Vocal learning-associated convergent evolution in mammalian proteins and regulatory elements.

Vocal production learning ("vocal learning") is a convergently evolved trait in vertebrates. To identify brain genomic elements associated with mammalian vocal learning, we integrated genomic, anatomical, and neurophysiological data from the Egyptian fruit bat (Rousettus aegyptiacus) with analyses of the genomes of 215 placental mammals. First, we identified a set of proteins evolving more slowly in vocal learners. Then, we discovered a vocal motor cortical region in the Egyptian fruit bat, an emergent vocal learner, and leveraged that knowledge to identify active cis-regulatory elements in the motor cortex of vocal learners. Machine learning methods applied to motor cortex open chromatin revealed 50 enhancers robustly associated with vocal learning whose activity tended to be lower in vocal learners. Our research implicates convergent losses of motor cortex regulatory elements in mammalian vocal learning evolution.

Myomatrix arrays for high-definition muscle recording.

Neurons coordinate their activity to produce an astonishing variety of motor behaviors. Our present understanding of motor control has grown rapidly thanks to new methods for recording and analyzing populations of many individual neurons over time. In contrast, current methods for recording the nervous system's actual motor output - the activation of muscle fibers by motor neurons - typically cannot detect the individual electrical events produced by muscle fibers during natural behaviors and scale poorly across species and muscle groups. Here we present a novel class of electrode devices ('Myomatrix arrays') that record muscle activity at unprecedented resolution across muscles and behaviors. High-density, flexible electrode arrays allow for stable recordings from the muscle fibers activated by a single motor neuron, called a 'motor unit,' during natural behaviors in many species, including mice, rats, primates, songbirds, frogs, and insects. This technology therefore allows the nervous system's motor output to be monitored in unprecedented detail during complex behaviors across species and muscle morphologies. We anticipate that this technology will allow rapid advances in understanding the neural control of behavior and identifying pathologies of the motor system.

Large-scale capture of hidden fluorescent labels for training generalizable markerless motion capture models.

Deep learning-based markerless tracking has revolutionized studies of animal behavior. Yet the generalizability of trained models tends to be limited, as new training data typically needs to be generated manually for each setup or visual environment. With each model trained from scratch, researchers track distinct landmarks and analyze the resulting kinematic data in idiosyncratic ways. Moreover, due to inherent limitations in manual annotation, only a sparse set of landmarks are typically labeled. To address these issues, we developed an approach, which we term GlowTrack, for generating orders of magnitude more training data, enabling models that generalize across experimental contexts. We describe: a) a high-throughput approach for producing hidden labels using fluorescent markers; b) a multi-camera, multi-light setup for simulating diverse visual conditions; and c) a technique for labeling many landmarks in parallel, enabling dense tracking. These advances lay a foundation for standardized behavioral pipelines and more complete scrutiny of movement.

Myomatrix arrays for high-definition muscle recording.

Neurons coordinate their activity to produce an astonishing variety of motor behaviors. Our present understanding of motor control has grown rapidly thanks to new methods for recording and analyzing populations of many individual neurons over time. In contrast, current methods for recording the nervous system's actual motor output - the activation of muscle fibers by motor neurons - typically cannot detect the individual electrical events produced by muscle fibers during natural behaviors and scale poorly across species and muscle groups. Here we present a novel class of electrode devices ("Myomatrix arrays") that record muscle activity at unprecedented resolution across muscles and behaviors. High-density, flexible electrode arrays allow for stable recordings from the muscle fibers activated by a single motor neuron, called a "motor unit", during natural behaviors in many species, including mice, rats, primates, songbirds, frogs, and insects. This technology therefore allows the nervous system's motor output to be monitored in unprecedented detail during complex behaviors across species and muscle morphologies. We anticipate that this technology will allow rapid advances in understanding the neural control of behavior and in identifying pathologies of the motor system.

Reverse-translational identification of a cerebellar satiation network.

