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Shared and specific signatures of locomotor ataxia in mutant mice

  1. Ana S Machado
  2. Hugo G Marques
  3. Diogo F Duarte
  4. Dana M Darmohray
  5. Megan R Carey  Is a corresponding author
  1. Champalimaud Neuroscience Program, Champalimaud Center for the Unknown, Portugal
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Cite this article as: eLife 2020;9:e55356 doi: 10.7554/eLife.55356

Abstract

Several spontaneous mouse mutants with deficits in motor coordination and associated cerebellar neuropathology have been described. Intriguingly, both visible gait alterations and neuroanatomical abnormalities throughout the brain differ across mutants. We previously used the LocoMouse system to quantify specific deficits in locomotor coordination in mildly ataxic Purkinje cell degeneration mice (pcd; Machado et al., 2015). Here, we analyze the locomotor behavior of severely ataxic reeler mutants and compare and contrast it with that of pcd. Despite clearly visible gait differences, direct comparison of locomotor kinematics and linear discriminant analysis reveal a surprisingly similar pattern of impairments in multijoint, interlimb, and whole-body coordination in the two mutants. These findings capture both shared and specific signatures of gait ataxia and provide a quantitative foundation for mapping specific locomotor impairments onto distinct neuropathologies in mice.

Introduction

Visibly ataxic mouse mutants exhibit varying patterns of neuropathology throughout the brain (Cendelin, 2014; Fortier et al., 1987; Goldowitz et al., 1997; Lalonde and Strazielle, 2007; Lalonde and Strazielle, 2019; Mullen et al., 1976; Walter et al., 2006). Although their motor coordination deficits are generally attributed to abnormal cell patterning within the cerebellum (Arshavsky et al., 1983; Orlovsky et al., 1999), these lines have distinct patterning defects within the cerebellum, varying degrees of extracerebellar involvement, and differences in age of onset (Cendelin, 2014; Lalonde and Strazielle, 2019). The nature of the motor deficits exhibited by these mice also varies, and can often be distinguished by trained observers (Berman, 2018; Brooks and Dunnett, 2009; Hoogland et al., 2015; Lalonde and Strazielle, 2019; Schiffmann et al., 1999; Stroobants et al., 2013; Vinueza Veloz et al., 2015). However, analysis of motor coordination is often limited to low dimensional descriptions of limited specificity that fail to distinguish between related behavioral phenotypes (Brooks and Dunnett, 2009; Lalonde and Strazielle, 2019). Analysis of locomotor kinematics can provide higher dimensional readouts of locomotor behavior (Cendelín et al., 2010; Gabriel et al., 2009; Zörner et al., 2010), but can still suffer from a lack of specificity due to an abundance of highly correlated measures that ultimately reflect non-specific features such as changes in walking speed or body size (Batka et al., 2014; Cendelín et al., 2010; Machado et al., 2015). A quantitative understanding of the specific nature of gait ataxia in mutants with well-described abnormalities in circuit architecture could provide important clues into neural mechanisms of motor coordination (Anderson and Perona, 2014; Bastian et al., 1996; Berman, 2018; Brown and de Bivort, 2018; Darmohray et al., 2019; Datta et al., 2019; Kiehn, 2016; Morton and Bastian, 2007; Powell et al., 2015; Sarnaik and Raman, 2018Udo et al., 1980).

We previously used the LocoMouse system (Machado et al., 2015) to analyze the locomotor coordination of mildly ataxic Purkinje cell degeneration (pcd) mice, in which neural degeneration, particularly early in postnatal development, is largely restricted to cerebellar Purkinje cells, effectively disconnecting the output of the cerebellar cortex (Chen et al., 1996; Fernandez-Gonzalez et al., 2002; Le Marec and Lalonde, 1997). We found that locomotor deficits in pcd were restricted to specific aspects of multijoint, interlimb, and whole-body coordination, while the forward trajectories of individual paws were spared (Machado et al., 2015). We further found that the tail movements of pcd mice reflected the passive consequences of limb movement (Machado et al., 2015). However, it remained unclear to what extent these features represented fundamental features of cerebellar ataxia, or were specific to pcd mice.

Reeler mice are a classic ataxic mutant (Cendelin, 2014; Curran and D'Arcangelo, 1998; D'Arcangelo et al., 1999; D'Arcangelo et al., 1995; Falconer, 1951) with an autosomal recessive mutation in the reelin gene, which is important for neural cell migration (Beckers et al., 1994; Hack et al., 2002). Its loss causes several defects, in particular aberrant localization of neurons and failure of neuronal layer formation. Several brain regions are affected, including cerebellum (Hamburgh, 1963; Terashima et al., 1983), hippocampus (Stanfield et al., 1979), neocortex (Mikoshiba et al., 1980), inferior olive (Blatt and Eisenman, 1988) and substantia nigra (Kang et al., 2010). Neuropathology in these mice is particularly striking within the cerebellum, where severe irregularities in cellular localization are also associated with corresponding aberrant synaptic connectivity between cell types, abnormal foliation, and hypoplasia. Although their locomotor kinematics and whole-body coordination have not been reported, homozygous reeler mutants have been described as having a severely ataxic, ‘reeling’ gait, with difficulties in maintaining their hindquarters upright (Cendelin, 2014; Lalonde et al., 2004; Lalonde and Strazielle, 2019). Like most ataxic mutants, reelers also exhibit poor performance in rotarod, stationary beam and water maze tests (Lalonde et al., 2004).

Thus, pcd and reeler mice share grossly abnormal cerebellar circuitry, but exhibit marked differences in synaptic connectivity within the cerebellum and across the brain. We wondered whether these similarities and differences on the anatomical level might be associated with similarly shared and distinct features of motor behavior. Here we analyze the locomotor behavior of reeler mutants and compare it quantitatively to that of the more mildly ataxic pcd mice (Machado et al., 2015). Detailed comparison of locomotor kinematics and linear discriminant analysis reveals both shared and distinct features of gait ataxia in these two mutants. This approach provides a quantitative foundation for mapping specific locomotor impairments onto distinct neuropathologies.

Results

Reeler mice have impaired hindlimb control and exhibit increased variability of movement

Reeler mice exhibited visible and severe gait ataxia when walking on the LocoMouse setup (Video 1). Like pcd mice (Machado et al., 2015), reelers were smaller and walked more slowly than control littermates (Materials and methods; Figure 1D). However, the locomotor phenotypes of reeler and pcd mice were clearly distinguishable by eye, with reeler mice appearing much more severely ataxic than the mildly ataxic pcd mice (Video 1Lalonde and Strazielle, 2007; Lalonde and Strazielle, 2019; Machado et al., 2015).

Intact forward motion of front paws, altered 3D paw trajectories, and impaired hindlimb control in reeler.

(A) Schematic of the LocoMouse setup with two dark boxes, glass corridor, motion sensors, high speed (400fps) camera, and mirror. Mice freely cross the corridor. (B) An example of side and bottom views captured in a single via mirror reflection. Continuous tracks (in x, y, z) for nose, paws and tail segments obtained from LocoMouse tracking are plotted on top of the frame. (C) Sagittal sections of mouse cerebellum from littermate control (left) and a reeler mouse (right) illustrate dramatic cerebellar reorganization in reeler. (D) Histogram of walking speeds for reeler (green N = 7 mice, n = 2439) and littermate controls (grey, N = 12, n = 2515). Walking speed distributions are significantly different, reelers mice walk slower (ind. t-test p=<0.001***). (E–G) Stride length (E), cadence (F, 1/stride duration) and stance duration (G) of the front right (FR) paw vs walking speed for reeler (green) and littermates (grey). For each parameter, thin lines with shadows represent median values ± 25th, 75th percentiles. Thick lines represent the predictions calculated using the equations previously derived from the mixed-effect models described in Machado et al., 2015. No significant difference was observed between littermate controls and reeler mice (main effects: stride length: F1,90=2.16, p=0.14; cadence: F1,90 = 0.7, p=0.4; stance duration: F1,90=2.97, p=0.09). (H) Average instantaneous forward (x) velocity of FR paw (top) and hind right (HR) paw (bottom), normalized to the swing phase. Line thickness represents increasing speed. Reeler (green), size-matched controls (black; N = 11; n = 3412). Reeler mice showed sig. higher avg. swing velocity (F1,104 = 4.59, p=0.03), but no difference in peak inst. velocity (F1,104 = 0.87, p=0.35). Hind paws showed lower peak velocity than size-matched controls (F1,103 = 14.1, p=<0.0001). (I) side-to-side (y)-excursion for FR and HR paws, relative to body midline. There are changes in peak to peak trajectories for both paws (FR: F1,96=197.4, p=<0.0001; HR: F1,103=353.9, p=<0.0001). (J) Average vertical (z) position of FR paw (top) and HR paw (bottom) relative to ground during swing. Reelers mice have larger vertical movement than size-matched controls (FR: F1,96=205.5, p=<0.0001; HR: F1,103=11.9, p=<0.0001). (K) x-y position of four paws relative to the body center during swing for reeler and size-matched controls. There was no significant difference in width of base of support (F1,101=2.4, p=0.12).

