Recording Forelimb Muscle Activity in Head-Fixed Mice with Chronically Implanted EMG Electrodes.

Powerful genetic and molecular tools available in mouse systems neuroscience research have enabled researchers to interrogate motor system function with unprecedented precision in head-fixed mice performing a variety of tasks. The small size of the mouse makes the measurement of motor output difficult, as the traditional method of electromyographic (EMG) recording of muscle activity was designed for larger animals like cats and primates. Pending commercially available EMG electrodes for mice, the current gold-standard method for recording muscle activity in mice is to make electrode sets in-house. This article describes a refinement of established procedures for hand fabrication of an electrode set, implantation of electrodes in the same surgery as headplate implantation, fixation of a connector on the headplate, and post-operative recovery care. Following recovery, millisecond-resolution EMG recordings can be obtained during head-fixed behavior for several weeks without noticeable changes in signal quality. These recordings enable precise measurement of forelimb muscle activity alongside in vivo neural recording and/or perturbation to probe mechanisms of motor control in mice.

Widespread innervation of motoneurons by spinal V3 neurons globally amplifies locomotor output in mice.

While considerable progress has been made in understanding the neuronal circuits that underlie the patterning of locomotor behaviours such as walking, less is known about the circuits that amplify motoneuron output to enable adaptable increases in muscle force across different locomotor intensities. Here, we demonstrate that an excitatory propriospinal neuron population (V3 neurons, Sim1 + ) forms a large part of the total excitatory interneuron input to motoneurons (∼20%) across all hindlimb muscles. Additionally, V3 neurons make extensive connections among themselves and with other excitatory premotor neurons (such as V2a neurons). These circuits allow local activation of V3 neurons at just one segment (via optogenetics) to rapidly depolarize and amplify locomotor-related motoneuron output at all lumbar segments in both the in vitro spinal cord and the awake adult mouse. Interestingly, despite similar innervation from V3 neurons to flexor and extensor motoneuron pools, functionally, V3 neurons exhibit a pronounced bias towards activating extensor muscles. Furthermore, the V3 neurons appear essential to extensor activity during locomotion because genetically silencing them leads to slower and weaker mice with a poor ability to increase force with locomotor intensity, without much change in the timing of locomotion. Overall, V3 neurons increase the excitability of motoneurons and premotor neurons, thereby serving as global command neurons that amplify the locomotion intensity.

Multimodal sensory control of motor performance by glycinergic interneurons of the mouse spinal cord deep dorsal horn.

Sensory feedback is integral for contextually appropriate motor output, yet the neural circuits responsible remain elusive. Here, we pinpoint the medial deep dorsal horn of the mouse spinal cord as a convergence point for proprioceptive and cutaneous input. Within this region, we identify a population of tonically active glycinergic inhibitory neurons expressing parvalbumin. Using anatomy and electrophysiology, we demonstrate that deep dorsal horn parvalbumin-expressing interneuron (dPV) activity is shaped by convergent proprioceptive, cutaneous, and descending input. Selectively targeting spinal dPVs, we reveal their widespread ipsilateral inhibition onto pre-motor and motor networks and demonstrate their role in gating sensory-evoked muscle activity using electromyography (EMG) recordings. dPV ablation altered limb kinematics and step-cycle timing during treadmill locomotion and reduced the transitions between sub-movements during spontaneous behavior. These findings reveal a circuit basis by which sensory convergence onto dorsal horn inhibitory neurons modulates motor output to facilitate smooth movement and context-appropriate transitions.

Distinct roles of spinal commissural interneurons in transmission of contralateral sensory information.

