Phenotypic Characterization of Speed-Associated Gait Changes in Mice Reveals Modular Organization of Locomotor Networks

“Speed of locomotion also regulates the visual cortical state [37–39] and cognitive functions like navigation [40], suggesting the need for considering locomotor network selection in a broader context.”

“The Need to Consider Gaits when Studying Locomotion The mouse is presently the prevailing model for studying the neuronal control of locomotion in mammals [26, 31–34].”

“The Need to Consider Gaits when Studying Locomotion The mouse is presently the prevailing model for studying the neuronal control of locomotion in mammals [26, 31–34].”

“Speed of locomotion also regulates the visual cortical state [37–39] and cognitive functions like navigation [40], suggesting the need for considering locomotor network selection in a broader context.”

“Mice Data were collected from five wild-type female mice at 2 months of age and from E1Ngn2::Cre [39]: Dbx1 (Dbx1) [41] and Vglut2::Cre [42]; Dbx1 (Dbx1) mice (n = 3 for each strain) aged 1 month.”

“Walk Walk was defined as a pattern of limb movement where three or four feet were simultaneously in contact with the ground (Movie S1; Figure 1A) [11].”

“We have previously proposed that left-right alternation is secured by both excitatory and inhibitory commissural interneurons [21–29], belonging to the V0 class of neurons [27, 30].”

“In this last cohort, step frequency and locomotor speed were significantly increased to values characteristic of boundgait (Figures 1F and 1G; Bellardita and Kiehn, 2015; Lemieux et al., 2016).”

“11) 55,56 .”

“Interestingly, mutant mice lacking excitatory glutamatergic V0v interneurons can walk and gallop, though they cannot trot [79,142].”

“In rodents, recent methods have made progress by incorporation of speed in gait analysis [23, 24] and determination of whole body coordination [25, 26].”

“Not having the body fixed (Musienko et al. 2020) reflects a more real-life scenario but will cause postural and strategic variation (Bellardita and Kiehn 2015; McLean et al. 2008), which can lead to a different sensory processing and thus different kinematics (Deliagina et al. 2012; Gerasimenko et…”

“This medial shift as the frequency of fictive locomotor activity increases thus provides support for the idea that the locomotor CPG is a modular network with speed-dependent recruitment of interneuronal components, as has been previously suggested (Bellardita and Kiehn, 2015).”

“Based on in vitro and in vivo experiments 19,21,25,28 , a network design has been suggested that constitutes two inhibitory commissural pathways involving the V0 and the V3 CINs to coordinate left–right stepping in a speed-dependent manner 26,28 .”

“V0-interneuron ablated mice, which were also uncoordinated and slower (Bellardita and Kiehn, 2015).”

“The identified neuron types include the commissural interneurons (CINs), such as V0 (V0D and V0V) [27,28] and V3 [29–31], and ipsilaterally-projecting V2a neurons [37,38] that are critically involved in the coordination of left–right rhythmic activities, as well as the ipsilaterally-projecting inhibitory V1 and V2b ne…”

“…that glycine and D-serine appear to have opposite roles in determining locomotor frequency, activity-dependent changes in their availability may provide a mechanism for speed control and perhaps contribute to gait selection (Bellardita and Kiehn 2015; Talpalar and Kiehn 2010; Talpalar et al. 2013).”

“In mammalian locomotion, spinal modules also control locomotor speed and gait, particularly those including V0 and V2a neurons (Crone et al., 2009; Zhong et al., 2010; Talpalar et al., 2013; Bellardita and Kiehn, 2015).”

“…the kinematics of FW locomotor movements have been studied in considerable detail in both humans and terrestrial quadrupeds (Barrey et al. 1993; Bellardita and Kiehn 2015; Carlson-Kuhta et al. 1998; Gambaryan 1974; Herbin et al. 2007; Leblond et al. 2003; Miller et al. 1975; Nilsson et al.…”

“…and demonstrated that the dorsal subset is inhibitory and responsible for coordinating left-right alternation at slow locomotor speeds, while the ventral subset is excitatory and responsible for this function when locomotor speed increases (Talpalar et al., 2013; Bellardita and Kiehn, 2015).”

“112 Also, the slip events used here consisted of an increase in the speed of translation by a factor of two in relation to the running speed of the animal.”

“As speed increases there is synchronous activity across the midline and two similar locomotor gaits, gallop and bound, dominate (Bellardita and Kiehn, 2015).”