Video 1
Visible gait ataxia in reeler and Purkinje cell degeneration mice walking on the LocoMouse setup.

Wild type (top), reeler (middle), and pcd (bottom) mice were recorded walking across the glass corridor of the LocoMouse setup. Side and bottom (via mirror reflection) views were captured by a single high-speed camera at 400fps and are shown here at 50 fps (slowed down 8x). Note the slower walking speeds of both mutants and the visible differences in their locomotor behavior.

We analyzed the locomotor phenotype of reeler mice using the quantitative framework for locomotor coordination that we established previously (Figure 1A–C; Machado et al., 2015). First, the equations we previously generated with mixed-effects linear models (Machado et al., 2015) to predict stride parameters based on walking speed and body size were able to accurately predict the individual stride parameters (including stride length, cadence, stance duration) of reeler mice (Figure 1. E-G), as we had previously shown for pcd (Machado et al., 2015). Moreover, the continuous forward trajectories of reeler front paws were similar to those of size-matched controls, across walking speeds (Figure 1H, top). Notably, the forward trajectories of reeler hind paws exhibited lower forward velocities compared to size and speed-matched controls (Figure 1H, bottom), and they had accompanying increases in swing duration (from size-matched controls = 98.78 ± 6.48 ms to reeler = 114.72 ± 8.21 ms). Comparison of 3D trajectories revealed clear differences between reeler and control mice in both the side-to-side and the vertical paw movements of both front and hind paws (Figure 1I–K). Finally, and perhaps surprisingly given their severe ataxia, reeler mice did not exhibit an increased width of base of support (Figure 1K).

Direct comparison of average paw kinematics in the two mutants reveals remarkable similarities (Figure 2). Both reeler and pcd exhibited altered 3D trajectories of all paws when compared to those of speed and size-matched controls (Figure 2E–G). In particular, the off-axis (side-to-side and vertical) movements of all paws showed nearly identical alterations in the two mutants (Figure 2F,G). In addition, forward hind paw trajectories were profoundly affected in reelers, while more subtle effects were seen on the forward movement of the front paws (Figure 2B–E).

Comparison of Reeler and pcd mice reveals overall similarity of averaged paw trajectories, with additional hind limb impairments and increased variability in reeler.

(A) Histogram of walking speeds, for reeler (green N = 7; n = 2439) and pcd (purple, N = 3; n = 3052; Machado et al., 2015). (B–D) Stride length (B), cadence (C), 1/stride duration) and stance duration (D) vs walking speed for reeler (green) and pcd (purple) mice. For each parameter, the thin lines with shadows represent median values ± 25th, 75th percentiles. Thick lines represent the predictions calculated based on the models derived in Machado et al., 2015. Reeler mice had sig. higher stride lengths (F1,52=5.23, p=0.03). No significant differences were observed between pcd and reeler in cadence: F1,55 = 0.01, p=0.92 or stance duration: F1,52=0.19, p=0.66). (E–G) The differences in averaged trajectories between each mutant size and speed-matched controls are plotted for reeler (green) and pcd (purple). Line thicknesses represent increasing walking speed. (E) The peak instantaneous forward (x) velocity of FR paws (top) was sig. higher in reeler (F1,8=50.23, p=<0.0001). Peak HR paw velocity is lower in reeler (bottom, F1,8 = 6.09, p=<0.0001). (F) Differences in side-to-side (y)-excursion for FR (top) and HR (bottom) paws during swing phase, relative to body midline. No significant difference is observed in peak excursion between pcd and reeler (FR: F1,8=4.81, p=0.06; HR: F1,8 = 0.04, p=0.84). (G) Differences in vertical (z) trajectory of FR paw (top) and HR paw (bottom) during swing phase. No significant difference in peak z was observed between pcd and reeler mice (FR: F1,8=0.91, p=0.37; HR: F1,8 = 1.98, p=0.2). (H,I) Coefficient of variation (CV) for peak forward velocity (H) and vertical displacement (I) for size-matched controls, reeler, and pcd. Hind paw velocity (H, bottom; F1,99=13, p=<0.0001) and both front (I, top; F1,101=45.1, p=<0.0001) and hind (I, bottom; F1,101=73.3, p=<0.0001) paw vertical movements were more variable in reeler.

In contrast to the broad similarities in averaged paw trajectories, there were clear differences in the variability of paw kinematics between reeler and pcd (Figure 2H,I). Despite their ataxia, neither the front nor hind limb trajectories of pcd mice exhibited increased variability (Figure 2H; Machado et al., 2015). Paw movement was generally more variable in reeler, including the forward motion of the hind paws (Figure 2H-bottom) and the vertical movements of both front and hind paws (Figure 2I-top and bottom).

Impaired interlimb coordination and increased front paw support in Reeler

Mice typically walk in a symmetrical trot pattern across a wide range of walking speeds (Figure 3AMachado et al., 2015). The normal pattern of interlimb coordination was markedly disrupted in reeler, due to specific and consistent changes in the phase relationship between front and hind limbs (Figure 3B–C). Remarkably, the alterations in front-hind limb stance phasing in reelers were identical on average to those of pcd mice; in both mutants, hind paw touch downs were delayed relative to their diagonal partners (Figure 3B,C; Machado et al., 2015). In contrast, relative left-right stance phasing of both the front and hind limbs was intact in both reelers and pcd (Figure 3DMachado et al., 2015). Consistent with the increased variability of hind limb movements, the front-hind limb phasing was more variable on average in reeler, but not pcd (size-matched controls = 0.25 ± 0.05%, pcd = 0.29 ± 0.09% F1,82 = 1.02, p=0.32, reeler = 0.48 ± 0.11% F1,82 = 5.79, **p=9.5×10−3), and left-right phasing was not more variable in either mutant (size-matched controls = 0.25 ± 0.06%, pcd = 0.19 ± 0.07% F1,54 = 0.23, p=0.63, reeler = 0.22 ± 0.07%, F1,74 = 0.11, p=0.75).

Impaired front-hind limb coordination and increased front paw support patterns in reeler.

(A–B) Polar plots indicating the phase of the step cycle in which each limb enters stance, aligned to stance onset of FR paw (red). Radial axis represents walking speed. Limbs are color coded according to the inset; large symbols represent averages across animals and small symbols represent individual mice. (A) size-matched control mice (N = 11) and (B) reeler mice (N = 7). (C,D) Relative front-hind (C) and left-right (D) stance phases across walking speeds for pcd (purple), reeler (green)r hases across walking spee(black). Each circle represents one animal. Lines show fit of linear-mixed effects model for each variable. Only front-hind phase is impaired in reeler and pcd mice (front-hind phase: F1,104 = 11.7, p=<0.0001; left-right phase: F1,104 = 0.7, p=0.41). (E) Both pcd and reeler have a higher percentage of 3 paw supports at all speeds (F1,104 = 115.1, p=<0.0001). (F) Non-diagonal 2-paw support configurations are increased in both pcd and reeler (F1,104 = 28.3, p=<0.0001). (G) Only reeler mice show an increase in 2-front paw support configurations (F1,101 = 207, p=<0.0001). (H,I) Average ). n increase in 2-front paw support configurations (th s one (H) and hind (I) paws of pcd (purple), reelers (green) and size-matched controls (black). Reeler mice have a higher % of front double support and lower % of hind double support when compared with size-matched controls (front double support: F1,99 = 71,9, p=<0.0001; hind double support: F1,103 = 27.2, p=<0.0001).

We also observed changes in support patterns (ie, the configuration of paws that are in stance at any given time, Gorska et al., 1998) in reeler mice. At most natural walking speeds, wildtype mice typically have a single diagonal pair of paws on the ground at any given time (Machado et al., 2015). Reeler mice exhibited an increase in 3-paw support patterns (Figure 3E). They also spent more time in unstable support configurations such as non-diagonal 2-paw support (Figure 3F) and 2-front paw supports (Figure 3G). This increased instability was also observed in pcd (Machado et al., 2015) and is consistent with impaired interlimb coordination rather than a simple switch to a different gait pattern (Bellardita and Kiehn, 2015).

Although the alterations in interlimb coordination were similar in reeler and pcd, there were some notable differences between the two mutants. In particular, reeler mice spent more time with both front paws on the ground than both controls and pcd, either as sole supports (Figure 3G), or as part of a 3 or four paw support configuration (Figure 3H). Interestingly, reelers also spent less time with both hind paws on the ground than pcd or control mice (Figure 3I). The ratio of front to hind paw double support was higher in reeler (front/hind = 0.43) than in pcd (front/hind = 0.14) and control mice (front/hind = 0.10). This decreased hind paw double support is likely to be a consequence of the impaired hindlimb control described in Figure 1 and Figure 2.