Crossed reflexes are mediated by commissural pathways transmitting sensory information to the contralateral side of the body, but the underlying network is not fully understood. Commissural pathways coordinating the activities of spinal locomotor circuits during locomotion have been characterized in mice, but their relationship to crossed reflexes is unknown. We show the involvement of two genetically distinct groups of commissural interneurons (CINs) described in mice, V0 and V3 CINs, in the crossed reflex pathways. Our data suggest that the exclusively excitatory V3 CINs are directly involved in the excitatory crossed reflexes and show that they are essential for the inhibitory crossed reflexes. In contrast, the V0 CINs, a population that includes excitatory and inhibitory CINs, are not directly involved in excitatory or inhibitory crossed reflexes but downregulate the inhibitory crossed reflexes. Our data provide insights into the spinal circuitry underlying crossed reflexes in mice, describing the roles of V0 and V3 CINs in crossed reflexes.

Distinct roles of spinal commissural interneurons in transmission of contralateral sensory information.

Crossed reflexes (CR) are mediated by commissural pathways transmitting sensory information to the contralateral side of the body, but the underlying network is not fully understood. Commissural pathways coordinating the activities of spinal locomotor circuits during locomotion have been characterized in mice, but their relationship to CR is unknown. We show the involvement of two genetically distinct groups of commissural interneurons (CINs) described in mice, V0 and V3 CINs, in the CR pathways. Our data suggest that the exclusively excitatory V3 CINs are directly involved in the excitatory CR, and show that they are essential for the inhibitory CR. In contrast, the V0 CINs, a population that includes excitatory and inhibitory CINs, are not directly involved in excitatory or inhibitory CRs but down-regulate the inhibitory CR. Our data provide insights into the spinal circuitry underlying CR in mice, describing the roles of V0 and V3 CINs in CR.

Muscle spindles and their role in maintaining robust locomotion.

Muscle spindles, one of the two main classes of proprioceptors together with Golgi tendon organs, are sensory structures that keep the central nervous system updated about the position and movement of body parts. Although they were discovered more than 150 years ago, their function during movement is not yet fully understood. Here, we summarize the morphology and known functions of muscle spindles, with a particular focus on locomotion. Although certain properties such as the sensitivity to dynamic and static muscle stretch are long known, recent advances in molecular biology have allowed the characterization of the molecular mechanisms for signal transduction in muscle spindles. Building upon classic literature showing that a lack of sensory feedback is deleterious to locomotion, we bring to the discussion more recent findings that support a pivotal role of muscle spindles in maintaining murine and human locomotor robustness, defined as the ability to cope with perturbations. Yet, more research is needed to expand the existing mechanistic understanding of how muscle spindles contribute to the production of robust, functional locomotion in real world settings. Future investigations should focus on combining different animal models to identify, in health and disease, those peripheral, spinal and brain proprioceptive structures involved in the fine tuning of motor control when locomotion happens in challenging conditions.

ERR2 and ERR3 promote the development of gamma motor neuron functional properties required for proprioceptive movement control.

The ability of terrestrial vertebrates to effectively move on land is integrally linked to the diversification of motor neurons into types that generate muscle force (alpha motor neurons) and types that modulate muscle proprioception, a task that in mammals is chiefly mediated by gamma motor neurons. The diversification of motor neurons into alpha and gamma types and their respective contributions to movement control have been firmly established in the past 7 decades, while recent studies identified gene expression signatures linked to both motor neuron types. However, the mechanisms that promote the specification of gamma motor neurons and/or their unique properties remained unaddressed. Here, we found that upon selective loss of the orphan nuclear receptors ERR2 and ERR3 (also known as ERRß, ERRγ or NR3B2, NR3B3, respectively) in motor neurons in mice, morphologically distinguishable gamma motor neurons are generated but do not acquire characteristic functional properties necessary for regulating muscle proprioception, thus disrupting gait and precision movements. Complementary gain-of-function experiments in chick suggest that ERR2 and ERR3 could operate via transcriptional activation of neural activity modulators to promote a gamma motor neuron biophysical signature of low firing thresholds and high firing rates. Our work identifies a mechanism specifying gamma motor neuron functional properties essential for the regulation of proprioceptive movement control.