“Notably, V0v and V2a, but not V0d, neurons residing in the lumbar enlargement have been implicated in securing left-right limb alternation at high speeds ( Bellardita and Kiehn, 2015 ; Crone et al., 2009 ; Talpalar et al., 2013 ; Ruder et al., 2016 ).”

“the signature of a quadruped walking gait (Hildebrand, 1965; Bellardita and Kiehn, 2015).”

“5 occurs at low burst frequencies (walk and trot gaits), whereas a phase lag ∆ close to 0 (or to 1, equivalently) occurs at high burst frequencies (gallop and bound gaits) [3, 23, 26].”

“Locomotor patterns can be dynamically turned on and off so that there is a change in gait, form, or direction (e.g., Mesce and PierceShimomura, 2010; Gruhn et al., 2011; Kawano et al., 2011; Bellardita and Kiehn, 2015).”

“During ambulatory locomotion, increases in speed are accompanied by increases in the cyclical frequency of muscles within limbs and changes in the timing and amplitude of muscle contractions within and between them [29,30].”

“…depending on context, the locomotor CPG produces either walking, or running or bounding where each form of locomotion is mutually exclusive (Bellardita & Kiehn, 2015; Grillner & El Manira, 2020; Kiehn, 2016); the oromotor CPG produces either chewing, or lapping or swallowing where, again,…”

“One can notice that previous studies have described a correlation between gait speed and a shorter paw contact time with the ground [67, 90].”

“They found that mice lacking V0-Dbx1 neurons were not able to present alternating limb movements, instead, the only gait they expressed was hopping [50,51,58].”

“In vertebrates, the neural circuits responsible for the coordinated limb movements include motor pools, a topographic map, and commissural neurons ( Romanes, 1951 ; Cruce, 1974 ; Tsuchida et al., 1994 ; Lanuza et al., 2004 ; Bellardita and Kiehn, 2015 ; Sengupta et al., 2021 ; Yang et al., 2023 ).”

“While the behavioral signatures of stereotypical velocity-dependent foot placement patterns across species [10, 11, 12, 13, 14, 15, 16] has helped solidify the existence of velocity-driven neural control during locomotion [17, 6], we lack analogous cross-species signatures of error-driven foot…”

“To achieve these slower velocities without changing gait, mice significantly decrease their stride length from approximately 7 cm to approximately 2.5–4 cm [30,31].”

“…locomotion to accommodate changes in gait (Bellardita and Kiehn, 2015), with for example bilateral limbs transitioning from alternation during trot to synchrony during bound (Figure 1A) (Bellardita and Kiehn, 2015; English and Lennard, 1982; Grillner and El Manira, 2020; Lemieux et al., 2016).”

“In mice, different groups of V0 spinal 35 interneurons are involved in mediating left-right limb alterations at different speeds [1, 2].”

“We chose the 300/500 ms ISI based on previous reports indicating this frequency as a proxy of the speed of forelimb movements in rodents when walking or trotting 9,25,26 .”

“…such as balance beam or horizontal ladder crossing tests (Jones and Roberts, 1968; Hartmann et al., 2004; Stanley et al., 2005; Zörner et al., 2010; Bellardita and Kiehn, 2015; Machado et al., 2015; Vinueza Veloz et al., 2015; Preisig et al., 2016; Darmohray et al., 2019; Timotius et al., 2019).”

“In addition to locomotor velocity, whose definition is the distance traveled over a fixed period of time (10 min in open field), EthoVision XT quantifies the frequency of hopping—a gait that is characterized by bilateral and symmetrical forelimb/hindlimb synchronous movement [63]—which is qualitatively different from…”

“It has been unclear how more gradual, overlapping sequences of muscle contractions, which are common to most movements, are generated (Bellardita and Kiehn, 2015; Berkowitz and Stein, 1994; Hinckley et al., 2015; Machado et al., 2015; Pratt et al., 1991).”

“33 Limbs are thus initially coupled to the tail rhythm, consistent with previous electrophysiological observations, 34 and, as the tail recedes, gain the ability to drive independent, synchronous, and fine-scale movement.”

“Naturalistic posture generates phenotypic trotting gait at high-speed VR navigation Rodents in the wild produce a specific set of gaits (walk, trot, gallop, and bound) that are expressed in distinct ranges of speed with phenotypic inter-limb and intra-limb coordination (Bellardita and Kiehn, 2015).”

“Since limbed animals adapt to higher locomotor speeds not only by increasing frequency but also by changing their gait, 5,21–24 we focused on a simpler rhythmic behavior, the scratch reflex.”

“Bellardita et al. found that wild-type mice had four ILCMs, namely, walk, trot, bound, and gallop (Bellardita and Kiehn, 2015).”

