis procedures have been used to quantify motor function in rodents for many years as well. Initial manual methods to obtain footprint patterns included dipping or painting the animal’s paws, and having them walk down a paper-covered aisle (for an extensive overview, see [8]). Originally, Rushton et al. [9] smeared the paws with vaseline, which was later replaced by ink ([10,11]), a method still frequently used today (e.g. [12–14]). The need for automated and objective behavioural measurements [15] inspired the development of computerized footprint recording techniques during spontaneous [7,16] or forced locomotion [17,18]. Forced locomotion on a treadmill may mask deficits in movement initiation [19], but the possibility to manipulate ambulatory speed allows experimenters to measure the influence of velocity on gait
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parameters sensitively and precisely [20,21]. Although still less thoroughly validated, gait analysis may therefore be the method of choice for detailed assessment of neuromotor performance [22]. Much more sensitive than any rotarod procedure, automated analysis of walking patterns has documented neuromotor impairment in rodent models of stroke, Huntington’s disease, amyotrophic lateral sclerosis and metachromatic leukodystrophy [7,23,24]. Since rotarod procedures are still widely accepted to assess cerebellum-based performance [25], we presently compare treadmill walking and an accelerating rotarod task in their ability to detect consequences of cerebellar cortex lesions in mice. The electrolytic methods used here resulted in small lesions that did not induce overt signs of ataxia or abnormal ambulatory activity. The cerebellum processes ascending sensory information and descending motor impulses, and projects to structures involved in the execution of movement to control motor coordination and equilibrium [25]. Accordingly, impaired cerebellar function results in gait disturbances and lack of coordination of voluntary movements, and mice with cerebellar defects display alterations in footprint and rotarod measures [21,26–30]. In view of the putative differences in sensitivity between these two methods, we presently hypothesize treadmill gait analysis is actually more likely to detect subtle motor alterations in cerebellar-lesioned mice than rotarod procedures. In addition, we define a subset of sensitive gait parameters that can be used to assess the effects of cerebellar defects in laboratory mice, and relate to clinical neurological signs in humans with cerebellar lesions. 2. Methods 2.1. Animals and surgical procedure A total of 23 adult female C57Bl/6 mice, approximately 2 months of age, were purchased from Elevage Janvier (Le-Genest-Saint-Isle, France) and were group housed (4–7 mice per cage) at standard laboratory conditions (12 h light/dark cycle, constant room temperature and humidity). Behavioural testing took place during the light phase of the cycle. Food and water were available at libitum. All procedures were approved by the ethical research committee of the university in accordance with the Declaration of Helsinki. The animals were randomly assigned into two procedure groups for receiving lesion of the cerebellum (n = 13) or sham surgery (n = 10). Both groups were mixed housed. Animals were anaesthetized by intraperitoneal injection of 5% chloral hydrate (1% of body weight). Mice were positioned on a stereotaxic frame (Narishige scientific instruments, Tokyo, Japan) and bilateral electrolytic lesions were produced by applying a 0.8 mA current for 30 s (Lesion making device 3500, Ugo Basile, Comerio, Italy). Sham animals were submitted to the same surgical procedure but no current was delivered after placement of the electrode (Teflon insulated Tungsten wire, advent research materials, Oxford, UK). Coordinates of lesions (2 lesion sites) were 5.5 mm and 6.5 mm posterior to bregma, 2.0 mm and 2.2 mm lateral to midline, 1.5 mm and 1.5 mm dorso-ventral [31]. The needle was left another 30 s before being slowly withdrawn from the brain. Mice were given a post-surgical 3 week recovery period prior to behavioural testing.
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Fig. 1. Footprint pattern and extracted gait parameters. Front base, distance between the front paws; Hind base, distance between the hind paws; Front/hind distance, distance between the front paw print and the subsequent corresponding hind paw print in a step cycle; Stride length, distance between unilateral front or hind prints.
During these latter trials, mice were placed on a rotating rod that accelerated from 4 to 40 rpm in 5 min, and the time until they dropped from the rod was recorded. Additionally, variability of performance was assessed by calculating the standard deviation of standardised drop latencies for each animal. 2.5. Treadmill gait analysis
For histology, mice were sacrificed after the last behavioural testing day by cervical dislocation. Following decapitation, the brains were stored in 0.15 M phosphate-buffered saline (PBS, pH 7.42). Coronal sections (50 m) were cut on a vibratome (Leica, VT1000 S, Nussloch, Germany) and one out of three sections were mounted onto 2% gelatin (AppliChem, Darmstadt, Germany)-coated slides. Sections were Nissl stained with 5% thionin acetate (Alfa Aesar, Karlsruhe, Germany) and size and location of the lesion were assessed under the microscope.
