Identification of a spinal circuit for light touch and fine motor control.

Sensory circuits in the dorsal spinal cord integrate and transmit multiple cutaneous sensory modalities including the sense of light touch. Here, we identify a population of excitatory interneurons (INs) in the dorsal horn that are important for transmitting innocuous light touch sensation. These neurons express the ROR alpha (RORα) nuclear orphan receptor and are selectively innervated by cutaneous low threshold mechanoreceptors (LTMs). Targeted removal of RORα INs in the dorsal spinal cord leads to a marked reduction in behavioral responsiveness to light touch without affecting responses to noxious and itch stimuli. RORα IN-deficient mice also display a selective deficit in corrective foot movements. This phenotype, together with our demonstration that the RORα INs are innervated by corticospinal and vestibulospinal projection neurons, argues that the RORα INs direct corrective reflex movements by integrating touch information with descending motor commands from the cortex and cerebellum.

Identification of spinal circuits transmitting and gating mechanical pain.

Pain information processing in the spinal cord has been postulated to rely on nociceptive transmission (T) neurons receiving inputs from nociceptors and Aß mechanoreceptors, with Aß inputs gated through feed-forward activation of spinal inhibitory neurons (INs). Here, we used intersectional genetic manipulations to identify these critical components of pain transduction. Marking and ablating six populations of spinal excitatory and inhibitory neurons, coupled with behavioral and electrophysiological analysis, showed that excitatory neurons expressing somatostatin (SOM) include T-type cells, whose ablation causes loss of mechanical pain. Inhibitory neurons marked by the expression of dynorphin (Dyn) represent INs, which are necessary to gate Aß fibers from activating SOM(+) neurons to evoke pain. Therefore, peripheral mechanical nociceptors and Aß mechanoreceptors, together with spinal SOM(+) excitatory and Dyn(+) inhibitory neurons, form a microcircuit that transmits and gates mechanical pain. PAPERCLIP:

Parallel ascending spinal pathways for affective touch and pain.

The anterolateral pathway consists of ascending spinal tracts that convey pain, temperature and touch information from the spinal cord to the brain1-4. Projection neurons of the anterolateral pathway are attractive therapeutic targets for pain treatment because nociceptive signals emanating from the periphery are channelled through these spinal projection neurons en route to the brain. However, the organizational logic of the anterolateral pathway remains poorly understood. Here we show that two populations of projection neurons that express the structurally related G-protein-coupled receptors (GPCRs) TACR1 and GPR83 form parallel ascending circuit modules that cooperate to convey thermal, tactile and noxious cutaneous signals from the spinal cord to the lateral parabrachial nucleus of the pons. Within this nucleus, axons of spinoparabrachial (SPB) neurons that express Tacr1 or Gpr83 innervate distinct sets of subnuclei, and strong optogenetic stimulation of the axon terminals induces distinct escape behaviours and autonomic responses. Moreover, SPB neurons that  express Gpr83 are highly sensitive to cutaneous mechanical stimuli and receive strong synaptic inputs from both high- and low-threshold primary mechanosensory neurons. Notably, the valence associated with activation of SPB neurons that express Gpr83 can be either positive or negative, depending on stimulus intensity. These findings reveal anatomically, physiologically and functionally distinct subdivisions of the SPB tract that underlie affective aspects of touch and pain.

Identifying the pathways required for coping behaviours associated with sustained pain.

Animals and humans display two types of response to noxious stimuli. The first includes reflexive defensive responses that prevent or limit injury; a well-known example of these responses is the quick withdrawal of one's hand upon touching a hot object. When the first-line response fails to prevent tissue damage (for example, a finger is burnt), the resulting pain invokes a second-line coping response-such as licking the injured area to soothe suffering. However, the underlying neural circuits that drive these two strings of behaviour remain poorly understood. Here we show in mice that spinal neurons marked by coexpression of TAC1Cre and LBX1Flpo drive coping responses associated with pain. Ablation of these spinal neurons led to the loss of both persistent licking and conditioned aversion evoked by stimuli (including skin pinching and burn injury) that-in humans-produce sustained pain, without affecting any of the reflexive defensive reactions that we tested. This selective indifference to sustained pain resembles the phenotype seen in humans with lesions of medial thalamic nuclei1-3. Consistently, spinal TAC1-lineage neurons are connected to medial thalamic nuclei by direct projections and via indirect routes through the superior lateral parabrachial nuclei. Furthermore, the anatomical and functional segregation observed at the spinal level also applies to primary sensory neurons. For example, in response to noxious mechanical stimuli, MRGPRD- and TRPV1-positive nociceptors are required to elicit reflexive and coping responses, respectively. Our study therefore reveals a fundamental subdivision within the cutaneous somatosensory system, and challenges the validity of using reflexive defensive responses to measure sustained pain.