The brain is the seat of body weight homeostasis. However, our inability to control the increasing prevalence of obesity highlights a need to look beyond canonical feeding pathways to broaden our understanding of body weight control1-3. Here we used a reverse-translational approach to identify and anatomically, molecularly and functionally characterize a neural ensemble that promotes satiation. Unbiased, task-based functional magnetic resonance imaging revealed marked differences in cerebellar responses to food in people with a genetic disorder characterized by insatiable appetite. Transcriptomic analyses in mice revealed molecularly and topographically -distinct neurons in the anterior deep cerebellar nuclei (aDCN) that are activated by feeding or nutrient infusion in the gut. Selective activation of aDCN neurons substantially decreased food intake by reducing meal size without compensatory changes to metabolic rate. We found that aDCN activity terminates food intake by increasing striatal dopamine levels and attenuating the phasic dopamine response to subsequent food consumption. Our study defines a conserved satiation centre that may represent a novel therapeutic target for the management of excessive eating, and underscores the utility of a 'bedside-to-bench' approach for the identification of neural circuits that influence behaviour.

Modulation of tactile feedback for the execution of dexterous movement.

Although dexterity relies on the constant transmission of sensory information, unchecked feedback can be disruptive. Yet how somatosensory feedback from the hands is regulated and whether this modulation influences movement remain unclear. We found that mouse tactile afferents recruit neurons in the brainstem cuneate nucleus, whose activity is modulated by distinct classes of local inhibitory neurons. Manipulation of these inhibitory circuits suppresses or enhances the transmission of tactile information, which affects manual behaviors. Top-down cortical pathways innervate cuneate in a complementary pattern, with somatosensory cortical neurons targeting the core tactile region of cuneate and a large rostral cortical population driving feed-forward inhibition of tactile transmission through an inhibitory shell. These findings identify a circuit basis for tactile feedback modulation that enables the effective execution of dexterous movement.

Anipose: A toolkit for robust markerless 3D pose estimation.

Quantifying movement is critical for understanding animal behavior. Advances in computer vision now enable markerless tracking from 2D video, but most animals move in 3D. Here, we introduce Anipose, an open-source toolkit for robust markerless 3D pose estimation. Anipose is built on the 2D tracking method DeepLabCut, so users can expand their existing experimental setups to obtain accurate 3D tracking. It consists of four components: (1) a 3D calibration module, (2) filters to resolve 2D tracking errors, (3) a triangulation module that integrates temporal and spatial regularization, and (4) a pipeline to structure processing of large numbers of videos. We evaluate Anipose on a calibration board as well as mice, flies, and humans. By analyzing 3D leg kinematics tracked with Anipose, we identify a key role for joint rotation in motor control of fly walking. To help users get started with 3D tracking, we provide tutorials and documentation at http://anipose.org/.

The Cerebellar Nuclei and Dexterous Limb Movements.

Dexterous forelimb movements like reaching, grasping, and manipulating objects are fundamental building blocks of the mammalian motor repertoire. These behaviors are essential to everyday activities, and their elaboration underlies incredible accomplishments by human beings in art and sport. Moreover, the susceptibility of these behaviors to damage and disease of the nervous system can lead to debilitating deficits, highlighting a need for a better understanding of function and dysfunction in sensorimotor control. The cerebellum is central to coordinating limb movements, as defined in large part by Joseph Babinski and Gordon Holmes describing motor impairment in patients with cerebellar lesions over 100 years ago (Babinski, 1902; Holmes, 1917), and supported by many important human and animal studies that have been conducted since. Here, with a focus on output pathways of the cerebellar nuclei across mammalian species, we describe forelimb movement deficits observed when cerebellar circuits are perturbed, the mechanisms through which these circuits influence motor output, and key challenges in defining how the cerebellum refines limb movement.

Gain control in the sensorimotor system.

Coordinated movement depends on constant interaction between neural circuits that produce motor output and those that report sensory consequences. Fundamental to this process are mechanisms for controlling the influence that sensory signals have on motor pathways - for example, reducing feedback gains when they are disruptive and increasing gains when advantageous. Sensory gain control comes in many forms and serves diverse purposes - in some cases sensory input is attenuated to maintain movement stability and filter out irrelevant or self-generated signals, or enhanced to facilitate salient signals for improved movement execution and adaptation. The ubiquitous presence of sensory gain control across species at multiple levels of the nervous system reflects the importance of tuning the impact that feedback information has on behavioral output.

Proprioception.