A standard treadmill set-up was adapted for the purpose of gait analysis. Mice were placed on a transparent treadmill belt and confined to the belt surface via a plexi-glass cuboid (39.5 cm × 7.2 cm × 5.5 cm; video area 13 × 5.2 cm). Following a 30 s habituation trial, mice were tested for 60 s at a constant speed of 22 cm/s. Mice were encouraged to keep pace with the treadmill using an electric grid placed at the end of the treadmill (0.8 mA). There was no difference between the groups in walking efficacy. Ventral mirror-reflected imagery was captured with a USB webcam (resolution 640 × 480 pixels; 30 Hz) mounted 12 cm sideways from the belt. 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 analysing the acquired images, equivalent as described [32,33]. Different basic gait parameters were extracted from these video data (Fig. 1): base-widths (distance between contralateral paw prints), front/hind distances (distance between front paw print and subsequent hind paw print) and stride lengths (distance between subsequent prints of the same paw). Furthermore, standard deviations of each parameter for each individual mouse were calculated as a measure of ataxia-induced gait variability. In addition, accordance of concurrent gait parameters reflecting gait uniformity was evaluated by a group-dependent correlation analysis for related parameters. Between-group comparison of correlation coefficients can become difficult when contrasting small groups or when confounding factors need to be controlled for. Therefore, coefficients of incongruity are also calculated as an alternative measure of gait uniformity which can be determined for each subject individually. These coefficients are defined as the absolute value of the difference between the standard scores or z-scores for the subject on two concurrent parameters. Dissimilar deviations of the group mean for the parameters will result in a higher score for incongruity. E.g., for base-width congruity, the formula would look as follows:
2.3. Cage activity
IC = |Z(FB) − Z(HB)| = |((FB − FBmean )/FBst dev ) − ((HB − HBmean )/HBst dev )|
2.2. Histology
Home cage circadian activity rhythm was analysed in 20 cm vs 30 cm transparent cages which were placed between 3 infrared beams. Total number of beam crossing was recorded during 23 h using 30 min intervals (start 4pm).
With IC = incongruity coefficient; FB = front base; HB = hind base; stdev = standard deviation. 2.6. Statistics
2.4. Rotarod Motor coordination and equilibrium were tested on an accelerating rotarod (MED Associates Inc., St. Albans, Vermont, US). After training at constant speed (4 rpm, 2 min), four 5-min trials were performed with 10-min intertrial intervals.
Data are presented as mean and standard error of the mean (SEM). Group comparisons were carried out using one-way analysis of variance (ANOVA). The significance threshold was set at ˛ = 0.05. Difference between correlation coefficients for concurrent parameters were evaluated using Fisher’s Z tests.
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Fig. 4. Drop latencies during 4 consecutive trials on the accelerating rotarod in sham (filled circles) and cerebellar (empty circles) lesioned mice. Both groups showed significant motor learning (p < 0.001), which was evident by their increasing performance over trials. Although cerebellar-lesioned mice generally performed worse, this difference was not significant. Fig. 2. Representative coronal sections (between −6.9 and −7 mm from Bregma) of cerebellar-lesioned (CEREB, left picture) and sham (SHAM, right picture) animals. Lesion is encircled by a dotted line in left picture.
3.2. Cage activity
3. Results
General activity levels were assessed in a home cage environment. Circadian rhythm including increased activity during the dark cycle was highlighted by a significant effect of Interval (Fig. 3; F = 35; p < 0.001). There were no differences in general activity or temporal patterns of activity.
3.1. Location and size of lesion Fig. 2 illustrates the size and location of the lesion in cerebellar cortex area viewed in a coronal section. Following histological analysis, the mean extent of the lesion along the axis of the brain was determined to be between 5.40 mm and 7.30 mm posterior to bregma for the largest lesion and between 5.50 mm and 6.30 mm posterior to bregma for the smallest lesion. Cerebellar lesions were located between 1.3 mm and 2.5 mm lateral to bregma and 1.5–3 mm ventral to bregma. Lesions in all animals were bilateral and mostly exclusive in cerebellar cortex areas (see dotted line Fig. 2, left panel), including Sim, Crus I and Crus II. Size of lesions differed between animals and between left and right side of the same animal. The cerebellar lesion of six animals included the most lateral part of the vermis (1.3–1.6 mm to bregma). None of the animals contained lesions including the nuclei. No animals were discarded from the lesioned (n = 13) and sham-operated animals (n = 10).
3.3. Rotarod Accelerated rotarod experiments were conducted to assess motor coordination and equilibrium (Fig. 4). Cerebellar- and shamlesioned mice showed successful motor learning across these four test trials (F = 10.4; p < 0.001). Although cerebellar-lesioned mice generally fell down earlier than sham lesioned mice, this difference was not significant (p = 0.115), neither was the genotype × trial interaction. Both groups showed similar levels of performance variability (not shown; p = 0.172). 3.4. Treadmill gait analysis Gait was analysed during step sequences recorded as the animals ran at constant speed on a motor-driven belt. All mice
Fig. 3. Beam crossings per 30 min interval measured in a home cage environment (23 h). Cerebellar-lesioned mice (empty circles) and sham lesioned controls (filled circles) displayed a normal circadian rhythm.