Impaired whole-body coordination in reeler reflects passive consequences of front paw motion

Like pcd (Machado et al., 2015), reeler mice exhibited large side-to-side oscillations of the tail and nose with respect to the stride cycle (Figure 4A–F, green) when compared to controls (black). Also like pcd, both tail (Figure 4A,C) and nose (Figure 4D,F) movements became increasingly phase-lagged relative to the locomotor cycle at faster speeds.

Figure 4 with 1 supplement see all
Tail and nose movements of reeler mice can be modeled as a passive consequence of the forward movement of the front paws.

(A) Compared to controls (black), reelers (green) display larger averaged side-to-side tail oscillations, and increased phase lags with increased walking speed (from thin to thick lines) (tail amplitude: F1,103 = 24.8, p=<0.0001; tail phase: F1,104 = 59.2, p=<0.0001). (B) Different phase relationships of reeler (green) and pcd (purple) tail oscillations relative to the hind limb stride cycle (walking speed 0.25–0.3 m/s). (C) When aligned to the front limbs, reeler (green) tail phases are very close to those of pcd (purple) aligned to the hind limbs (lines show fits to the data; F1,56 = 1.3, p=0.26). Inset shows the tail trajectories of reelers aligned to the front paws (green) and pcd (purple) aligned to the hind limbs (mid-tail segment for animals walking at 0.25–0.3 m/s). (D) Reelers also show larger nose oscillations and phase-lags that increase with speed increases when compared with controls (amplitude: F1,104 = 5.1, p=0.03 ; phase:F1,104 = 42.1, p=<0.0001). (E) Different phases of reeler nose (green) and pcd (purple), aligned to front paws. (F) The reeler nose (orange) is nearly perfectly out of phase with the base of the tail (green), suggesting oscillation of a single body axis (circles represent data points, solid lines show fits to the data, dashed line shows a prediction of the nose phases with respect to the same body axis as the tail. Inset shows the trajectories of the base of the tail and nose aligned to front limbs. (G) Interpretation of tail and nose movements observed in control (left), reeler (middle) and pcd (right) mice. (H) Average side-to-side (y)-excursion of the body center during strides (F1,99=1072.5, p=<0.0001). (I) Geometric interpretation of the analytical model (see Materials and methods). The forward movement of front limbs (AB) is transformed into lateral oscillations of a body axis (AC). The lateral oscillations of tail and nose are then given by a time delay relative to the movement of the body axis. (J) Phase (relative to front limb oscillation) of nose (orange), base of the tail (dark green), mid-tail segment (intermediate green) and tip of the tail (light green), plotted as a function of walking speed. Circles represent data, lines are the predictions of the analytical model.

We previously showed that the tail and nose movements of pcd mice could be successfully modelled as a passive consequence of hind limb movement (Machado et al., 2015). A model that converted hindlimb oscillation into nose and tail trajectories using solely the geometric relationships between body parts moving with fixed time delays accurately predicted the side-to-side tail and nose movements of pcd mice across walking speeds.

At first glance, the similarities in averaged tail trajectories suggest that the reeler tail and nose movements, like pcd, might also reflect passive consequences of limb movement during locomotion. However, the specific phase relationships of both the tail and nose with respect to the locomotor cycle were dramatically different in reeler compared to pcd (Figure 4B,E). We wondered whether these phase differences could be accounted for by the shift in support patterns towards the front limbs in reelers (Figure 3F–H). To our surprise, simply aligning the reeler tail oscillations to the front limbs, rather than hind limbs (Figure 4C), immediately revealed that the phase relationship between the reeler tail and the front limbs was nearly identical to that of the pcd tail to the hind limbs (Figure 4C). In other words, the tail movements of reeler mice have the same quantitative relationship to front limb movement that the pcd tail had to the hind limbs.

Nose trajectories in reeler were similarly pronounced, but also phase-shifted, compared to those of pcd (Figure 4D,E). Side-to-side nose movements in reeler were almost perfectly out of phase with the base of the tail (Figure 4F), suggesting that both the tail and nose movements of reeler mice oscillate along a single, straight body axis with each stride (Figure 4G). One possible explanation for the differences in relative tail and nose phasing between pcd and reeler (Figure 4E) is that the shift of supports to front paws leads to the loss of a front-limb steering component in reeler (Figure 4G). Consistent with this idea, reelers, but not pcd, exhibited larger side-to-side movements of the body center while walking (Figure 4H, Video 1).

To test the idea that the nose and tail movements of reeler mice might reflect the passive consequence of front, rather than hind, limb movement, we built an analytical model that computed predicted lateral trajectories of the tail and nose directly from the forward-backward oscillations of the front limbs (Figure 4I; Materials and methods). The model and its parameters were analogous to the geometrical model described in Machado et al., 2015, but with a shift to the front limbs and a single body axis for reeler (see Materials and methods). The model output accurately reproduced the phases as well as the actual trajectories of the tail and nose of reeler mice, across a range of walking speeds (Figure 4J and Figure 4—figure supplement 1A–C). Notably, hindlimb-driven models, although optimal for pcd (Figure 4—figure supplement 1H,I), performed less well at predicting reeler tail and nose movements, even allowing for the possibility of substantially longer time delays (Figure 4—figure supplement 1D-F). Similarly, unlike reeler, a single body-axis model was not appropriate for the pcd data.

Thus, in reeler, like pcd, the tail and nose appear to move as a passive consequence of forward limb motion. The altered phasing in the two mutants suggests that the shift in the support patterns towards the front paws in reeler (Figure 3, and presumably resulting from impaired hindlimb control shown in Figure 1) causes the tail and body axis to oscillate as a passive consequence of the front, rather than hind limbs.

Linear discriminant analysis reveals shared and specific features of gait ataxia

The results so far have described a comprehensive set of locomotor features and highlighted similarities and differences between reeler and pcd mice. Finally, we sought an unbiased way to summarize and conceptualize these findings (Brown and de Bivort, 2018; Berman, 2018; Datta et al., 2019). To do this we turned to linear discriminant analysis (LDA; Fisher, 1936; James et al., 2013), as a means to quantitatively distinguish the three groups of mice (wildtype, pcd and reeler; Figure 5).

Figure 5 with 2 supplements see all
Linear discriminant analysis separates ataxic mutants and reveals shared and distinct features of gait ataxia.

Linear discriminant analysis of locomotor kinematics reveals two axes, which separate ataxic mutants from controls (LD1) and from each other (LD2). Each dot represents a single animal walking at a particular speed. Faster speeds are shown with larger marker sizes. Speeds ranged from 0.05 to 0.35 m/s and were binned with a binwidth of 0.05 m/s. Size-matched controls are in grey (N = 11 for all speed bins; n = ~ 3288), reeler in green (N = 7 for 0.05–0.15 m/s; N = 6 for 0.25–0.35 m/s; n = ~ 2387), and pcd in purple (N = 3 for all speed bins except 0.25–0.30 m/s N = 2; n = ~ 3066). The bars along each axis are ranked by the contribution scores (LD coefficients) of each variable to that axis (larger bars indicate higher contributions). Features contributing strongly to LD1 (which accounts for 84% of the total between-group variance) include interlimb and whole-body coordination, as well as off-axis paw trajectories. For LD2 (which accounts for 16% of the between-group variance), they also include variability, front paw supports, and relative phasing of tail/nose movements.

Forty-five variables representing the various features of paw, nose, and tail movements that we measured during locomotion were extracted for this analysis (Figure 5—figure supplement 1). Because this relatively high-dimensional dataset contained many highly correlated variables (Figure 5—figure supplement 2A), we first applied principal component analysis (PCA) to account for inter-variable correlations and avoid overfitting (see Materials and methods, Figure 5—figure supplement 2B–F).

LDA successfully captured meaningful differences in an abstract feature subspace to separate the three genotypes based on their locomotor phenotypes across walking speeds (Figure 5). Unexpectedly, this analysis revealed that two distinct, orthogonal axes effectively separate controls from mutants (LD1), and the two mutants from each other (LD2) (Figure 5). The locomotor features contributing most strongly to these two linear discriminants thus correspond to the shared (LD1) and distinct (LD2) features of ataxia in reeler and pcd mice.

Inspection of the contributions of each gait parameter to the two LDs reveals that LD1, which separates controls from ataxic mutants, is highly influenced by variables representing 3D paw trajectories and interlimb and whole-body coordination (Figure 5, x-axis bar graph). The features contributing most strongly to LD2, which captured the differences in locomotor phenotype between the two mutants, included measurements of hindpaw movement (and subsequent changes in support patterns), variability, and relative phase of tail/nose movements (Figure 5, y-axis bar graph). Most variables relating to the averaged forward motion of individual limbs, which are largely spared in both mutants, do not strongly influence either LD.