The brain integrates proprioceptive information to ensure robust locomotion.

Robust locomotion relies on information from proprioceptors: sensory organs that communicate the position of body parts to the spinal cord and brain. Proprioceptive circuits in the spinal cord are known to coarsely regulate locomotion in the presence of perturbations. Yet, the regulatory importance of the brain in maintaining robust locomotion remains less clear. Here, through mouse genetic studies and in vivo electrophysiology, we examined the role of the brain in integrating proprioceptive information during perturbed locomotion. The systemic removal of proprioceptors left the mice in a constantly perturbed state, similar to that observed during mechanically perturbed locomotion in wild-type mice and characterised by longer and less accurate synergistic activation patterns. By contrast, after surgically interrupting the ascending proprioceptive projection to the brain through the dorsal column of the spinal cord, wild-type mice showed normal walking behaviour, yet lost the ability to respond to external perturbations. Our findings provide direct evidence of a pivotal role for ascending proprioceptive information in achieving robust, safe locomotion. KEY POINTS: Whether brain integration of proprioceptive feedback is crucial for coping with perturbed locomotion is not clear. We showed a crucial role of the brain for responding to external perturbations and ensure robust locomotion. We used mouse genetics to remove proprioceptors and a spinal lesion model to interrupt the flow of proprioceptive information to the brain through the dorsal column in wild-type animals. Using a custom-built treadmill, we administered sudden and random mechanical perturbations to mice during walking. External perturbations affected locomotion in wild-type mice similar to the absence of proprioceptors in genetically modified mice. Proprioceptive feedback from muscle spindles and Golgi tendon organs contributed to locomotor robustness. Wild-type mice lost the ability to respond to external perturbations after interruption of the ascending proprioceptive projection to the brainstem.

Crossed reflex responses to flexor nerve stimulation in mice.

Motor responses in one leg to sensory stimulation of the contralateral leg have been named "crossed reflexes" and are extensively investigated in cats and humans. Despite this effort, a circuit-level understanding of the crossed reflexes has remained missing. In mice, advances in molecular genetics enabled insights into the "commissural spinal circuitry" that ensures coordinated leg movements during locomotion. Despite some common features between the commissural spinal circuitry and the circuit for the crossed reflexes, the degree to which they overlap has remained obscure. Here, we describe excitatory crossed reflex responses elicited by electrically stimulating the common peroneal nerve that mainly innervates ankle flexor muscles and the skin on anterolateral aspect of the hind leg. Stimulation of the peroneal nerve with low current intensity evoked low-amplitude motor responses in the contralateral flexor and extensor muscles. At higher current strengths, stimulation of the same nerve evoked stronger and more synchronous responses in the same contralateral muscles. In addition to the excitatory crossed reflex pathway indicated by muscle activation, we demonstrate the presence of an inhibitory crossed reflex pathway, which was modulated when the motor pools were active during walking. The results are compared with the crossed reflex responses initiated by stimulating proprioceptors from extensor muscles and cutaneous afferents from the posterior part of the leg. We anticipate that these findings will be essential for future research combining the in vivo experiments presented here with mouse genetics to understand crossed reflex pathways at the network level in vivo.NEW & NOTEWORTHY Insights into the mechanisms of crossed reflexes are essential for understanding coordinated leg movements that maintain stable locomotion. Advances in mouse genetics allow for the selective manipulation of spinal interneurons and provide opportunities to understand crossed reflexes. Crossed reflexes in mice, however, are poorly described. Here, we describe crossed reflex responses in mice initiated by stimulation of the common peroneal nerve, which serves as a starting point for investigating crossed reflexes at the cellular level.

Control of Mammalian Locomotion by Somatosensory Feedback.