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Fig. 5. (a) Basic gait parameters for sham (black bars) and cerebellar (grey bars) lesioned mice. There is no difference between the groups. (b) Within-mice standard deviations of basic gait parameters for sham (black bars) and cerebellar (grey bars) lesioned mice. Cerebellar-lesioned mice show significantly increased standard deviations for FB and LHS. Abbreviations: 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. Asterisk indicates significant difference between sham group and cerebellar-lesioned group: *p < 0.05.
displayed sufficient ambulatory episodes for gait analysis. There was no difference between the experimental groups concerning base-widths, front/hind distances or stride lengths (Fig. 5a). In contrast, they showed amplified variability of front base (Fig. 5b; F = 5.1; p < 0.05) and hind stride lengths, which reached significance for the left hind paw (F = 5.3; p < 0.05). Correlation analysis of related parameters revealed weaker relationships between stride lengths of different paws in mice with cerebellar lesions (Table I). Several parameter correlations were significantly lower in the experimental group (RFS-LHS: Z = 2.77; p < 0.01–RFS-RHS: Z = 1.67; p < 0.05–LHS-RHS: Z = 2.00; p < 0.05). On the other hand, there was no difference in correlations between base-widths or front/hind distances. Equivalently, cerebellar-lesioned mice showed a significantly increased incongruity coefficient for front vs hind stride lengths (Fig. 6; F = 5.0; p < 0.05) and a tendency for left vs right stride lengths (p = 0.07), but no incongruity of base-widths and front/hind distances. Stride length incongruity in cerebellar-lesioned mice is further illustrated in Fig. 7. Sham lesioned controls generally show evenly distributed stride lengths. In contrast, individual stride lengths of cerebellarlesioned mice show a rather distorted distribution, despite group averages being similar.
study, we compared accelerating rotarod and treadmill gait procedures in mice with bilateral lesions of the cerebellar cortex. A carefully tuned electrolytic technique produced localized bilateral lesions extending less than 2 mm along the anterior-posterior axis in cerebellar cortex. Lateral and medial cerebellar cortical lesions might influence motor coordination and equilibrium in a different way [34,35], but the lesions described here included both lateral and medial segments. Importantly, lesioned mice showed no obvious signs of ataxia, which was corroborated by normal ambulation during 23 h cage activity recordings. Therefore, the possible occurrence of subtle deficits in motor coordination was further examined in this small lesion model for mild cerebellar dysfunction. Although cerebellar defects have been shown to affect rotarod performance (e.g., [36]), the cerebellar-lesioned mice in this study were not significantly impaired in the accelerating rotarod in comparison with sham-lesioned controls. Strong rotarod deficits follow bilateral ablation of the cerebellum in rodents [37–39], but mice in this study showed intact average drop latencies as well as motor learning over sequential trials, and similar performance variability. Furthermore, basic gait parameters (base-widths, front/hind distances, stride lengths) were also unaltered in these mice. Despite this lack of gross neuromotor impairments, mice with small
4. Discussion Although the rotarod assay remains a practical and wellvalidated tool for assessing neuromotor performance in rodents, it may lack the sensitivity to detect subtle motor deficits. Conversely, automated footprint-based gait procedures assess neuromotor performance objectively, precisely and sensitively. In the present Table I Intercorrelations of concurrent gait parameters. Correlation
Sham (n = 10)
Lesion (n = 13)
FB-HB LFHD-RFHD LFS-RFS LFS-LHS LFS-RHS RFS-LHS RFS-RHS LHS-RHS
0.35 0.02 0.80 0.86 0.93 0.97 0.87 0.92
0.30 0.30 0.65 0.60 0.78 0.62** 0.47* 0.54*
Asterisks indicate significant difference between sham group and cerebellarlesioned group: *p < 0.05 and **p < 0.01. Abbreviations: 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.
Fig. 6. Incongruity coefficients of concurrent gait parameters for sham (black bars) and cerebellar (grey bars) lesioned mice. Cerebellar-lesioned mice showed increased incongruity of stride lengths. Abbreviations: FB, front base; HB, hind base; LFHD, left front/hind distance; RFHD, right front/hind distance; FS, front stride; HS, hind stride; LS, left stride; RS, right stride. Asterisk indicates significant difference between sham group and cerebellar-lesioned group: *p < 0.05.
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Fig. 7. Illustration of stride length congruity levels in sham (left panel; n = 10) and cerebellar- (right panel; n = 10) lesioned mice. Scatter plots represent mean stride lengths for individual mice, which are connected with dotted lines. Average group values are indicated by dashed lines in bold. Group averages are similar, but distribution of stride values clearly differentiates cerebellar-lesioned mice and sham lesioned controls. Strong correspondence is evident in sham lesioned mice, in contrast to the scrambled profile in cerebellar-lesioned mice. Abbreviations: LFS, left front stride; RFS, right front stride; LHS, left hind stride; RHS, right hind stride.