The LDA provides a quantitative summary that captures the essential elements of the similarities and differences in locomotor behavior across groups of mice. In so doing, it shows that a high-dimensional set of gait parameters contains a subspace of mixed features in which multiple neuropathologies are represented behaviorally. The results, together with our previous description of the locomotor phenotype of pcd mice (Machado et al., 2015), reveal multijoint, interlimb, and whole-body coordination as shared, and possibly fundamental, features of locomotor ataxia.

Discussion

When assessing motor coordination deficits in mice there is often a tradeoff between specificity and interpretability. For example, approaches such as rotarod testing provide measurable and intuitive low dimensional outputs, but lack specificity. Others, such as the CatWalk system (Gabriel et al., 2009) can provide many detailed measurements of locomotor behavior, but their meaning is not always readily apparent. With LocoMouse we have tried to provide both a comprehensive, quantitative description of locomotor behavior as well as a conceptual framework within which to interpret that high-dimensional data.

Here we analyzed the locomotor behavior of severely ataxic reeler mice and quantitatively compared it with that of Purkinje cell degeneration mutants (Machado et al., 2015). Detailed comparison of locomotor kinematics and linear discriminant analysis revealed both shared and distinct features of gait ataxia in the two mouse lines. Although the generality of the conclusions that can be reached from the analysis of two mutants is of course limited, the approach described here provides a comprehensive and quantitative way to map complex patterns of locomotor features onto partially overlapping neuropathologies. Extending it to additional models and manipulations could enable association of specific movement features with increasingly precise alterations in underlying neural circuits.

Our first finding lies in capturing specific quantitative differences in gait ataxia between pcd and reeler mice. Rather than simple differences in the level of severity in the same set of affected features, the visible gait differences in the two mutants (captured by the second linear discriminant) appear to stem specifically from an increase in movement variability and additional hindlimb involvement (Cendelin, 2014) in reelers. As a likely consequence of those deficits, analysis of support patterns and nose and tail movements suggests that reelers, unlike controls and pcd, use their front paws as their main supports while walking. This difference in support patterns fully accounts for differences in tail movements observed in the two mutants (Figure 4). It could also explain the inability of reelers to walk in a straight line (Video 1, Figure 4H), likely because the front limbs are unable to provide both support and steering control to keep the body moving forward. Thus, while all of these features in combination contribute to the ability of human observers to visually distinguish the overall differences in walking patterns in the two mice, the quantitative LocoMouse analysis is able to distill them down to reveal fundamental underlying alterations in variability and hindlimb control.

Capturing the essential differences in locomotor control between ataxic phenotypes provides a necessary starting point for understanding the contributions of individual circuit elements to this complex, whole-body behavior. In pcd, the main anatomical phenotype is cerebellar, with striking degeneration of Purkinje cells that effectively removes cerebellar cortical input to the cerebellar nuclei. In contrast, reeler mice exhibit aberrant cell localization throughout the brain (Terashima et al., 1983; Stanfield et al., 1979; Mikoshiba et al., 1980; Blatt and Eisenman, 1988; Kang et al., 2010), which could also contribute to their gait abnormalities. Moreover, in reeler, abnormal cell migration during development results in aberrant circuit wiring that alters, rather than removes, Purkinje cell activity (Dupont et al., 1983; Curran and D'Arcangelo, 1998; D'Arcangelo et al., 1995; Lalonde and Strazielle, 2019).

Locomotor differences between the two mutants (captured by LD2 in Figure 5) thus likely represent a combination of specific differences in circuitry within the cerebellum itself plus extracerebellar effects. The differences in variability may be of cerebellar origin (Walter et al., 2006; Medina and Lisberger, 2007). Movement variability is often considered to be a hallmark of cerebellar ataxia, and yet surprisingly, pcd mice were less variable than controls on several movement measures (Machado et al., 2015). We speculate that the opposite effects on variability in the two mutants may arise as a consequence of altered Purkinje cell output in reeler, compared to a lack of Purkinje cell output in pcd (Medina and Lisberger, 2007). Meanwhile, differences in hindlimb involvement, and the resulting reliance on front paw supports (Figure 3G,H), may be extracerebellar, perhaps resulting from cell positioning defects in reeler spinal cord (Yip et al., 2000; Phelps et al., 2002). Future experiments analyzing locomotor phenotypes in additional cerebellar and extracerebellar models will be crucial for establishing mechanistic links between specific features of locomotor behavior and precise alterations in underlying neural circuits.

Despite the more severe behavioral and anatomical phenotype in reeler, we found the overall pattern of affected locomotor features in pcd and reeler to be surprisingly similar. The first linear discriminant pulled out a set of shared impairments in multi-joint, interlimb, and whole-body coordination that may reflect core quantitative features of mouse cerebellar ataxia. We propose that these features comprise the gestalt impression of clearly cerebellar gait phenotypes that trained observers can readily identify, despite substantial variation in specific manifestations across models.

In particular, like pcd (Machado et al., 2015), the tail and nose movements of reelers were also successfully modeled as passive consequences of limb movement, with a shift of supports to the front, rather than hind, paws. We had previously interpreted the pattern of coordination deficits in pcd mice, and particularly the passive tail oscillation, as consistent with the lack of an internal forward model that predicts and compensates for the consequences of movement (Bastian et al., 1996; Ebner and Pasalar, 2008; Ito, 2008; Kennedy et al., 2014; Wolpert et al., 1998). Specifically for locomotor control, a forward model can predict how the movement of one part of the body (e.g., a limb or a paw) will affect the movement of another part (e.g., another limb, or the tail), and inject a compensatory control signal to eliminate those consequences.

The idea that the cerebellum could provide a forward model for motor control is often invoked when considering the role of the cerebellum in motor learning (Ito, 2008; Shadmehr and Krakauer, 2008; Wolpert et al., 1998). Recently, we showed that both pcd and reeler mice were unable to learn to restore gait symmetry on a locomotor learning task that requires predictive control (Darmohray et al., 2019; Morton and Bastian, 2006; Reisman et al., 2005). That finding, together with the passive tail oscillations of both mutants, reinforces the idea that these animals lack the ability, provided by an intact cerebellum, to predict and compensate unintended movements during locomotion. The consistent lack of compensatory predictive mechanisms across mutants and behavioral paradigms suggests that they may represent core features of cerebellar contributions to coordinated locomotion. Extending the current findings and applying a similar approach across a broader range of circuit manipulations could become a key element in understanding how various neural components work together to control complex, whole-body behaviors.

Materials and methods

Animals

All procedures were reviewed and performed in accordance with the Champalimaud Centre for the Unknown Ethics Committee guidelines and approved by the Portuguese Direcção Geral de Veterinária (Ref. No. 0421/000/000/2015).

Heterozygous reeler (Relnrl) mice on a C57BL7/6 background were obtained from Jackson labs (#000235 B6C3Fe a/a-Relnrl/J). Data were collected from homozygous reeler mice (n = 9392 strides; N = 7 mice; two females; five males; 8–18 g; 35–52 days old; average weight = 13.57 ± 3.3 g) and their littermates (n = 9524 strides; N = 12 mice; eight females, four males; 12–25 g; 36–52 days old; average weight = 18.67 ± 3.5 g). Data from Purkinje cell degeneration mice (#0537 B6.BRAgtpbp1pcd/J) and additional wildtype C57BL7/6 (Jackson #000664) mice used for size-matching was previously collected and described in Machado et al., 2015. Mice were housed on a reversed light cycle 12 hr light/12 hr dark cycle, in standard cages with no more than five animals per cage. They had access to water and food ad libitum.

Experimental procedures

LocoMouse overground walking setup

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The LocoMouse system for overground locomotion was composed by a glass corridor, 66.5 cm long and 4.5 cm wide with a mirror placed at 45 deg under the corridor. A single high-speed camera (AVT Bonito, 1440 × 250 pixels, 400 frames per second) recorded both bottom and side views of walking mice. Infrared sensors were used to automatically detect when mice entered and exited the corridor and trigger data collection, which was performed in LABVIEW 2012 (Machado et al., 2015).

Data collection

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Animals were handled and acclimated to the overground setup for several sessions before data collection. Individual trials consisted of single crossings of the corridor, in which mice walked freely between two dark boxes. No food or water restriction or reward was used. Ten to twenty-five corridor trials were collected in each session for five consecutive days. An average of n = 1342 ± 637 strides were collected per reeler mouse (n = 348 ± 116 strides per paw) and n = 635 ± 341 strides per littermate mouse (n = 162 ± 86 strides per animal per paw) were collected.