When animals walk overground, mechanical stimuli activate various receptors located in muscles, joints, and skin. Afferents from these mechanoreceptors project to neuronal networks controlling locomotion in the spinal cord and brain. The dynamic interactions between the control systems at different levels of the neuraxis ensure that locomotion adjusts to its environment and meets task demands. In this article, we describe and discuss the essential contribution of somatosensory feedback to locomotion. We start with a discussion of how biomechanical properties of the body affect somatosensory feedback. We follow with the different types of mechanoreceptors and somatosensory afferents and their activity during locomotion. We then describe central projections to locomotor networks and the modulation of somatosensory feedback during locomotion and its mechanisms. We then discuss experimental approaches and animal models used to investigate the control of locomotion by somatosensory feedback before providing an overview of the different functional roles of somatosensory feedback for locomotion. Lastly, we briefly describe the role of somatosensory feedback in the recovery of locomotion after neurological injury. We highlight the fact that somatosensory feedback is an essential component of a highly integrated system for locomotor control. © 2021 American Physiological Society. Compr Physiol 11:1-71, 2021.

The Role of Muscle Spindle Feedback in the Guidance of Hindlimb Movement by the Ipsilateral Forelimb during Locomotion in Mice.

Safe and efficient locomotion relies on placing the foot on a reliable surface at the end of each leg swing movement. Visual information has been shown to be important for determining the location of foot placement in humans during walking when precision is required. Yet in quadrupedal animals where the hindlimbs are outside of the visual field, such as in mice, the mechanisms by which precise foot placement is achieved remain unclear. Here we show that the placement of the hindlimb paw is determined by the position of the forelimb paw during normal locomotion and in the presence of perturbations. When a perturbation elicits a stumbling corrective reaction, we found that the forelimb paw shifts posteriorly relative to body at the end of stance, and this spatial shift is echoed in hindlimb paw placement at the end of the swing movement. Using a mutant mouse line in which muscle spindle feedback is selectively removed, we show that this posterior shift of paw placement is dependent on muscle spindle feedback in the hindlimb but not in the forelimb. These findings uncover a neuronal mechanism that is independent of vision to ensure safe locomotion during perturbation. This mechanism adds to our general knowledge of how the nervous system controls targeted limb movements and could inform the development of autonomous walking machines.

C-Boutons and Their Influence on Amyotrophic Lateral Sclerosis Disease Progression.

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease with progressive motor neuron death, where patients usually die within 5 years of diagnosis. Previously, we showed that the C-boutons, which are large cholinergic synapses to motor neurons that modulate motor neuron activity, are necessary for behavioral compensation in mSOD1G93A mice, a mouse model for ALS. We reasoned that, since the C-boutons likely increase the excitability of surviving motor neurons to compensate for motor neuron loss during ALS disease progression, then amplitude modulation through the C-boutons likely increases motor neuron stress and worsens disease progression. By comparing male and female mSOD1G93A mice to mSOD1G93A mice with genetically silenced C-boutons [mSOD1G93A ; Dbx1::cre; ChATfl/fl (mSOD1G93A/Coff )], we show that the C-boutons do not influence the humane end point of mSOD1G93A mice; however, our histologic analysis shows that C-bouton silencing significantly improves fast-twitch muscle innervation over time. Using immunohistology, we also show that the C-boutons are active in a task-dependent manner, and that symptomatic mSOD1G93A mice show significantly higher C-bouton activity than wild-type mice during low-intensity walking. Last, by using behavioral analysis, we provide evidence that C-bouton silencing in combination with swimming is beneficial for the behavioral capabilities of mSOD1G93A mice. Our observations suggest that manipulating the C-boutons in combination with a modulatory-targeted training program may therefore be beneficial for ALS patients and could result in improved mobility and quality of life.SIGNIFICANCE STATEMENT Despite decades of research on amyotrophic lateral sclerosis (ALS), there have been little improvements in treatments and therapies. We sought to better understand how the activation of C-boutons, which are large cholinergic modulatory synapses on motor neurons, change and affect the disease as it progresses. When these C-boutons are genetically silenced and exercises designed to otherwise activate the C-boutons are frequently performed in ALS model mice, the mice perform better than their untreated counterparts over time. C-bouton-targeted therapies could therefore be beneficial for ALS patients and could result in improved mobility and quality of life.