Spinal Interneurons as Gatekeepers to Neuroplasticity after Injury or Disease.

Spinal interneurons are important facilitators and modulators of motor, sensory, and autonomic functions in the intact CNS. This heterogeneous population of neurons is now widely appreciated to be a key component of plasticity and recovery. This review highlights our current understanding of spinal interneuron heterogeneity, their contribution to control and modulation of motor and sensory functions, and how this role might change after traumatic spinal cord injury. We also offer a perspective for how treatments can optimize the contribution of interneurons to functional improvement.

Spinal Circuits for Touch, Pain, and Itch.

The exteroceptive somatosensory system is important for reflexive and adaptive behaviors and for the dynamic control of movement in response to external stimuli. This review outlines recent efforts using genetic approaches in the mouse to map the spinal cord circuits that transmit and gate the cutaneous somatosensory modalities of touch, pain, and itch. Recent studies have revealed an underlying modular architecture in which nociceptive, pruritic, and innocuous stimuli are processed by distinct molecularly defined interneuron cell types. These include excitatory populations that transmit information about both innocuous and painful touch and inhibitory populations that serve as a gate to prevent innocuous stimuli from activating the nociceptive and pruritic transmission pathways. By dissecting the cellular composition of dorsal-horn networks, studies are beginning to elucidate the intricate computational logic of somatosensory transformation in health and disease.

Molecular layer perforant path-associated cells contribute to feed-forward inhibition in the adult dentate gyrus.

New neurons, which have been implicated in pattern separation, are continually generated in the dentate gyrus in the adult hippocampus. Using a genetically modified rabies virus, we demonstrated that molecular layer perforant pathway (MOPP) cells innervated newborn granule neurons in adult mouse brain. Stimulating the perforant pathway resulted in the activation of MOPP cells before the activation of dentate granule neurons. Moreover, activation of MOPP cells by focal uncaging of glutamate induced strong inhibition of granule cells. Together, these results indicate that MOPP cells located in the molecular layer of the dentate gyrus contribute to feed-forward inhibition of granule cells via perforant pathway activation.

Neurogenin3 restricts serotonergic neuron differentiation to the hindbrain.

The development of the nervous system is critically dependent on the production of functionally diverse neuronal cell types at their correct locations. In the embryonic neural tube, dorsoventral signaling has emerged as a fundamental mechanism for generating neuronal diversity. In contrast, far less is known about how different neuronal cell types are organized along the rostrocaudal axis. In the developing mouse and chick neural tube, hindbrain serotonergic neurons and spinal glutamatergic V3 interneurons are produced from ventral p3 progenitors, which possess a common transcriptional identity but are confined to distinct anterior-posterior territories. In this study, we show that the expression of the transcription factor Neurogenin3 (Neurog3) in the spinal cord controls the correct specification of p3-derived neurons. Gain- and loss-of-function manipulations in the chick and mouse embryo show that Neurog3 switches ventral progenitors from a serotonergic to V3 differentiation program by repressing Ascl1 in spinal p3 progenitors through a mechanism dependent on Hes proteins. In this way, Neurog3 establishes the posterior boundary of the serotonergic system by actively suppressing serotonergic specification in the spinal cord. These results explain how equivalent p3 progenitors within the hindbrain and the spinal cord produce functionally distinct neuron cell types.

Renshaw cell interneuron specialization is controlled by a temporally restricted transcription factor program.

The spinal cord contains a diverse array of physiologically distinct interneuron cell types that subserve specialized roles in somatosensory perception and motor control. The mechanisms that generate these specialized interneuronal cell types from multipotential spinal progenitors are not known. In this study, we describe a temporally regulated transcriptional program that controls the differentiation of Renshaw cells (RCs), an anatomically and functionally discrete spinal interneuron subtype. We show that the selective activation of the Onecut transcription factors Oc1 and Oc2 during the first wave of V1 interneuron neurogenesis is a key step in the RC differentiation program. The development of RCs is additionally dependent on the forkhead transcription factor Foxd3, which is more broadly expressed in postmitotic V1 interneurons. Our demonstration that RCs are born, and activate Oc1 and Oc2 expression, in a narrow temporal window leads us to posit that neuronal diversity in the developing spinal cord is established by the composite actions of early spatial and temporal determinants.

Functional subpopulations of V3 interneurons in the mature mouse spinal cord.

