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.

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.

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.

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.

Directed differentiation of functional corticospinal-like neurons from endogenous SOX6+/NG2+ cortical progenitors.

Corticospinal neurons (CSN) centrally degenerate in amyotrophic lateral sclerosis (ALS), along with spinal motor neurons, and loss of voluntary motor function in spinal cord injury (SCI) results from damage to CSN axons. For functional regeneration of specifically affected neuronal circuitry in vivo , or for optimally informative disease modeling and/or therapeutic screening in vitro , it is important to reproduce the type or subtype of neurons involved. No such appropriate in vitro models exist with which to investigate CSN selective vulnerability and degeneration in ALS, or to investigate routes to regeneration of CSN circuitry for ALS or SCI, critically limiting the relevance of much research. Here, we identify that the HMG-domain transcription factor Sox6 is expressed by a subset of NG2+ endogenous cortical progenitors in postnatal and adult cortex, and that Sox6 suppresses a latent neurogenic program by repressing inappropriate proneural Neurog2 expression by progenitors. We FACS-purify these genetically accessible progenitors from postnatal mouse cortex and establish a pure culture system to investigate their potential for directed differentiation into CSN. We then employ a multi-component construct with complementary and differentiation-sharpening transcriptional controls (activating Neurog2, Fezf2 , while antagonizing Olig2 with VP16:Olig2 ). We generate corticospinal-like neurons from SOX6+/NG2+ cortical progenitors, and find that these neurons differentiate with remarkable fidelity compared with corticospinal neurons in vivo . They possess appropriate morphological, molecular, transcriptomic, and electrophysiological characteristics, without characteristics of the alternate intracortical or other neuronal subtypes. We identify that these critical specifics of differentiation are not reproduced by commonly employed Neurog2 -driven differentiation. Neurons induced by Neurog2 instead exhibit aberrant multi-axon morphology and express molecular hallmarks of alternate cortical projection subtypes, often in mixed form. Together, this developmentally-based directed differentiation from genetically accessible cortical progenitors sets a precedent and foundation for in vitro mechanistic and therapeutic disease modeling, and toward regenerative neuronal repopulation and circuit repair.

The Dorsal Column Nuclei Scale Mechanical Sensitivity in Naive and Neuropathic Pain States.

Tactile perception relies on reliable transmission and modulation of low-threshold information as it travels from the periphery to the brain. During pathological conditions, tactile stimuli can aberrantly engage nociceptive pathways leading to the perception of touch as pain, known as mechanical allodynia. Two main drivers of peripheral tactile information, low-threshold mechanoreceptors (LTMRs) and postsynaptic dorsal column neurons (PSDCs), terminate in the brainstem dorsal column nuclei (DCN). Activity within the DRG, spinal cord, and DCN have all been implicated in mediating allodynia, yet the DCN remains understudied at the cellular, circuit, and functional levels compared to the other two. Here, we show that the gracile nucleus (Gr) of the DCN mediates tactile sensitivity for low-threshold stimuli and contributes to mechanical allodynia during neuropathic pain in mice. We found that the Gr contains local inhibitory interneurons in addition to thalamus-projecting neurons, which are differentially innervated by primary afferents and spinal inputs. Functional manipulations of these distinct Gr neuronal populations resulted in bidirectional changes to tactile sensitivity, but did not affect noxious mechanical or thermal sensitivity. During neuropathic pain, silencing Gr projection neurons or activating Gr inhibitory neurons was able to reduce tactile hypersensitivity, and enhancing inhibition was able to ameliorate paw withdrawal signatures of neuropathic pain, like shaking. Collectively, these results suggest that the Gr plays a specific role in mediating hypersensitivity to low-threshold, innocuous mechanical stimuli during neuropathic pain, and that Gr activity contributes to affective, pain-associated phenotypes of mechanical allodynia. Therefore, these brainstem circuits work in tandem with traditional spinal circuits underlying allodynia, resulting in enhanced signaling of tactile stimuli in the brain during neuropathic pain.