Freely Behaving Mice Can Brake and Turn During Optogenetic Stimulation of the Mesencephalic Locomotor Region.

A key function of the mesencephalic locomotor region (MLR) is to control the speed of forward symmetrical locomotor movements. However, the ability of freely moving mammals to integrate environmental cues to brake and turn during MLR stimulation is poorly documented. Here, we investigated whether freely behaving mice could brake or turn, based on environmental cues during MLR stimulation. We photostimulated the cuneiform nucleus (part of the MLR) in mice expressing channelrhodopsin in Vglut2-positive neurons in a Cre-dependent manner (Vglut2-ChR2-EYFP) using optogenetics. We detected locomotor movements using deep learning. We used patch-clamp recordings to validate the functional expression of channelrhodopsin and neuroanatomy to visualize the stimulation sites. In the linear corridor, gait diagram and limb kinematics were similar during spontaneous and optogenetic-evoked locomotion. In the open-field arena, optogenetic stimulation of the MLR evoked locomotion, and increasing laser power increased locomotor speed. Mice could brake and make sharp turns (~90°) when approaching a corner during MLR stimulation in the open-field arena. The speed during the turn was scaled with the speed before the turn, and with the turn angle. Patch-clamp recordings in Vglut2-ChR2-EYFP mice show that blue light evoked short-latency spiking in MLR neurons. Our results strengthen the idea that different brainstem neurons convey braking/turning and MLR speed commands in mammals. Our study also shows that Vglut2-positive neurons of the cuneiform nucleus are a relevant target to increase locomotor activity without impeding the ability to brake and turn when approaching obstacles, thus ensuring smooth and adaptable navigation. Our observations may have clinical relevance since cuneiform nucleus stimulation is increasingly considered to improve locomotion function in pathological states such as Parkinson's disease, spinal cord injury, or stroke.

Relative Contribution of Proprioceptive and Vestibular Sensory Systems to Locomotion: Opportunities for Discovery in the Age of Molecular Science.

Locomotion is a fundamental animal behavior required for survival and has been the subject of neuroscience research for centuries. In terrestrial mammals, the rhythmic and coordinated leg movements during locomotion are controlled by a combination of interconnected neurons in the spinal cord, referred as to the central pattern generator, and sensory feedback from the segmental somatosensory system and supraspinal centers such as the vestibular system. How segmental somatosensory and the vestibular systems work in parallel to enable terrestrial mammals to locomote in a natural environment is still relatively obscure. In this review, we first briefly describe what is known about how the two sensory systems control locomotion and use this information to formulate a hypothesis that the weight of the role of segmental feedback is less important at slower speeds but increases at higher speeds, whereas the weight of the role of vestibular system has the opposite relation. The new avenues presented by the latest developments in molecular sciences using the mouse as the model system allow the direct testing of the hypothesis.

Fractal analysis of muscle activity patterns during locomotion: pitfalls and how to avoid them.