Although familiar to each of us, the sensation of inhabiting a body is ineffable. Traditional senses like vision and hearing monitor the external environment, allowing humans to have shared sensory experiences. But proprioception, the sensation of body position and movement, is fundamentally personal and typically absent from conscious perception. Nonetheless, this 'sixth sense' remains critical to human experience, a fact that is most apparent when one considers those who have lost it. Take, for example, the case of Ian Waterman who, at the age of 19, suffered a rare autoimmune response to a flu infection that attacked the sensory neurons from his neck down. This infection deprived him of the sense of position, movement and touch in his body. With this loss of feedback came a complete inability to coordinate his movements. While he could compel his muscles to contract, he lost the ability to orchestrate these actions into purposeful behaviors, in essence leaving him immobile, unable to stand, walk, or use his body to interact with the world. Only after years of dedicated training was he able to re-learn to move his body entirely under visual control.

Reduction of aberrant NF-κB signalling ameliorates Rett syndrome phenotypes in Mecp2-null mice.

Mutations in the transcriptional regulator Mecp2 cause the severe X-linked neurodevelopmental disorder Rett syndrome (RTT). In this study, we investigate genes that function downstream of MeCP2 in cerebral cortex circuitry, and identify upregulation of Irak1, a central component of the NF-κB pathway. We show that overexpression of Irak1 mimics the reduced dendritic complexity of Mecp2-null cortical callosal projection neurons (CPN), and that NF-κB signalling is upregulated in the cortex with Mecp2 loss-of-function. Strikingly, we find that genetically reducing NF-κB signalling in Mecp2-null mice not only ameliorates CPN dendritic complexity but also substantially extends their normally shortened lifespan, indicating broader roles for NF-κB signalling in RTT pathogenesis. These results provide new insight into both the fundamental neurobiology of RTT, and potential therapeutic strategies via NF-κB pathway modulation.

Direct and indirect spino-cerebellar pathways: shared ideas but different functions in motor control.

The impressive precision of mammalian limb movements relies on internal feedback pathways that convey information about ongoing motor output to cerebellar circuits. The spino-cerebellar tracts (SCT) in the cervical, thoracic and lumbar spinal cord have long been considered canonical neural substrates for the conveyance of internal feedback signals. Here we consider the distinct features of an indirect spino-cerebellar route, via the brainstem lateral reticular nucleus (LRN), and the implications of this pre-cerebellar "detour" for the execution and evolution of limb motor control. Both direct and indirect spino-cerebellar pathways signal spinal interneuronal activity to the cerebellum during movements, but evidence suggests that direct SCT neurons are mainly modulated by rhythmic activity, whereas the LRN also receives information from systems active during postural adjustment, reaching and grasping. Thus, while direct and indirect spino-cerebellar circuits can both be regarded as internal copy pathways, it seems likely that the direct system is principally dedicated to rhythmic motor acts like locomotion, while the indirect system also provides a means of pre-cerebellar integration relevant to the execution and coordination of dexterous limb movements.

Skilled forelimb movements and internal copy motor circuits.

Mammalian skilled forelimb movements are remarkable in their precision, a feature that emerges from the continuous adjustment of motor output. Here we discuss recent progress in bridging the gap between theory and neural implementation in understanding the basis of forelimb motor refinement. One influential theory is that feedback from internal copy motor pathways enables fast prediction, through a forward model of the limb, an idea supported by behavioral studies that have explored how forelimb movements are corrected online and can adapt to changing conditions. In parallel, neural substrates of forelimb internal copy pathways are coming into clearer focus, in part through the use of genetically tractable animal models to isolate spinal and cerebellar circuits and explore their contributions to movement.

Presynaptic inhibition of spinal sensory feedback ensures smooth movement.

The precision of skilled movement depends on sensory feedback and its refinement by local inhibitory microcircuits. One specialized set of spinal GABAergic interneurons forms axo-axonic contacts with the central terminals of sensory afferents, exerting presynaptic inhibitory control over sensory-motor transmission. The inability to achieve selective access to the GABAergic neurons responsible for this unorthodox inhibitory mechanism has left unresolved the contribution of presynaptic inhibition to motor behaviour. We used Gad2 as a genetic entry point to manipulate the interneurons that contact sensory terminals, and show that activation of these interneurons in mice elicits the defining physiological characteristics of presynaptic inhibition. Selective genetic ablation of Gad2-expressing interneurons severely perturbs goal-directed reaching movements, uncovering a pronounced and stereotypic forelimb motor oscillation, the core features of which are captured by modelling the consequences of sensory feedback at high gain. Our findings define the neural substrate of a genetically hardwired gain control system crucial for the smooth execution of movement.