cerebellar lesions did show signs of increased gait variability, indicating subtle alterations in limb movement control and coordination. Notably, increased variability in the distance between the forepaws as well as in hind stride length was measured in these animals. Increased base-width and/or stride length variability has been observed in various motor-impaired mice [40–43]. Using correlational and standardisation approaches [7,44], we additionally showed that stride lengths from singular paws were less congruent in cerebellar-lesioned mice, possibly due to deficient interlimb coordination, in contrast to the robust uniformity of these gait features in controls. Notably, incongruity of stride lengths was also observed in LAMAN−/− mice, a murine model of alphamannosidosis showing progressive degeneration of Purkinje cells [45]. Gait analysis is one of few rodent behaviour tests that can be directly translated to human behaviour. In humans, gait variability is a typical feature of cerebellar ataxia [46], which has also been associated with an increased risk of falls (e.g., [47,48]). It is interesting to note that Wuehr et al. [49] examined the occurrence of spatiotemporal gait variability in patients with cerebellar ataxia, and showed increased stride length and stride time variability, which correspond to the increased hind stride variability in our cerebellar-lesioned mice. Base-width variability was unaltered in patients, but elevation of lateral variability in the forepaws of cerebellar-lesioned mice is difficult to relate to changes in bipedal gait of human subjects. Cerebellum is known to play a crucial role in sensorimotor integration processes including the adaptation of locomotor patterns to novel and varying walking conditions [50]. Notably, stride variability was lowest when cerebellar patients moved forward at their preferred walking speed [49]. We also failed to observe atactic features in cerebellar-lesioned mice during spontaneous movement or cage activity. It should be noted that the mechanics of treadmill locomotion differ substantially from normal horizontal locomotion [51,52]. Our mice were video-tracked at a (relatively high) speed of 22 cm/s during forced treadmill locomotion, which might have evoked or amplified gait variability. The present results also suggest that automated gait analysis is a more sensitive method to detect subtle neuromotor effects of cerebellar dysfunction. This finding converges with observations in different models of motor impairment [7,23,24]. Although coordinated paw movements and equilibrium are definitely involved in rotarod performance, the commonly used read-out of drop latency is by all standards a rather crude motor performance measure. The sensitivity of rotarod assays could be enhanced by changes
in the test protocol [53], which often render the test more time consuming [22]. In our experiments we used an established accelerating rotarod protocol [26,54], whereas fixed-speed protocols might be more sensitive ([55], but this was challenged by Pallier et al. [56]). By and large, however, detailed parameters of coordinated movement can be more readily extracted from automated gait analysis. Automated gait analysis appears to be able to uncover subtle changes in motor coordination, which could be valuable in the study of animal models of progressive diseases and their use in time-dependent evaluation of therapeutic strategies. In conclusion, we have compared mice with small cerebellar cortex lesions to sham-lesioned mice using treadmill gait analysis and an accelerating rotarod assay. Although our cerebellar-lesioned mice show no signs of overt ataxia, different parameters extracted from gait analysis distinguish them from control mice, contrary to the rotarod task. Cerebellar-lesioned mice showed an increase of inter- and intra-stride length variability, comparable to findings in genetic models with cerebellar deficits [45]. The neuromotor profile described here could relate to gait variability observed in human patients with cerebellar ataxia. Acknowledgements The authors would like to thank Leen Van Aerschot for excellent technical assistance. Financial support was received from EU FP7 grant ALPHA-MAN and MM Delacroix Foundation. References [1] Bailey KR, Rustay NR, Crawley JN. Behavioral phenotyping of transgenic and knockout mice: practical concerns and potential pitfalls. ILAR Journal 2006;47:124–31. [2] Dunham NW, Miya TS. A note on a simple apparatus for detecting neurological deficit in rats and mice. Journal of the American Pharmaceutical Association (Baltimore) 1957;46:208–9. [3] Gunn RK, Keenan ME, Brown RE. Analysis of sensory, motor and cognitive functions of the coloboma (C3Sn.Cg-Cm/J) mutant mouse. Genes, Brain and Behavior 2011;10:579–88. [4] Truong DT, Venna VR, McCullough LD, Fitch RH. Deficits in auditory, cognitive, and motor processing following reversible middle cerebral artery occlusion in mice. Experimental Neurology 2012;238:114–21. [5] Yokoi F, Dang MT, Yang G, Li J, Doroodchi A, Zhou T, et al. Abnormal nuclear envelope in the cerebellar Purkinje cells and impaired motor learning in DYT11 myoclonus-dystonia mouse models. Behavioural Brain Research 2012;227:12–20. [6] Chang M, Cooper JD, Sleat DE, Cheng SH, Dodge JC, Passini MA, et al. Intraventricular enzyme replacement improves disease phenotypes in a mouse model of late infantile neuronal ceroid lipofuscinosis. Molecular Therapy 2008;16:649–56.
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Research report
Increased gait variability in mice with small cerebellar cortex lesions and normal rotarod performance Stijn Stroobants ∗ , Ilse Gantois, Tine Pooters, Rudi D’Hooge Laboratory of Biological Psychology, University of Leuven, Belgium
h i g h l i g h t s
Bilateral lesions of the cerebellar cortex were electrolytically produced in mice. Cerebellar lesioned mice showed no overt signs of ataxia or abnormal ambulation. Accelerating rotarod did not distinguish cerebellar lesioned mice from sham controls. Treadmill gait analysis distinguished cerebellar lesioned mice from sham controls. Cerebellar lesions evoked an increase of gait variability in mice.