To track the paws, nose and tail of locomoting mice we used the previously described noninvasive, markerless LocoMouse tracking system (Machado et al., 2015; https://github.com/careylab/LocoMouse), with additional, subsequent updates to the tail tracking, in order to handle the more erratic tail movements of the reeler mice, which often left the field of view of the videos. The new tail tracking algorithm was implemented using Matlab and the Signal Processing, Image Processing and Statistics and Machine Learning toolboxes. Tail tracking started with the side view using binary thresholding followed by a skeletonization operation for finding candidate tail segments based on length and position. These points were then projected onto the bottom view starting from the distal tail segment. The bottom view image was convoluted with a hamming window with a kernel width representative of a mouse tail. Subsequent points were identified iteratively towards the proximal tail, stopping at the base of the tail. In the case of tail segmentation after skeletonization the additional step of looking for tail points towards the distal tail was taken. The tail was then divided into 15 tail points (referred to as segments) with constant Euclidian distance (in 3D) between them, similarly to Machado et al., 2015. Matlab code for the updated tail tracker is available at https://github.com/careylab/LocoMouse_Dev.

Data analysis and statistics

Matlab 2012b and 2015a were used to process and analyze the data. Paw, nose and tail tracks (x,y,z) were obtained from the LocoMouse tracker (Machado et al., 2015). All tracks were divided in strides cycles. Stride cycle was defined as the period from stance onset to subsequent stance onset. For each stride, average walking speed was calculated. All data were sorted into speed bins (0.05 m/s bin width). Individual limb movements and interlimb coordination were calculated as follows:

Individual limb parameters

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  • Walking speed: x displacement of the body center during that stride divided by the stride duration.

  • Stride duration: time between two consecutive stance onsets.

  • Cadence: inverse of stride duration.

  • Swing velocity: x displacement of single limb during swing phase divided by swing duration.

  • Stride length: x displacement from touchdown to touchdown of single limb.

  • Stance duration: time in milliseconds that foot is on the ground during stride.

  • Duty factor: stance duration divided by stride duration.

  • Trajectories: (x,y,z) trajectories were aligned to swing onset and resampled to 100 equidistant points using linear interpolation. Interpolated trajectories were then binned by speed and the average trajectory was computed for each individual animal and smoothed with a Savitzky-Golay first-order filter with a 3-point window size.

  • Instantaneous swing velocity: the derivative of swing trajectory.

  • Model predictions: equations that were previously generated with mixed-effects models (Machado et al., 2015) to predict basic stride parameters.

Interlimb and whole-body coordination parameters

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  • Base of support: width between the two front and two hind paws during stance phase.

  • Body y displacement: y displacement of the body center during that stride.

  • Stance phase: relative timing of limb touchdowns to stride cycle of reference paw (FR). Calculated as: stance time - stance timereference paw/stride duration.

  • Supports: Support types were categorized by how many and which paws were on the ground, expressed as a percentage of the total stride duration for each stride. Paw support categories include 3-paw, 2-paw diagonal, 2-paw other/non diagonal (homolateral and homologous), and 2-paw front (only)supports.

  • Double support for each limb is defined as the percentage of the stride cycle between the touch down of a reference paw to lift-off of the contralateral paw. Because at higher speeds (running), the opposing limb lifts off before the reference paw touches down, we included negative double support by looking backwards in time, up to 25% of the stride cycle duration. Positive values of double support indicate that contralateral lift-off occurred after reference paw touch down, and negative values indicate that contralateral lift-off occurred before reference paw touch down. Note that front paw double support percentages include 2-paw front (only) support patterns as well as 3 and 4-paw support patterns in which both front paws were on the ground.

  • Tail and nose phases: For each speed bin we correlate the stridewise tail and nose trajectories with the trajectory given by the difference between the forward position of the right paw and the forward position of the left paw (also normalized to the stride). We do this both for front limbs (for the analysis of reeler mice) and hind limbs (for the analysis of previous pcd data). The phase is then calculated by the delay in which this correlation is maximized.

  • Tail and nose peak-to-peak amplitude: the change between peak (highest amplitude value) and trough (lowest amplitude value) in y or z during a stride duration.

  • Variability: All variability analyses were based on coefficients of variation (CV).

Geometric model of the tail and nose

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The analytical model of the nose and tail was a simpler version of our previously described geometric model (Machado et al., 2015). The current model transforms the forward movements of the front limbs into predicted lateral oscillations of tail and nose. The model is described by the equation:

yts=Gs(XtDsrXtDsl) and ytn=Gn(XtDnlXtDnr)

where yst and ynt are the lateral positions of tail segment s and nose, respectively; Gs and Gn are gains, obtained from fitting the data, that transform the limb oscillation amplitude to the amplitude of tail segment s and nose movements, respectively; xrt and xlt are the positions of front right and front left limbs at time t obtained from average trajectories of limb movements during strides at different speeds. Ds and Dn are the delays of tail segment s and nose, where the delay D1 is the delay of the base of the tail obtained by fitting the data.

As in Machado et al., 2015, delays between subsequent tail segments decreased according to the equation:

Ds=0.23s+3.97

where s is the segment number, starting at the base of the tail. The delay of the nose was the same as the delay of the base of the tail (e.g. Dn = D1).

Principal component and linear discriminant analyses

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The dataset consisted of a matrix of 109 observations of 45 features. Each observation was data from one mouse locomoting at a certain speed (binned) and features are z-scored gait parameters. LDA assumes independence within the feature space, which we knew to be violated due to the high speed-dependence of many gait features (Machado et al., 2015). Therefore, PCA was applied to address inter-variable correlation and avoid overfitting in LDA. PCA was performed by eigenvalue decomposition of the data covariance matrix. The first 10 PCs explained 88% of the variance and the data projected onto these 10 PCs were used as input to the LDA. The end contributions of the initial gait parameters to the two LD axes were obtained by multiplying the PCA mapping by the LDA mapping. LDA output is presented for each speed bin to verify that the pattern of differences across groups was captured, across all walking speeds.

Statistical analyses

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All statistics can be found in Table S1. Statistical analyses were done in Matlab with the Statistics toolbox. An independent samples t-test was used to test for differences in walking speed distributions (Figure 1D and Figure 2A). For all other gait parameters, analysis was performed on animal averages binned by speed using mixed-effects models (Bates et al., 2013). Fixed-effects terms included speed and genotype; animals were included as random terms. We report F statistics from mixed ANOVAs with Satterthwaite degrees of freedom correction. Differences were considered significant at *p < 0.05, **p < 0.01, and ***p < 0.001; asterisks report main effects of genotype.

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Decision letter

  1. Ronald L Calabrese
    Senior and Reviewing Editor; Emory University, United States

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]

Thank you for submitting your work entitled "Shared and specific signatures of locomotor ataxia in mutant mice" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The reviewers have opted to remain anonymous.

Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.

The work seems like a useful extension of Machado et al., 2015, from a benchmarking perspective – this paper helps to show that LocoMouse "works" at least in the sense of being able to identify commonalities and differences between mutants and to make semantic sense of those similarities and differences. However, after extensive consultation the consensus was reached that the paper does not contain enough new and impactful science for an advance on Machado et al., 2015, especially given reeler data in Darmohray et al., 2019, and the methodology used is that previously used by the authors, albeit in a different context and with a different mutant. The attempt to tie the behavioral defects quantified to circuit level defects were not convincing.

Reviewer #1:

This manuscript is a very well written manuscript describing the differences and similarities in the ataxic leg movements during walking in two mutant mouse models that have cerebellar neuropathy. Namely these mice are the Purkinje Cell Generation mice and the Reeler mice. They did a nice set of recordings and data analysis. However, this manuscript is purely descriptive, without any conceptual conclusion. I don't think this work, although well done, is not appropriate for eLife.

Reviewer #2:

We are still at the proof-of-concept phase for computational neuroethology. One goal of these methods is to identify relationships between complex patterns of behavior by – in a task or context specific manner – balancing (as the discussion says) specificity (e.g., high dimensional measures) with interpretability (e.g., a conceptual framework). This paper puts this hypothesis to the test using the LocoMouse framework developed by this lab to compare ataxic phenotypes among pcd and reeler mice. Here the authors do a very nice job of demonstrating that – like the pcd mice – many of the apparent phenotypes in the reeler mouse are a simple consequence of the slowed speed and smaller size of the mutants. In addition, the observations that phenotypes are now largely found in the front paws – and that tail and nose dynamics can be predicted from this effect – effectively distinguishes the two mutants. The data are clear, and the findings of significant import and utility to those interested in ataxias and in motor control.

Given that the technology and analytics associated with LocoMouse are at this point well vetted, I have no specific comments or concerns about the data in the paper. I would like only a few small adjustments. It would be nice to move some of the data from Figure 1—figure supplement 1 into main Figure 1 (or maybe to make Figure 1—figure supplement 1 a new Figure 2) so that the baseline comparisons between the mutants is more front stage in the figures. In addition, it would be clarifying to generate some simplified graphs (maybe focused on specific speeds) that, in at least some cases, enable comparisons of reeler, pcd and controls all at once. The compromises used in Figure 3 could work here (where you have adjacent panels with two of the three comparisons), but on the whole it would be generally nice to see more direct comparisons across the data in the main figures. Finally, I am left with a sense of the power of the approach to both relate and distinguish, but the one thing that isn't made completely explicit is why reeler mice are so much more apparently ataxic when you just look at them. Is this largely the collective consequence of the "high-stepping" phenotype, the increased variability and the steering phenotype? There is obviously not a simple answer to this, but I would appreciate some discussion of what might account for the fact that the reeler mice look worse by eye, and how quantitative measures might highlight or obscure the gestalt we have as researchers inspecting our mice.