Ultraflexible electrodes for recording neural activity in the mouse spinal cord during motor behavior.

Implantable electrode arrays are powerful tools for directly interrogating neural circuitry in the brain, but implementing this technology in the spinal cord in behaving animals has been challenging due to the spinal cord's significant motion with respect to the vertebral column during behavior. Consequently, the individual and ensemble activity of spinal neurons processing motor commands remains poorly understood. Here, we demonstrate that custom ultraflexible 1-µm-thick polyimide nanoelectronic threads can conduct laminar recordings of many neuronal units within the lumbar spinal cord of unrestrained, freely moving mice. The extracellular action potentials have high signal-to-noise ratio, exhibit well-isolated feature clusters, and reveal diverse patterns of activity during locomotion. Furthermore, chronic recordings demonstrate the stable tracking of single units and their functional tuning over multiple days. This technology provides a path for elucidating how spinal circuits compute motor actions.

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.

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.

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.

SOX5 controls the sequential generation of distinct corticofugal neuron subtypes.

The molecular mechanisms controlling the development of distinct subtypes of neocortical projection neurons, and CNS neuronal diversity more broadly, are only now emerging. We report that the transcription factor SOX5 controls the sequential generation of distinct corticofugal neuron subtypes by preventing premature emergence of normally later-born corticofugal neurons. SOX5 loss-of-function causes striking overlap of the identities of the three principal sequentially born corticofugal neuron subtypes: subplate neurons, corticothalamic neurons, and subcerebral projection neurons. In Sox5(-/-) cortex, subplate neurons aberrantly develop molecular hallmarks and connectivity of subcerebral projection neurons; corticothalamic neurons are imprecisely differentiated, while differentiation of subcerebral projection neurons is accelerated. Gain-of-function analysis reinforces the critical role of SOX5 in controlling the sequential generation of corticofugal neurons--SOX5 overexpression at late stages of corticogenesis causes re-emergence of neurons with corticofugal features. These data indicate that SOX5 controls the timing of critical fate decisions during corticofugal neuron production and thus subtype-specific differentiation and neocortical neuron diversity.

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/.

Lmo4 and Clim1 progressively delineate cortical projection neuron subtypes during development.

Molecular controls over the development of the exceptional neuronal subtype diversity of the cerebral cortex are now beginning to be identified. The initial subtype fate decision early in the life of a neuron, and the malleability of this fate when the balance of key postmitotic signals is modified, reveals not only that a neuron is deterministically set on a general developmental path at its birth, but also that this program must be precisely executed during postmitotic differentiation. Here, we show that callosal projection neurons (CPN) and subcerebral projection neurons (subcerebral PN) in layer V of the neocortex share aspects of molecular identity after their birth that are progressively resolved during differentiation. The LIM-homeodomain-related genes Lmo4 and Clim1 are initially expressed by both CPN and subcerebral PN in layer V, and only during mid to late differentiation does expression of Lmo4 and Clim1 become largely segregated into distinct neuronal subtypes. This progressive postmitotic resolution of molecular identity reveals similarities and possibly shared evolutionary origin between layer V CPN and subcerebral PN, and provides insight into how and when these neuronal subtypes achieve their distinct identities during cortical development.

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.

Humor modulates the mesolimbic reward centers.

Humor plays an essential role in many facets of human life including psychological, social, and somatic functioning. Recently, neuroimaging has been applied to this critical human attribute, shedding light on the affective, cognitive, and motor networks involved in humor processing. To date, however, researchers have failed to demonstrate the subcortical correlates of the most fundamental feature of humor-reward. In an effort to elucidate the neurobiological substrate that subserves the reward components of humor, we undertook a high-field (3 Tesla) event-related functional MRI study. Here we demonstrate that humor modulates activity in several cortical regions, and we present new evidence that humor engages a network of subcortical regions including the nucleus accumbens, a key component of the mesolimbic dopaminergic reward system. Further, the degree of humor intensity was positively correlated with BOLD signal intensity in these regions. Together, these findings offer new insight into the neural basis of salutary aspects of humor.

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.