“…duration of the L2 ENG in comparison to the WT. Left-right and flexor-extensor coordination during neonatal locomotor-like activity Although some mutant mice can produce a more variable locomotor coordination (Zhang et al., 2008), others produce a less variable one (Bellardita and Kiehn, 2015).”

“298 Genetic ablation and inactivation experiments [32,63–67] implicate certain neurons 299 to be part of the CPG.”

“We focused on continuous locomotor bouts with a speed >15 cm s-1, which corresponds to fast walk or trot (46).”

“More recently, investigators have gathered data in support of hypotheses suggesting more complex multipartite modular organization of spinal CPGs (Ampatzis et al. 2014; Bagnall and McLean 2014; Bellardita and Kiehn 2015; Bizzi et al. 2008; Duysens et al. 2013; Giszter 2015; Giszter and Hart 2013; Grillner 1981; Grilln…”

“By reproducing the speed-dependent gait patterns of intact mice ( Bellardita and Kiehn, 2015 ; Lemieux et al., 2016 ) and simulating the effects of silencing or ablating various classes of commissural ( Talpalar et al., 2013 ; Bellardita and Kiehn, 2015 ) and long propriospinal interneurons ( Ruder…”

“2,5 Similarly, V2a neurons have been shown to selectively contribute to maintaining left-right alternation at high speeds.”

“Some weights are obtained from the previous models [27] , while others are selected from numerous simulation tests that replicate the locomotor features observed in experiments [2 , 14 , 18] .”

“24 The design of the head-fixation bars only permits vertical adjustment of the mouse head without the capability for lateral movement, restricting the range of brain regions accessible for imaging.”

“Given the involvement of LPDNs in the diagonal synchronisation of fore and hindlimb during locomotion (Bellardita and Kiehn, 2015; Ruder et al., 2016; Sherrington et al., 1906), we first injected contralateral FMs and GS (Figure S4A).”

“…examine this, each limb was tracked using DeepLabCut ((Mathis et al., 2018), Figure 3A-B, see Materials and Methods) and their coordinates were used to register the time of footfall as well as the stance and the swing phases that define the locomotor cycle ((Bellardita and Kiehn, 2015), Figure 3C).”

“…walking mice at slow walking speeds similar to the imposed speed, paws spent larger fractions of the total stride time in the stance compared to swing phase (all mice and FL paw : swing duration [0.055, 0.09, 0.14] s and stance duration [0.075, 0.205, 0.365] s for [25, 50, 75]th percentile) (Fig.”

“Spinal V0v INs, for instance, are known to be involved in trotting, though their ablation preserves walking (51, 52).”

“This critical calibration allowed us to translate the fi eld data into biologically relevant locomotion patterns, revealing stride frequencies of 3 – 5 Hz, consistent with established rat walking and trotting gaits 18 .”

“As spinal neural circuits have been considered to contribute to high-speed locomotion as well as low-speed walking, at least for animals 20,21 , human sprinters are also hypothesized to show plastic changes in spinal neural circuits that innervate the thigh muscles, rather than cortical neural…”

“However, gait selection is also known to be influenced by locomotor speed and the descending pathway from the CnF that at least partly mediates it [5, 9, 10].”

“Such differences can be found in both the building blocks and the activity of these networks (Swensen and Bean, 2005; Schulz et al., 2006; Bellardita and Kiehn, 2015; Pulver et al., 2015), and also between cells of the same cell type (Ransdell et al., 2013).”

“This rules out proton-gated channels like acid-sensing ion channels (ASICs) as the sour taste receptor since they are activated by H+ but have Na+ currents.”

“…systems that incorporate machine learning algorithms ( e.g., NeuroCube) and lateral view analysis ( e.g., Mo-toRater, Locomouse (Machado et al., 2015)) to analyze gait more comprehensively (Alexandrov et al., 2015; Bellardita and Kiehn, 2015; de Bruin et al., 2016; Talpalar et al., 2013).”

“…analysis of locomotor data at different walking speeds revealed systematic variation in the temporal ( Figure 1F ) and spatial ( Figure 1G, H ) components of limb movement, consistent with prior reports ( Akay et al., 2006 ; Bellardita and Kiehn, 2015 ; Machado et al., 2015 ; Mendes et al., 2015 ).”

“Locomotion analyses span a range of granularities, including joint movement reconstructions (22, 23), limb coordination pat­ terns (gaits) (21), and more complex analyses grouping basic fea­ tures in motor synergies (28) or behavior syllables (24).”