Reviewer #3:

The authors use established methods from their lab to compare locomotor kinematics in wildtype mice and two mutant mouse lines: pcd and reeler. This work builds on some of their impressive previous work where they already showed the results for the pcd mice (Machado et al., 2015), as well as evaluations of both pcd and reeler mice in a different paradigm testing locomotor kinematics, the split-belt treadmill (Darmohray et al., 2019). With the current manuscript, the authors aim to provide a quantitative foundation for mapping specific locomotor impairments onto distinct neuropathologies. The experiments seem to be competently performed, and the manuscript is well written. However, the limitation of the current manuscript is that it does not provide much new insight to the analysis of body part kinematics which hasn't already largely been covered in their previous work.

The main concerns are:

1) Lack of novelty compared to their previous papers (Machado et al., 2015; Darmohray et al., 2019). Basic locomotor kinematics of both pcd and reeler mice (as shown here in Figures 1-3) are already described in these papers. Figure 4 does contain new information, although it largely depends on the results from Figure 2. Similar observations to those in Figure 2 of the current manuscript, e.g. different paw supports for the two mutant lines compared to controls, have already been shown in Figure 3D and H of Darmohray et al., 2019, albeit in a different behavioral paradigm.

2) The authors claim to provide a quantitative foundation for mapping specific locomotor impairments onto distinct neuropathologies. However, there is not enough biological validation for the claim that the data presented in the current manuscript can link behavioral phenotypes with underlying neural circuits, as there is no detailed description of the anatomical differences between the two mutant lines within the cerebellum. Certainly the found behavioral differences between the two are not linked to their neural circuits. The authors would need to revise their conclusions based on the data they present here.

3) An attempt to possibly rescue the phenotype would help to improve the biological understanding of which behavioral phenotypes are linked to specifically which anatomical underlying structures. I.e., is it possible to intervene in one of the animal models, so that their phenotype switches on the LD1 axis presented in Figure 4 to the side of the control mice? Such conclusions would then truly link behavioral phenotypes to neural circuits, and would help to significantly improve the biological understanding of mouse locomotor kinematics.

[Editors’ note: further revisions were suggested prior to acceptance, as described below.]

Thank you for submitting your article "Shared and specific signatures of locomotor ataxia in mutant mice" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Ronald Calabrese as the Senior Editor and Reviewing Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, when editors judge that a submitted work as a whole belongs in eLife but that some conclusions require a modest amount of additional new data, as they do with your paper, we are asking that the manuscript be revised to either limit claims to those supported by data in hand, or to explicitly state that the relevant conclusions require additional supporting data.

Our expectation is that the authors will eventually carry out the additional experiments and report on how they affect the relevant conclusions either in a preprint on bioRxiv or medRxiv, or if appropriate, as a Research Advance in eLife, either of which would be linked to the original paper.

Summary:

In this manuscript, Machado and colleagues use LocoMouse, their behavioural tracking software package published previously as part of a study in eLife (Machado et al., 2015), to reveal similarities and differences in the locomotor kinematic deficits of two mouse models of ataxia, specifically pcd and reeler mice. A behavioural description of locomotion in pcd mice was already included in Machado et al., 2015, so the additional data provided here are for reeler mice. The analyses provided reveal that some locomotor parameters are similarly impaired in these two mutant models while other parameters are unique to each mutant.

Revisions for this paper:

The reviewers found considerable merit in this advance but have further revisions they recommend that can be accomplished quickly and evaluated by the RE. Two of the reviewers thought that further experiments were needed to sustain the authors claims but realizing that this advance was considerable recommend revision in consultation.

Reviewer 3:

"The analyses provided reveal that some locomotor parameters are similarly impaired in these two mutant models while other parameters are unique to each mutant. While the additional findings presented in this study are sound, it is unclear how much we can generalize from analysis of one additional ataxia model, particularly because the neuropathology in these two models is quite different. Without a deeper evaluation of the generality and specificity of locomotor deficits in mouse models of ataxia, this study only provides a relatively slim additional advance over their previous study, and the more general conclusions they draw are not sufficiently justified. I have the following suggestions for strengthening the present manuscript. The authors show nicely in Figure 5 that combinations of behavioral features can be used to distinguish not only wild type from mutant mice but also pcd and reeler mice from each other. The authors' interpretation of these results is that the locomotor features that commonly distinguish wild type animals from the two mutants have a common underlying neuropathological origin (i.e. a cerebellar deficit) while the features that distinguish pcd mice from reeler mice reflect differences between these two mutants (i.e. absence of Purkinje cell output vs. miswiring in the cerebellum plus the additional extracerebellar defects in reeler mice). This is an important idea that could be bolstered with some additional data/analysis: 1. To show that aberrant cerebellar function is the root cause of the variability along LD1, the authors could use perturbation experiments to specifically affect locomotion by manipulating activity in interpositus-projecting Purkinje cells. This could be done acutely (as in Sarnaik and Raman, 2018) and/or chronically (as in Darmohray et al., 2018). 2. To test if LD1 does indeed distinguish wild type from ataxic mice and LD2 (or higher dimensions) distinguish different types of ataxic mice from each other, the authors could analyze locomotion in other models of ataxia that have different neuropathologies."

Reviewer 2:

"On a related note, a major claim is that the LD analysis defines a core set of behavioral features associated with cerebellar dysfunction. These are: impairments in multijoint, interlimb, and whole-body coordination, as well as in predictive mechanisms (i.e. forward models). It is true that these behavioral impairments are observed in both pcd and reeler mice, which share cerebellar neuropathology. However, it is possible that pathology in other brain circuits (basal ganglia, for example) may also be associated with some of the same behavioral impairments observed in the pcd and reeler mice, which would indicate that these behavioral impairments are not unique to the cerebellum after all. The impact of the paper, and the degree to which it is possible to claim that particular abnormalities are hallmarks of cerebellar dysfunction, would be much higher if the LD analysis could be repeated in mice with gait abnormalities caused by non-cerebellar dysfunction: Amende et al., 2005. Gait dynamics in mouse models of Parkinson's disease and Huntington's disease. If it is not feasible at this time to use the Locomouse system to analyze gait abnormalities in the mouse models in the Amende paper, the paper should at least discuss some of the limitations in terms of interpreting results as capturing behavioral hallmarks of cerebellar dysfunction. The distinction is between the impairments being "specific" markers of cerebellar dysfunction (which is supported by the data) vs "unique" markers of cerebellar dysfunction (which is not supported by the data). In other words, if a mouse has cerebellar dysfunction then we would be able to predict, based on the current paper, some of the abnormalities we may expect. On the other hand, if a mouse displays some of the abnormalities, it would not be possible to assume, based on the current results, that the underlying neuropathology is cerebellar."

In light of these reservations, the authors should scale back some of their claims and discuss the limitations of the analysis more.

https://doi.org/10.7554/eLife.55356.sa1

Author response

[Editors’ note: The authors appealed the original decision. What follows is the authors’ response to the first round of review.]

Reviewer #1:

This manuscript is a very well written manuscript describing the differences and similarities in the ataxic leg movements during walking in two mutant mouse models that have cerebellar neuropathy. Namely these mice are the Purkinje Cell Generation mice and the Reeler mice. They did a nice set of recordings and data analysis. However, this manuscript is purely descriptive, without any conceptual conclusion. I don't think this work, although well done, is not appropriate for eLife.

The current manuscript was submitted to eLife as a Research Advance – a format unique to eLife in which a paper is closely associated with a previous eLife paper. In this case, the current manuscript describing reeler mice is a follow up of our 2015 paper that developed the LocoMouse analysis framework to quantify the gait impairments of Purkinje cell degeneration mice (Machado et al., 2015). Yes, the data in both papers are descriptive. But this does not mean that they have no conceptual conclusion. Many researchers have told us that our 2015 paper fundamentally transformed the way they think about mouse locomotion and the role of the cerebellum. The current manuscript provides solid evidence for some of the ideas we could only speculate about then, as well as capturing shared and specific features of two forms of gait ataxia in a broadly applicable way, for the first time. We have revised the paper, with substantive changes to the Abstract, Introduction, and Discussion, to make our motivation and conceptual points more clear. In particular, the Discussion now highlights conceptual conclusions that are only made possible by taking both of Machado et al., 2015 and the current study into account.