Time-dependent physiological data sets are often difficult to interpret objectively. Biosignals such as electromyogram, electroencephalogram, or single-neuron recordings can be interpreted using various linear and nonlinear methods. Each analysis technique aims at the explanation of different data features that might be visible or not to the naked eye. Here, we used linear decomposition based on machine learning to extract motor primitives (the time-dependent coefficients of muscle synergies) from the hindlimb electromyographic activity of mice during normal and mechanically perturbed locomotion. We set out to investigate the effects of calculation parameters and data quality on two nonlinear metrics derived from fractal analysis: the Higuchi's fractal dimension (HFD) and the Hurst exponent (H). Both HFD and H proved to be exceptionally sensitive to changes in motor primitives induced by external perturbations to locomotion. We discuss the potential pitfalls that might arise from fractal analysis by using examples based on surrogate data. We conclude giving some simple, data-driven suggestions to reduce the chance of misinterpretations when metrics such as HFD and H are applied to any biological signal containing elements of periodicity.NEW & NOTEWORTHY Despite the lack of consensus on how to perform fractal analysis of physiological time series, many studies rely on this technique. Here, we shed light on the potential pitfalls of using the Higuchi's fractal dimension and the Hurst exponent. We expose and suggest how to solve the drawbacks of such methods when applied to data from normal and perturbed locomotion by combining in vivo recordings and computational approaches.

Sensory Feedback Control of Locomotor Pattern Generation in Cats and Mice.

Traditionally, research aimed at the understanding of the sensory control of terrestrial mammalian locomotion has focused on cats as the animal model. But advances in molecular genetics and new methods to record movement in small animals have moved mice into the forefront of locomotor research. In this review article, I will first give an overview of what is known about sensory feedback control of locomotion, mainly emerged from experiments performed on cats. This overview will not be an exhaustive overview, but will rather aim to give a broad picture of what has been learned about the sensory control of locomotion using cats as the animal model. I will then give a brief summary of how the mouse is adding to these insights.

Cholinergic modulation of motor neurons through the C-boutons are necessary for the locomotor compensation for severe motor neuron loss during amyotrophic lateral sclerosis disease progression.

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterized by progressive motor neuron (MN) death that leads to muscle weakness, paralysis, and eventually death. When symptoms become clinically evident, patients and ALS model animals (mSod1G93A mice) have already lost a large portion of motor units, suggesting the existence of a compensatory mechanism that allows for reactively normal movement despite denervation. Furthermore, it has been shown that specialized cholinergic synapses, the C-boutons, regulate activity strength of motor output in a task dependent manner. We hypothesized that the cholinergic modulation of motor neurons through C-boutons increases motor neuron excitability, and that this C-bouton associated activity increase in surviving motor neurons could compensate for motor unit loss during ALS disease progression. We first provide a thorough analysis of the muscle denervation and behavioral changes in the mSod1G93A mice using immunohistology, electrophysiology, and quantitative analysis of locomotor behavior. Then, in support of our hypothesis, we show that task dependent modulation of hindlimb muscle activation that relies on C-bouton activation diminishes as the disease progresses. Furthermore, the capability of mSod1G93A mice to walk at higher speeds on a treadmill decreases significantly at younger ages when C-boutons are silenced. Our observations that C-bouton modulation of motor neurons is involved in compensation during ALS disease progression can have significant therapeutic implications for sustaining mobility and preserving the quality of life in human ALS patients.

Modular organization of murine locomotor pattern in the presence and absence of sensory feedback from muscle spindles.

KEY POINTS: Locomotion on land and in water requires the coordination of a great number of muscle activations and joint movements. Constant feedback about the position of own body parts in relation to the surrounding environment and the body itself (proprioception) is required to maintain stability and avoid failure. The central nervous system may follow a modular type of organization by controlling muscles in orchestrated groups (muscle synergies) rather than individually. We used this concept on genetically modified mice lacking muscle spindles, one of the two main classes of proprioceptors. We provide evidence that proprioceptive feedback is required by the central nervous system to accurately tune the modular organization of locomotion. ABSTRACT: For exploiting terrestrial and aquatic locomotion, vertebrates must build their locomotor patterns based on an enormous amount of variables. The great number of muscles and joints, together with the constant need for sensory feedback information (e.g. proprioception), make the task of controlling movement a problem with overabundant degrees of freedom. It is widely accepted that the central nervous system may simplify the creation and control of movement by generating activation patterns common to muscle groups, rather than specific to individual muscles. These activation patterns, called muscle synergies, describe the modular organization of movement. We extracted synergies through electromyography from the hind limb muscle activities of wild-type and genetically modified mice lacking sensory feedback from muscle spindles. Muscle spindle-deficient mice underwent a modification of the temporal structure (motor primitives) of muscle synergies that resulted in diminished functionality during walking. In addition, both the temporal and spatial (motor modules) components of synergies were severely affected when external perturbations were introduced or when animals were immersed in water. These findings show that sensory feedback from group Ia/II muscle spindles regulates motor function in normal and perturbed walking. Moreover, when group Ib Golgi tendon organ feedback is lacking due to enhanced buoyancy, the modular organization of swimming is almost completely compromised.

