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.
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, 2018; Udo 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.
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 1; Lalonde 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=