Reviewer #2:

We are still at the proof-of-concept phase for computational neuroethology. One goal of these methods is to identify relationships between complex patterns of behavior by – in a task or context specific manner – balancing (as the discussion says) specificity (e.g., high dimensional measures) with interpretability (e.g., a conceptual framework). This paper puts this hypothesis to the test using the LocoMouse framework developed by this lab to compare ataxic phenotypes among pcd and reeler mice. Here the authors do a very nice job of demonstrating that – like the pcd mice – many of the apparent phenotypes in the reeler mouse are a simple consequence of the slowed speed and smaller size of the mutants. In addition, the observations that phenotypes are now largely found in the front paws – and that tail and nose dynamics can be predicted from this effect – effectively distinguishes the two mutants. The data are clear, and the findings of significant import and utility to those interested in ataxias and in motor control.

Given that the technology and analytics associated with LocoMouse are at this point well vetted, I have no specific comments or concerns about the data in the paper. I would like only a few small adjustments. It would be nice to move some of the data from Figure 1—figure supplement 1 into main Figure 1 (or maybe to make Figure 1—figure supplement 1 a new Figure 2) so that the baseline comparisons between the mutants is more front stage in the figures.

We thank the reviewer for this suggestion. We have converted Figure 1—figure supplement 1 into a main figure (now Figure 2).

In addition, it would be clarifying to generate some simplified graphs (maybe focused on specific speeds) that, in at least some cases, enable comparisons of reeler, pcd and controls all at once. The compromises used in Figure 3 could work here (where you have adjacent panels with two of the three comparisons), but on the whole it would be generally nice to see more direct comparisons across the data in the main figures.

We agree. What makes this tricky is the need to both speed and size-match the data from the two mutants with the controls. In Figure 1—figure supplement 1 (now Figure 2), we had accomplished that by plotting the differences between each mutant and their respective size-matched controls, but this does not work for every feature. To do our best to satisfy this reviewer’s request, which is similar to some of the points raised by reviewer 3, we have a) included Figure 1—figure supplement 1 as a main figure (now Figure 2), and b) included Summary plots like those from Figure 2 for the 43 features that provided inputs to the LDA, in the new Figure 5—figure supplement 1.

Finally, I am left with a sense of the power of the approach to both relate and distinguish, but the one thing that isn't made completely explicit is why reeler mice are so much more apparently ataxic when you just look at them. Is this largely the collective consequence of the "high-stepping" phenotype, the increased variability and the steering phenotype? There is obviously not a simple answer to this, but I would appreciate some discussion of what might account for the fact that the reeler mice look worse by eye, and how quantitative measures might highlight or obscure the gestalt we have as researchers inspecting our mice.

We thank the reviewer for this suggestion and have expanded our discussion of this issue, and of the relationship between hindlimb involvement, variability, and front paw supports, along with their possible neural basis, in the Discussion.

Reviewer #3:

The authors use established methods from their lab to compare locomotor kinematics in wildtype mice and two mutant mouse lines: pcd and reeler. This work builds on some of their impressive previous work where they already showed the results for the pcd mice (Machado et al., 2015), as well as evaluations of both pcd and reeler mice in a different paradigm testing locomotor kinematics, the split-belt treadmill (Darmohray et al., 2019). With the current manuscript, the authors aim to provide a quantitative foundation for mapping specific locomotor impairments onto distinct neuropathologies. The experiments seem to be competently performed, and the manuscript is well written. However, the limitation of the current manuscript is that it does not provide much new insight to the analysis of body part kinematics which hasn't already largely been covered in their previous work.

We thank the reviewer for the nice comments about our previous work. Below, we clarify some misunderstandings about what we have and have not shown before. We also note that this paper’s tight relationship to Machado et al., 2015 is precisely why we submitted this paper as a Research Advance – a format, unique to eLife, that allows a manuscript to be formally associated with a previous eLife paper.

The main concerns are:

1) Lack of novelty compared to their previous papers (Machado et al., 2015; Darmohray et al., 2019). Basic locomotor kinematics of both pcd and reeler mice (as shown here in Figures 1-3) are already described in these papers. Figure 4 does contain new information, although it largely depends on the results from Figure 2. Similar observations to those in Figure 2 of the current manuscript, e.g. different paw supports for the two mutant lines compared to controls, have already been shown in Figure 3D and H of Darmohray et al., 2019, albeit in a different behavioral paradigm.

First, yes, the pcd data were published in Machado et al., 2015 (which is part of why we are submitting this as a Research Advance). But all of the reeler data in this paper is entirely new. The statement “Basic locomotor kinematics of both pcd and reeler mice (as shown here in Figures 1-3) are already described in these papers” is just not true.

That claim appears to be supported by the statement “Similar observations to those in Figure 2 of the current manuscript, e.g. different paw supports for the two mutant lines compared to controls, have already been shown in Figure 3D and H of Darmohray et al., 2019, albeit in a different behavioral paradigm.” We know that the locomotor terminology can be confusing, and so here we aim to clarify an unfortunate misunderstanding. Darmohray et al., 2019 dealt exclusively with locomotor learning. Figure 3D and H of that paper quantified the extent of spatial vs. temporal components of learning in those mutants. Changes in double support symmetry after learning were one way that we assessed the temporal component of learning, but that measure has no correspondence to any parameter reported in the current manuscript. We invite reviewers to consider of Figure 3D and H of Darmohray et al. alongside Figure 2H from our current manuscript. Immediate inspection of these two plots reveals that while they both contain the words “% double support”, they contain entirely different data. While the reeler datapoints hover around 0 in the learning experiments (because there was no temporal learning in these mice), the opposite is true in the current manuscript – the reelers never approach 0 double support.

The reason for this discrepancy is that double support aftereffects, which measure learning, are a completely different measure from double support itself. Given that reeler mice were not included at all in Machado et al., 2015, and that the only reeler data in Darmohray et al., 2019 were a few datapoints concerned only with learning, there is simply no basis for the claim that we have previously reported the locomotor kinematics of reeler mice, or that the reeler data in the first 3 figures of this paper are not new.

To avoid additional confusion, we have added a statement in the Introduction to clarify that “the locomotor kinematics and whole-body coordination of reeler mice have not been reported”.

Finally, the reviewer acknowledges that “Figure 4 does contain new information, although it largely depends on the results from Figure 2.” In fact, Figure 4 incorporates results from all figures of this paper and those of Machado et al., 2015, in order to synthesize this vast amount of locomotor analysis into a digestible format. This approach is entirely new and, as we discuss in the Discussion, not only does it allow us to capture shared and specific features of gait ataxia here, it will form an essential component of our ability to extract meaning from the kinds of behavioral quantification that we first described in Machado et al., 2015 and that have become much more common since.

2) The authors claim to provide a quantitative foundation for mapping specific locomotor impairments onto distinct neuropathologies. However, there is not enough biological validation for the claim that the data presented in the current manuscript can link behavioral phenotypes with underlying neural circuits, as there is no detailed description of the anatomical differences between the two mutant lines within the cerebellum. Certainly the found behavioral differences between the two are not linked to their neural circuits. The authors would need to revise their conclusions based on the data they present here.

We sincerely apologize if we mistakenly gave the impression that we were trying to link specific aspects of the altered cerebellar circuitry to specific aspects of behavior. This was completely unintentional! We have made a number of revisions to the text to more clearly convey our actual meaning, including:

– Major changes to Abstract and Introduction to provide additional context and motivation for the current study .

– Changes in language throughout the paper to favor use of “structures”,

“neuropathologies”, “anatomical features”, and similar in place of “circuitry”, “circuits”, etc.

– A clarified and extended discussion of how we think the gait alterations we observe may related to gross pathology (related to the final comment of reviewer 2).

We hope that these changes have helped clarify that rather than claiming to have mapped specific locomotor features onto specific circuit abnormalities within the cerebellum, we are trying to demonstrate the ability to map shared and specific gait abnormalities onto shared and specific anatomical features. The shared locomotor phenotypes between the two mutants are almost by definition cerebellar, for example, as the neuropathology in pcd is largely specific to the cerebellum (particularly at the youngest ages we tested in Machado et al., 2015) and that is the only overlapping anatomical feature between the two lines. We speculate in the Discussion (and have now expanded this section) about possible anatomical foundations for the differences between the two mutants.

Finally, although we hope it is now clear that we are not attempting to make specific claims about their altered cerebellar circuitry giving rise to specific behavioral differences (other than speculating in the Discussion about the difference between a lack of Purkinje cell output (pcd) vs. aberrant Purkinje cell output (reeler)), we note that reeler and pcd are two of the bestdescribed mutants in the literature – their neuropathologies within the cerebellum and beyond have been described and replicated in countless papers, many of which we cite and describe in some detail here.

3) An attempt to possibly rescue the phenotype would help to improve the biological understanding of which behavioral phenotypes are linked to specifically which anatomical underlying structures. I.e., is it possible to intervene in one of the animal models, so that their phenotype switches on the LD1 axis presented in Figure 4 to the side of the control mice? Such conclusions would then truly link behavioral phenotypes to neural circuits, and would help to significantly improve the biological understanding of mouse locomotor kinematics.