Excitatory and inhibitory crossed reflex pathways in mice.

Sensory information from one leg has been known to elicit reflex responses in the contralateral leg, known as "crossed reflexes," and these have been investigated extensively in cats and humans. Furthermore, experiments with mice have shown commissural pathways in detail by using in vitro and in vivo physiological approaches combined with genetics. However, the relationship between these commissural pathways discovered in mice and crossed reflex pathways described in cats and humans is not known. In this study, we analyzed the crossed reflex in mice by using in vivo electromyographic recording techniques combined with peripheral nerve stimulation protocols to provide a detailed description of the crossed reflex pathways. We show that excitatory crossed reflexes are mediated by both proprioceptive and cutaneous afferent activation. In addition, we provide evidence for a short-latency inhibitory crossed reflex pathway likely mediated by cutaneous feedback. Furthermore, the short-latency crossed inhibition is downregulated in the knee extensor muscle and the ankle flexor muscle during locomotion. In conclusion, this article provides an analysis of excitatory and inhibitory crossed reflex pathways during resting and locomoting mice in vivo. The data presented in this article pave the way for future research aimed at understanding crossed reflexes using genetics in mice. NEW & NOTEWORTHY We describe for the first time excitatory and inhibitory crossed reflex pathways in mouse spinal cord in vivo and show that the inhibitory pathways are modulated during walking. This is a first step toward an understanding of crossed reflexes and their function during walking using in vivo recording techniques combined with mouse genetics.

Role of muscle spindle feedback in regulating muscle activity strength during walking at different speed in mice.

Terrestrial animals increase their walking speed by increasing the activity of the extensor muscles. However, the mechanism underlying how this speed-dependent amplitude modulation is achieved remains obscure. Previous studies have shown that group Ib afferent feedback from Golgi tendon organs that signal force is one of the major regulators of the strength of muscle activity during walking in cats and humans. In contrast, the contribution of group Ia/II afferent feedback from muscle spindle stretch receptors that signal angular displacement of leg joints is unclear. Some studies indicate that group II afferent feedback may be important for amplitude regulation in humans, but the role of muscle spindle feedback in regulation of muscle activity strength in quadrupedal animals is very poorly understood. To examine the role of feedback from muscle spindles, we combined in vivo electrophysiology and motion analysis with mouse genetics and gene delivery with adeno-associated virus. We provide evidence that proprioceptive sensory feedback from muscle spindles is important for the regulation of the muscle activity strength and speed-dependent amplitude modulation. Furthermore, our data suggest that feedback from the muscle spindles of the ankle extensor muscles, the triceps surae, is the main source for this mechanism. In contrast, muscle spindle feedback from the knee extensor muscles, the quadriceps femoris, has no influence on speed-dependent amplitude modulation. We provide evidence that proprioceptive feedback from ankle extensor muscles is critical for regulating muscle activity strength as gait speed increases. NEW & NOTEWORTHY Animals upregulate the activity of extensor muscles to increase their walking speed, but the mechanism behind this is not known. We show that this speed-dependent amplitude modulation requires proprioceptive sensory feedback from muscle spindles of ankle extensor muscle. In the absence of muscle spindle feedback, animals cannot walk at higher speeds as they can when muscle spindle feedback is present.