a r t i c l e
i n f o
Article history: Received 18 October 2012 Received in revised form 22 November 2012 Accepted 24 November 2012 Available online 3 December 2012 Keywords: Cerebellar-lesioned mice Treadmill gait analysis Rotarod Ataxia Behavioural testing
a b s t r a c t The physiological and pathophysiological role of the cerebellum in neuromotor performance and gait is a prominent research topic in contemporary brain research. However, it has proven difficult to measure subtle neuromotor changes and cerebellar dysfunction in laboratory rodents with some of the common behavioural assays. Rotarod assays and gait analyses have been used extensively as indicators of neuromotor performance, and more specifically, cerebellar function. Standard rotarod procedures fail to reveal subtle motor alterations, whereas automated gait analysis could be more sensitive in this respect. In the present study, we compared detailed treadmill gait analysis to the standard accelerating rotarod assay in its ability to reveal neuromotor alterations in mice with small bilateral lesions in the cerebellar cortex. This small lesion model showed no readily observable signs of ataxia or abnormal activity. In the rotarod test, cerebellar-lesioned mice performed at the level of control animals, and basic gait parameters were not altered. However, cerebellar-lesioned mice did show increased front base-width and hind stride length variability, as well as increased stride length incongruity between different paws. We conclude that small cerebellar lesions lead to increased gait variability as it does in humans with cerebellar dysfunction. Treadmill gait analysis is better suited than accelerating rotarod assays to measure such subtle neuromotor defects. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Different behavioural assays have been described to evaluate neuromotor performance in rodents, most of which involve the ability to balance on a rotating cylinder [1]. The rotarod test has been used for more than 50 years as an easy way to test effects of drugs, brain damage or disease states [2]. Most protocols not only assess motor coordination proper, but various other aspects of neuromotor functioning as well, such as motor learning manifested by improved performance over sequential trials (e.g., [3–5]).
∗ Corresponding author at: Laboratory of Biological Psychology, University of Leuven, Tiensestraat 102, B-3000 Leuven, Belgium. Tel.: +32 16 325639; fax: +32 16 326099. E-mail address: [email protected] (S. Stroobants). 0166-4328/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbr.2012.11.034
However, several authors have questioned the sensitivity of rotarod procedures to detect subtle alterations in neuromotor function, especially in the early stages of progressive disease (e.g., [6,7]). Footprint gait analysis procedures have been used to quantify motor function in rodents for many years as well. Initial manual methods to obtain footprint patterns included dipping or painting the animal’s paws, and having them walk down a paper-covered aisle (for an extensive overview, see [8]). Originally, Rushton et al. [9] smeared the paws with vaseline, which was later replaced by ink ([10,11]), a method still frequently used today (e.g. [12–14]). The need for automated and objective behavioural measurements [15] inspired the development of computerized footprint recording techniques during spontaneous [7,16] or forced locomotion [17,18]. Forced locomotion on a treadmill may mask deficits in movement initiation [19], but the possibility to manipulate ambulatory speed allows experimenters to measure the influence of velocity on gait
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parameters sensitively and precisely [20,21]. Although still less thoroughly validated, gait analysis may therefore be the method of choice for detailed assessment of neuromotor performance [22]. Much more sensitive than any rotarod procedure, automated analysis of walking patterns has documented neuromotor impairment in rodent models of stroke, Huntington’s disease, amyotrophic lateral sclerosis and metachromatic leukodystrophy [7,23,24]. Since rotarod procedures are still widely accepted to assess cerebellum-based performance [25], we presently compare treadmill walking and an accelerating rotarod task in their ability to detect consequences of cerebellar cortex lesions in mice. The electrolytic methods used here resulted in small lesions that did not induce overt signs of ataxia or abnormal ambulatory activity. The cerebellum processes ascending sensory information and descending motor impulses, and projects to structures involved in the execution of movement to control motor coordination and equilibrium [25]. Accordingly, impaired cerebellar function results in gait disturbances and lack of coordination of voluntary movements, and mice with cerebellar defects display alterations in footprint and rotarod measures [21,26–30]. In view of the putative differences in sensitivity between these two methods, we presently hypothesize treadmill gait analysis is actually more likely to detect subtle motor alterations in cerebellar-lesioned mice than rotarod procedures. In addition, we define a subset of sensitive gait parameters that can be used to assess the effects of cerebellar defects in laboratory mice, and relate to clinical neurological signs in humans with cerebellar lesions. 2. Methods 2.1. Animals and surgical procedure A total of 23 adult female C57Bl/6 mice, approximately 2 months of age, were purchased from Elevage Janvier (Le-Genest-Saint-Isle, France) and were group housed (4–7 mice per cage) at standard laboratory conditions (12 h light/dark cycle, constant room temperature and humidity). Behavioural testing took place during the light phase of the cycle. Food and water were available at libitum. All procedures were approved by the ethical research committee of the university in accordance with the Declaration of Helsinki. The animals were randomly assigned into two procedure groups for receiving lesion of the cerebellum (n = 13) or sham surgery (n = 10). Both groups were mixed housed. Animals were anaesthetized by intraperitoneal injection of 5% chloral hydrate (1% of body weight). Mice were positioned on a stereotaxic frame (Narishige scientific instruments, Tokyo, Japan) and bilateral electrolytic lesions were produced by applying a 0.8 mA current for 30 s (Lesion making device 3500, Ugo Basile, Comerio, Italy). Sham animals were submitted to the same surgical procedure but no current was delivered after placement of the electrode (Teflon insulated Tungsten wire, advent research materials, Oxford, UK). Coordinates of lesions (2 lesion sites) were 5.5 mm and 6.5 mm posterior to bregma, 2.0 mm and 2.2 mm lateral to midline, 1.5 mm and 1.5 mm dorso-ventral [31]. The needle was left another 30 s before being slowly withdrawn from the brain. Mice were given a post-surgical 3 week recovery period prior to behavioural testing.
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Fig. 1. Footprint pattern and extracted gait parameters. Front base, distance between the front paws; Hind base, distance between the hind paws; Front/hind distance, distance between the front paw print and the subsequent corresponding hind paw print in a step cycle; Stride length, distance between unilateral front or hind prints.