Skilled reaching relies on a V2a propriospinal internal copy circuit.

The precision of skilled forelimb movement has long been presumed to rely on rapid feedback corrections triggered by internally directed copies of outgoing motor commands, but the functional relevance of inferred internal copy circuits has remained unclear. One class of spinal interneurons implicated in the control of mammalian forelimb movement, cervical propriospinal neurons (PNs), has the potential to convey an internal copy of premotor signals through dual innervation of forelimb-innervating motor neurons and precerebellar neurons of the lateral reticular nucleus. Here we examine whether the PN internal copy pathway functions in the control of goal-directed reaching. In mice, PNs include a genetically accessible subpopulation of cervical V2a interneurons, and their targeted ablation perturbs reaching while leaving intact other elements of forelimb movement. Moreover, optogenetic activation of the PN internal copy branch recruits a rapid cerebellar feedback loop that modulates forelimb motor neuron activity and severely disrupts reaching kinematics. Our findings implicate V2a PNs as the focus of an internal copy pathway assigned to the rapid updating of motor output during reaching behaviour.

Internal and External Feedback Circuits for Skilled Forelimb Movement.

Skilled motor behavior emerges from interactions between efferent neural pathways that induce muscle contraction and feedback systems that report and refine movement. Two broad classes of feedback projections modify motor output, one from the periphery and a second that originates within the central nervous system. The mechanisms through which these pathways influence movement remain poorly understood, however. Here we discuss recent studies that delineate spinal circuitry that binds external and internal feedback pathways to forelimb motor behavior. A spinal presynaptic inhibitory circuit regulates the strength of external feedback, promoting limb stability during goal-directed reaching. A distinct excitatory propriospinal circuit conveys copies of motor commands to the cerebellum, establishing an internal feedback loop that rapidly modulates forelimb motor output. The behavioral consequences of manipulating these two circuits reveal distinct controls on motor performance and provide an initial insight into feedback strategies that underlie skilled forelimb movement.

Edging toward entelechy in motor control.

The organization and functional logic of corticospinal motor neurons and their target connections remains unclear, despite their evident influence on movement. Spinal interneurons mediate much of this influence, yet we know little about the way in which corticospinal neurons engage spinal interneurons. This is perhaps not surprising given that the principles of organization of local spinal microcircuits remain elusive--we have glimpses of an underlying order but lack a comprehensive view of their functional architecture. In this brief essay we make a case that a new focus on the intersection of cortical and spinal circuits may provide clarity to the interpretation of corticospinal motor neuron firing patterns and help specify the logic of corticospinal motor neuronal function.

Lmo4 establishes rostral motor cortex projection neuron subtype diversity.

The mammalian neocortex is parcellated into anatomically and functionally distinct areas. The establishment of area-specific neuronal diversity and circuit connectivity enables distinct neocortical regions to control diverse and specialized functional outputs, yet underlying molecular controls remain largely unknown. Here, we identify a central role for the transcriptional regulator Lim-only 4 (Lmo4) in establishing the diversity of neuronal subtypes within rostral mouse motor cortex, where projection neurons have particularly diverse and multi-projection connectivity compared with caudal motor cortex. In rostral motor cortex, we report that both subcerebral projection neurons (SCPN), which send projections away from the cerebrum, and callosal projection neurons (CPN), which send projections to contralateral cortex, express Lmo4, whereas more caudal SCPN and CPN do not. Lmo4-expressing SCPN and CPN populations are comprised of multiple hodologically distinct subtypes. SCPN in rostral layer Va project largely to brainstem, whereas SCPN in layer Vb project largely to spinal cord, and a subset of both rostral SCPN and CPN sends second ipsilateral caudal (backward) projections in addition to primary projections. Without Lmo4 function, the molecular identity of neurons in rostral motor cortex is disrupted and more homogenous, rostral layer Va SCPN aberrantly project to the spinal cord, and many dual-projection SCPN and CPN fail to send a second backward projection. These molecular and hodological disruptions result in greater overall homogeneity of motor cortex output. Together, these results identify Lmo4 as a central developmental control over the diversity of motor cortex projection neuron subpopulations, establishing their area-specific identity and specialized connectivity.