While we agree that it would be quite powerful if such a rescue were possible, it would essentially amount to curing ataxia, which is clearly outside the scope of the current manuscript. Instead, what we propose is that applying this same approach to a broad range of mutants and circuit manipulations could achieve a similar goal. As we outline in the Discussion, we would argue that LD1 here represents “cerebellar dysfunction”, which is the shared anatomical feature in the two mutants, whereas LD2 would represent a combination of a) specific differences in the circuitry within the cerebellum itself, and b) extracerebellar effects. With the future addition of more mouse models and manipulations, the LDA would acquire more axes that could capture the contributions of other brain areas or cell types. We absolutely were not trying to imply that we had already achieved that broader goal, and we have tried to be more circumspect in our conclusions as suggested in major point #2. However, we emphasize that our approach has successfully captured and distilled the shared and specific locomotor phenotypes of two mutants with very clear, and previously well described, shared and specific neuropathologies – and to our knowledge this is the first time that this has been done. We hope to share the success of this approach with a wide audience in the hopes that others will build on the foundation we have established here – as they have already started to do with the LocoMouse system.

[Editors’ note: what follows is the authors’ response to the second round of review.]

Revisions for this paper:

The reviewers found considerable merit in this advance but have further revisions they recommend that can be accomplished quickly and evaluated by the Reviewing Editor. Two of the reviewers thought that further experiments were needed to sustain the authors claims but realizing that this advance was considerable recommend revision in consultation.

Reviewer 3:

"The analyses provided reveal that some locomotor parameters are similarly impaired in these two mutant models while other parameters are unique to each mutant. While the additional findings presented in this study are sound, it is unclear how much we can generalize from analysis of one additional ataxia model, particularly because the neuropathology in these two models is quite different. Without a deeper evaluation of the generality and specificity of locomotor deficits in mouse models of ataxia, this study only provides a relatively slim additional advance over their previous study, and the more general conclusions they draw are not sufficiently justified. I have the following suggestions for strengthening the present manuscript. The authors show nicely in Figure 5 that combinations of behavioral features can be used to distinguish not only wild type from mutant mice but also pcd and reeler mice from each other. The authors' interpretation of these results is that the locomotor features that commonly distinguish wild type animals from the two mutants have a common underlying neuropathological origin (i.e. a cerebellar deficit) while the features that distinguish pcd mice from reeler mice reflect differences between these two mutants (i.e. absence of Purkinje cell output vs. miswiring in the cerebellum plus the additional extracerebellar defects in reeler mice). This is an important idea that could be bolstered with some additional data/analysis: 1. To show that aberrant cerebellar function is the root cause of the variability along LD1, the authors could use perturbation experiments to specifically affect locomotion by manipulating activity in interpositus-projecting Purkinje cells. This could be done acutely (as in Sarnaik and Raman, 2018) and/or chronically (as in Darmohray et al., 2018). 2. To test if LD1 does indeed distinguish wild type from ataxic mice and LD2 (or higher dimensions) distinguish different types of ataxic mice from each other, the authors could analyze locomotion in other models of ataxia that have different neuropathologies."

Reviewer 2:

"On a related note, a major claim is that the LD analysis defines a core set of behavioral features associated with cerebellar dysfunction. These are: impairments in multijoint, interlimb, and whole-body coordination, as well as in predictive mechanisms (i.e. forward models). It is true that these behavioral impairments are observed in both pcd and reeler mice, which share cerebellar neuropathology. However, it is possible that pathology in other brain circuits (basal ganglia, for example) may also be associated with some of the same behavioral impairments observed in the pcd and reeler mice, which would indicate that these behavioral impairments are not unique to the cerebellum after all. The impact of the paper, and the degree to which it is possible to claim that particular abnormalities are hallmarks of cerebellar dysfunction, would be much higher if the LD analysis could be repeated in mice with gait abnormalities caused by non-cerebellar dysfunction: Amende et al., 2005. Gait dynamics in mouse models of Parkinson's disease and Huntington's disease. If it is not feasible at this time to use the Locomouse system to analyze gait abnormalities in the mouse models in the Amende paper, the paper should at least discuss some of the limitations in terms of interpreting results as capturing behavioral hallmarks of cerebellar dysfunction. The distinction is between the impairments being "specific" markers of cerebellar dysfunction (which is supported by the data) vs "unique" markers of cerebellar dysfunction (which is not supported by the data). In other words, if a mouse has cerebellar dysfunction then we would be able to predict, based on the current paper, some of the abnormalities we may expect. On the other hand, if a mouse displays some of the abnormalities, it would not be possible to assume, based on the current results, that the underlying neuropathology is cerebellar." In light of these reservations, the authors should scale back some of their claims and discuss the limitations of the analysis more.

Thank you for these insightful comments and suggestions. We absolutely acknowledge that the conclusions that can be drawn from two mutants are limited and fully agree that the inclusion of additional cerebellar and non-cerebellar models within the framework we provide here will be hugely informative as to the specific contributions of both cerebellar and other circuits to locomotor phenotypes. Indeed, this has always been our plan, and we hope to follow up on this study with exactly those analyses in the near future. We have revised our language throughout the manuscript to soften our claims, address limitations, and make more explicit distinctions between what we have actually shown here, vs. predictions for future studies.

https://doi.org/10.7554/eLife.55356.sa2

Article and author information

Author details

  1. Ana S Machado

    Champalimaud Neuroscience Program, Champalimaud Center for the Unknown, Lisbon, Portugal
    Contribution
    Conceptualization, Data curation, Software, Formal analysis, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing
    Competing interests
    No competing interests declared
  2. Hugo G Marques

    Champalimaud Neuroscience Program, Champalimaud Center for the Unknown, Lisbon, Portugal
    Contribution
    Conceptualization, Formal analysis, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8709-4841
  3. Diogo F Duarte

    Champalimaud Neuroscience Program, Champalimaud Center for the Unknown, Lisbon, Portugal
    Contribution
    Conceptualization, Software, Formal analysis, Visualization, Writing - original draft, Writing - review and editing
    Competing interests
    No competing interests declared
  4. Dana M Darmohray

    Champalimaud Neuroscience Program, Champalimaud Center for the Unknown, Lisbon, Portugal
    Contribution
    Conceptualization, Software, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing
    Competing interests
    No competing interests declared
  5. Megan R Carey

    Champalimaud Neuroscience Program, Champalimaud Center for the Unknown, Lisbon, Portugal
    Contribution
    Conceptualization, Resources, Supervision, Funding acquisition, Validation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing
    For correspondence
    megan.carey@neuro.fchampalimaud.org
    Competing interests
    Reviewing Editor, eLife
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4499-1657

Funding

H2020 European Research Council (Starting Grant 640093)

  • Megan R Carey

Howard Hughes Medical Institute (International Early Career Scientist 55007413)

  • Megan R Carey

Fundação para a Ciência e a Tecnologia (FCT-PTDC/MED-NEU/30890/2017)

  • Megan R Carey

Fundação para a Ciência e a Tecnologia (SFRH/BD/51210/2010)

  • Ana S Machado

Fundação para a Ciência e a Tecnologia (SFRH/BPD/119404/2016)

  • Hugo G Marques

Fundação para a Ciência e a Tecnologia (PD/BD/141643/2018)

  • Diogo F Duarte

Fundação para a Ciência e a Tecnologia (SFRH/BD/86265/2012)

  • Dana M Darmohray

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Tracy Pritchett for maintenance of mouse lines and help with histology and imaging and João Fayad for optimization and maintenance of the LocoMouse tracker. Olivia Carmo assisted with data acquisition and Alex Azinheira made the video. We are grateful to the members of Carey Lab and the Champalimaud Neuroscience Program for helpful discussion.

This work was supported by a Howard Hughes Medical Institute International Early Career Scientist Grant #55007413 (to MRC), European Research Council Starting Grant #640093 (to MRC), FCT grant FCT-PTDC/MED-NEU/30890/2017 (to MRC), Congento LISBOA-01–0145-FEDER-022170, and fellowships from the Portuguese Fundação para a Ciência e a Tecnologia: SFRH/BD/51210/2010 (to ASM), SFRH/BD/86265/2012 (to DMD), PD/BD/141643/2018 (to DFD), and SFRH/BPD/119404/2016 (to HGM).

Ethics

Animal experimentation: All procedures were reviewed and performed in accordance with the Champalimaud Centre for the Unknown Ethics Committee guidelines and approved by the Portuguese Direcção Geral de Veterinária (Ref. No. 0421/000/000/2015).

Senior and Reviewing Editor

  1. Ronald L Calabrese, Emory University, United States

Publication history

  1. Received: January 23, 2020
  2. Accepted: July 9, 2020
  3. Version of Record published: July 28, 2020 (version 1)

Copyright

© 2020, Machado et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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