During these latter trials, mice were placed on a rotating rod that accelerated from 4 to 40 rpm in 5 min, and the time until they dropped from the rod was recorded. Additionally, variability of performance was assessed by calculating the standard deviation of standardised drop latencies for each animal. 2.5. Treadmill gait analysis
For histology, mice were sacrificed after the last behavioural testing day by cervical dislocation. Following decapitation, the brains were stored in 0.15 M phosphate-buffered saline (PBS, pH 7.42). Coronal sections (50 m) were cut on a vibratome (Leica, VT1000 S, Nussloch, Germany) and one out of three sections were mounted onto 2% gelatin (AppliChem, Darmstadt, Germany)-coated slides. Sections were Nissl stained with 5% thionin acetate (Alfa Aesar, Karlsruhe, Germany) and size and location of the lesion were assessed under the microscope.
A standard treadmill set-up was adapted for the purpose of gait analysis. Mice were placed on a transparent treadmill belt and confined to the belt surface via a plexi-glass cuboid (39.5 cm × 7.2 cm × 5.5 cm; video area 13 × 5.2 cm). Following a 30 s habituation trial, mice were tested for 60 s at a constant speed of 22 cm/s. Mice were encouraged to keep pace with the treadmill using an electric grid placed at the end of the treadmill (0.8 mA). There was no difference between the groups in walking efficacy. Ventral mirror-reflected imagery was captured with a USB webcam (resolution 640 × 480 pixels; 30 Hz) mounted 12 cm sideways from the belt. 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 analysing the acquired images, equivalent as described [32,33]. Different basic gait parameters were extracted from these video data (Fig. 1): base-widths (distance between contralateral paw prints), front/hind distances (distance between front paw print and subsequent hind paw print) and stride lengths (distance between subsequent prints of the same paw). Furthermore, standard deviations of each parameter for each individual mouse were calculated as a measure of ataxia-induced gait variability. In addition, accordance of concurrent gait parameters reflecting gait uniformity was evaluated by a group-dependent correlation analysis for related parameters. Between-group comparison of correlation coefficients can become difficult when contrasting small groups or when confounding factors need to be controlled for. Therefore, coefficients of incongruity are also calculated as an alternative measure of gait uniformity which can be determined for each subject individually. These coefficients are defined as the absolute value of the difference between the standard scores or z-scores for the subject on two concurrent parameters. Dissimilar deviations of the group mean for the parameters will result in a higher score for incongruity. E.g., for base-width congruity, the formula would look as follows:
2.3. Cage activity
IC = |Z(FB) − Z(HB)| = |((FB − FBmean )/FBst dev ) − ((HB − HBmean )/HBst dev )|
2.2. Histology
Home cage circadian activity rhythm was analysed in 20 cm vs 30 cm transparent cages which were placed between 3 infrared beams. Total number of beam crossing was recorded during 23 h using 30 min intervals (start 4pm).
With IC = incongruity coefficient; FB = front base; HB = hind base; stdev = standard deviation. 2.6. Statistics
2.4. Rotarod Motor coordination and equilibrium were tested on an accelerating rotarod (MED Associates Inc., St. Albans, Vermont, US). After training at constant speed (4 rpm, 2 min), four 5-min trials were performed with 10-min intertrial intervals.
Data are presented as mean and standard error of the mean (SEM). Group comparisons were carried out using one-way analysis of variance (ANOVA). The significance threshold was set at ˛ = 0.05. Difference between correlation coefficients for concurrent parameters were evaluated using Fisher’s Z tests.
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Fig. 4. Drop latencies during 4 consecutive trials on the accelerating rotarod in sham (filled circles) and cerebellar (empty circles) lesioned mice. Both groups showed significant motor learning (p < 0.001), which was evident by their increasing performance over trials. Although cerebellar-lesioned mice generally performed worse, this difference was not significant. Fig. 2. Representative coronal sections (between −6.9 and −7 mm from Bregma) of cerebellar-lesioned (CEREB, left picture) and sham (SHAM, right picture) animals. Lesion is encircled by a dotted line in left picture.
3.2. Cage activity
3. Results
General activity levels were assessed in a home cage environment. Circadian rhythm including increased activity during the dark cycle was highlighted by a significant effect of Interval (Fig. 3; F = 35; p < 0.001). There were no differences in general activity or temporal patterns of activity.
3.1. Location and size of lesion Fig. 2 illustrates the size and location of the lesion in cerebellar cortex area viewed in a coronal section. Following histological analysis, the mean extent of the lesion along the axis of the brain was determined to be between 5.40 mm and 7.30 mm posterior to bregma for the largest lesion and between 5.50 mm and 6.30 mm posterior to bregma for the smallest lesion. Cerebellar lesions were located between 1.3 mm and 2.5 mm lateral to bregma and 1.5–3 mm ventral to bregma. Lesions in all animals were bilateral and mostly exclusive in cerebellar cortex areas (see dotted line Fig. 2, left panel), including Sim, Crus I and Crus II. Size of lesions differed between animals and between left and right side of the same animal. The cerebellar lesion of six animals included the most lateral part of the vermis (1.3–1.6 mm to bregma). None of the animals contained lesions including the nuclei. No animals were discarded from the lesioned (n = 13) and sham-operated animals (n = 10).
3.3. Rotarod Accelerated rotarod experiments were conducted to assess motor coordination and equilibrium (Fig. 4). Cerebellar- and shamlesioned mice showed successful motor learning across these four test trials (F = 10.4; p < 0.001). Although cerebellar-lesioned mice generally fell down earlier than sham lesioned mice, this difference was not significant (p = 0.115), neither was the genotype × trial interaction. Both groups showed similar levels of performance variability (not shown; p = 0.172). 3.4. Treadmill gait analysis Gait was analysed during step sequences recorded as the animals ran at constant speed on a motor-driven belt. All mice
Fig. 3. Beam crossings per 30 min interval measured in a home cage environment (23 h). Cerebellar-lesioned mice (empty circles) and sham lesioned controls (filled circles) displayed a normal circadian rhythm.
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Fig. 5. (a) Basic gait parameters for sham (black bars) and cerebellar (grey bars) lesioned mice. There is no difference between the groups. (b) Within-mice standard deviations of basic gait parameters for sham (black bars) and cerebellar (grey bars) lesioned mice. Cerebellar-lesioned mice show significantly increased standard deviations for FB and LHS. Abbreviations: 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. Asterisk indicates significant difference between sham group and cerebellar-lesioned group: *p < 0.05.
displayed sufficient ambulatory episodes for gait analysis. There was no difference between the experimental groups concerning base-widths, front/hind distances or stride lengths (Fig. 5a). In contrast, they showed amplified variability of front base (Fig. 5b; F = 5.1; p < 0.05) and hind stride lengths, which reached significance for the left hind paw (F = 5.3; p < 0.05). Correlation analysis of related parameters revealed weaker relationships between stride lengths of different paws in mice with cerebellar lesions (Table I). Several parameter correlations were significantly lower in the experimental group (RFS-LHS: Z = 2.77; p < 0.01–RFS-RHS: Z = 1.67; p < 0.05–LHS-RHS: Z = 2.00; p < 0.05). On the other hand, there was no difference in correlations between base-widths or front/hind distances. Equivalently, cerebellar-lesioned mice showed a significantly increased incongruity coefficient for front vs hind stride lengths (Fig. 6; F = 5.0; p < 0.05) and a tendency for left vs right stride lengths (p = 0.07), but no incongruity of base-widths and front/hind distances. Stride length incongruity in cerebellar-lesioned mice is further illustrated in Fig. 7. Sham lesioned controls generally show evenly distributed stride lengths. In contrast, individual stride lengths of cerebellarlesioned mice show a rather distorted distribution, despite group averages being similar.
study, we compared accelerating rotarod and treadmill gait procedures in mice with bilateral lesions of the cerebellar cortex. A carefully tuned electrolytic technique produced localized bilateral lesions extending less than 2 mm along the anterior-posterior axis in cerebellar cortex. Lateral and medial cerebellar cortical lesions might influence motor coordination and equilibrium in a different way [34,35], but the lesions described here included both lateral and medial segments. Importantly, lesioned mice showed no obvious signs of ataxia, which was corroborated by normal ambulation during 23 h cage activity recordings. Therefore, the possible occurrence of subtle deficits in motor coordination was further examined in this small lesion model for mild cerebellar dysfunction. Although cerebellar defects have been shown to affect rotarod performance (e.g., [36]), the cerebellar-lesioned mice in this study were not significantly impaired in the accelerating rotarod in comparison with sham-lesioned controls. Strong rotarod deficits follow bilateral ablation of the cerebellum in rodents [37–39], but mice in this study showed intact average drop latencies as well as motor learning over sequential trials, and similar performance variability. Furthermore, basic gait parameters (base-widths, front/hind distances, stride lengths) were also unaltered in these mice. Despite this lack of gross neuromotor impairments, mice with small
4. Discussion Although the rotarod assay remains a practical and wellvalidated tool for assessing neuromotor performance in rodents, it may lack the sensitivity to detect subtle motor deficits. Conversely, automated footprint-based gait procedures assess neuromotor performance objectively, precisely and sensitively. In the present Table I Intercorrelations of concurrent gait parameters. Correlation
Sham (n = 10)
Lesion (n = 13)
FB-HB LFHD-RFHD LFS-RFS LFS-LHS LFS-RHS RFS-LHS RFS-RHS LHS-RHS
0.35 0.02 0.80 0.86 0.93 0.97 0.87 0.92
0.30 0.30 0.65 0.60 0.78 0.62** 0.47* 0.54*
Asterisks indicate significant difference between sham group and cerebellarlesioned group: *p < 0.05 and **p < 0.01. Abbreviations: 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.
Fig. 6. Incongruity coefficients of concurrent gait parameters for sham (black bars) and cerebellar (grey bars) lesioned mice. Cerebellar-lesioned mice showed increased incongruity of stride lengths. Abbreviations: FB, front base; HB, hind base; LFHD, left front/hind distance; RFHD, right front/hind distance; FS, front stride; HS, hind stride; LS, left stride; RS, right stride. Asterisk indicates significant difference between sham group and cerebellar-lesioned group: *p < 0.05.
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Fig. 7. Illustration of stride length congruity levels in sham (left panel; n = 10) and cerebellar- (right panel; n = 10) lesioned mice. Scatter plots represent mean stride lengths for individual mice, which are connected with dotted lines. Average group values are indicated by dashed lines in bold. Group averages are similar, but distribution of stride values clearly differentiates cerebellar-lesioned mice and sham lesioned controls. Strong correspondence is evident in sham lesioned mice, in contrast to the scrambled profile in cerebellar-lesioned mice. Abbreviations: LFS, left front stride; RFS, right front stride; LHS, left hind stride; RHS, right hind stride.
cerebellar lesions did show signs of increased gait variability, indicating subtle alterations in limb movement control and coordination. Notably, increased variability in the distance between the forepaws as well as in hind stride length was measured in these animals. Increased base-width and/or stride length variability has been observed in various motor-impaired mice [40–43]. Using correlational and standardisation approaches [7,44], we additionally showed that stride lengths from singular paws were less congruent in cerebellar-lesioned mice, possibly due to deficient interlimb coordination, in contrast to the robust uniformity of these gait features in controls. Notably, incongruity of stride lengths was also observed in LAMAN−/− mice, a murine model of alphamannosidosis showing progressive degeneration of Purkinje cells [45]. Gait analysis is one of few rodent behaviour tests that can be directly translated to human behaviour. In humans, gait variability is a typical feature of cerebellar ataxia [46], which has also been associated with an increased risk of falls (e.g., [47,48]). It is interesting to note that Wuehr et al. [49] examined the occurrence of spatiotemporal gait variability in patients with cerebellar ataxia, and showed increased stride length and stride time variability, which correspond to the increased hind stride variability in our cerebellar-lesioned mice. Base-width variability was unaltered in patients, but elevation of lateral variability in the forepaws of cerebellar-lesioned mice is difficult to relate to changes in bipedal gait of human subjects. Cerebellum is known to play a crucial role in sensorimotor integration processes including the adaptation of locomotor patterns to novel and varying walking conditions [50]. Notably, stride variability was lowest when cerebellar patients moved forward at their preferred walking speed [49]. We also failed to observe atactic features in cerebellar-lesioned mice during spontaneous movement or cage activity. It should be noted that the mechanics of treadmill locomotion differ substantially from normal horizontal locomotion [51,52]. Our mice were video-tracked at a (relatively high) speed of 22 cm/s during forced treadmill locomotion, which might have evoked or amplified gait variability. The present results also suggest that automated gait analysis is a more sensitive method to detect subtle neuromotor effects of cerebellar dysfunction. This finding converges with observations in different models of motor impairment [7,23,24]. Although coordinated paw movements and equilibrium are definitely involved in rotarod performance, the commonly used read-out of drop latency is by all standards a rather crude motor performance measure. The sensitivity of rotarod assays could be enhanced by changes
in the test protocol [53], which often render the test more time consuming [22]. In our experiments we used an established accelerating rotarod protocol [26,54], whereas fixed-speed protocols might be more sensitive ([55], but this was challenged by Pallier et al. [56]). By and large, however, detailed parameters of coordinated movement can be more readily extracted from automated gait analysis. Automated gait analysis appears to be able to uncover subtle changes in motor coordination, which could be valuable in the study of animal models of progressive diseases and their use in time-dependent evaluation of therapeutic strategies. In conclusion, we have compared mice with small cerebellar cortex lesions to sham-lesioned mice using treadmill gait analysis and an accelerating rotarod assay. Although our cerebellar-lesioned mice show no signs of overt ataxia, different parameters extracted from gait analysis distinguish them from control mice, contrary to the rotarod task. Cerebellar-lesioned mice showed an increase of inter- and intra-stride length variability, comparable to findings in genetic models with cerebellar deficits [45]. The neuromotor profile described here could relate to gait variability observed in human patients with cerebellar ataxia. Acknowledgements The authors would like to thank Leen Van Aerschot for excellent technical assistance. Financial support was received from EU FP7 grant ALPHA-MAN and MM Delacroix Foundation. References [1] Bailey KR, Rustay NR, Crawley JN. Behavioral phenotyping of transgenic and knockout mice: practical concerns and potential pitfalls. ILAR Journal 2006;47:124–31. [2] Dunham NW, Miya TS. A note on a simple apparatus for detecting neurological deficit in rats and mice. Journal of the American Pharmaceutical Association (Baltimore) 1957;46:208–9. [3] Gunn RK, Keenan ME, Brown RE. Analysis of sensory, motor and cognitive functions of the coloboma (C3Sn.Cg-Cm/J) mutant mouse. Genes, Brain and Behavior 2011;10:579–88. [4] Truong DT, Venna VR, McCullough LD, Fitch RH. Deficits in auditory, cognitive, and motor processing following reversible middle cerebral artery occlusion in mice. Experimental Neurology 2012;238:114–21. [5] Yokoi F, Dang MT, Yang G, Li J, Doroodchi A, Zhou T, et al. Abnormal nuclear envelope in the cerebellar Purkinje cells and impaired motor learning in DYT11 myoclonus-dystonia mouse models. Behavioural Brain Research 2012;227:12–20. [6] Chang M, Cooper JD, Sleat DE, Cheng SH, Dodge JC, Passini MA, et al. Intraventricular enzyme replacement improves disease phenotypes in a mouse model of late infantile neuronal ceroid lipofuscinosis. Molecular Therapy 2008;16:649–56.
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