V3 interneurons (INs) are a major group of excitatory commissural interneurons in the spinal cord, and they are essential for producing a stable and robust locomotor rhythm. V3 INs are generated from the ventral-most progenitor domain, p3, but migrate dorsally and laterally during postmitotic development. At birth, they are located in distinctive clusters in the ventral horn and deep dorsal horn. To assess the heterogeneity of this genetically identified group of spinal INs, we combined patch-clamp recording and anatomical tracing with cluster analysis. We examined electrophysiological and morphological properties of mature V3 INs identified by their expression of tdTomato fluorescent proteins in Sim1(Cre/+); Rosa(floxstop26TdTom) mice. We identified two V3 subpopulations with distinct intrinsic properties and spatial distribution patterns. Ventral V3 INs, primarily located in lamina VIII, possess a few branching processes and were capable of generating rapid tonic firing spikes. By contrast, dorsal V3 INs exhibited a more complex morphology and relatively slow average spike frequency with strong adaptation, and they also displayed large sag voltages and post-inhibitory rebound potentials. Our data suggested that hyperpolarization-activated cation channel currents and T-type calcium channel currents may account for some of the membrane properties of V3 INs. Finally, we observed that ventral and dorsal V3 INs were active in different ways during running and swimming, indicating that ventral V3 INs may act as premotor neurons and dorsal V3 INs as relay neurons mediating sensory inputs. Together, we detected two physiologically and topographically distinct subgroups of V3 INs, each likely playing different roles in locomotor activities.

Circuits controlling vertebrate locomotion: moving in a new direction.

Neurobiologists have long sought to understand how circuits in the nervous system are organized to generate the precise neural outputs that underlie particular behaviours. The motor circuits in the spinal cord that control locomotion, commonly referred to as central pattern generator networks, provide an experimentally tractable model system for investigating how moderately complex ensembles of neurons generate select motor behaviours. The advent of novel molecular and genetic techniques coupled with recent advances in our knowledge of spinal cord development means that a comprehensive understanding of how the motor circuitry is organized and operates may be within our grasp.

A spinal synergy of excitatory and inhibitory neurons coordinates ipsilateral body movements.

Innate and goal-directed movements require a high-degree of trunk and appendicular muscle coordination to preserve body stability while ensuring the correct execution of the motor action. The spinal neural circuits underlying motor execution and postural stability are finely modulated by propriospinal, sensory and descending feedback, yet how distinct spinal neuron populations cooperate to control body stability and limb coordination remains unclear. Here, we identified a spinal microcircuit composed of V2 lineage-derived excitatory (V2a) and inhibitory (V2b) neurons that together coordinate ipsilateral body movements during locomotion. Inactivation of the entire V2 neuron lineage does not impair intralimb coordination but destabilizes body balance and ipsilateral limb coupling, causing mice to adopt a compensatory festinating gait and be unable to execute skilled locomotor tasks. Taken together our data suggest that during locomotion the excitatory V2a and inhibitory V2b neurons act antagonistically to control intralimb coordination, and synergistically to coordinate forelimb and hindlimb movements. Thus, we suggest a new circuit architecture, by which neurons with distinct neurotransmitter identities employ a dual-mode of operation, exerting either synergistic or opposing functions to control different facets of the same motor behavior.

Identification of an essential spinoparabrachial pathway for mechanical itch.

The sensation of itch is a protective response that is elicited by either mechanical or chemical stimuli. The neural pathways for itch transmission in the skin and spinal cord have been characterized previously, but the ascending pathways that transmit sensory information to the brain to evoke itch perception have not been identified. Here, we show that spinoparabrachial neurons co-expressing Calcrl and Lbx1 are essential for generating scratching responses to mechanical itch stimuli. Moreover, we find that mechanical and chemical itch are transmitted by separate ascending pathways to the parabrachial nucleus, where they engage separate populations of FoxP2PBN neurons to drive scratching behavior. In addition to revealing the architecture of the itch transmission circuitry required for protective scratching in healthy animals, we identify the cellular mechanisms underlying pathological itch by showing the ascending pathways for mechanical and chemical itch function cooperatively with the FoxP2PBN neurons to drive chronic itch and hyperknesis/alloknesis.

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.

V1 spinal neurons regulate the speed of vertebrate locomotor outputs.

The neuronal networks that generate vertebrate movements such as walking and swimming are embedded in the spinal cord. These networks, which are referred to as central pattern generators (CPGs), are ideal systems for determining how ensembles of neurons generate simple behavioural outputs. In spite of efforts to address the organization of the locomotor CPG in walking animals, little is known about the identity and function of the spinal interneuron cell types that contribute to these locomotor networks. Here we use four complementary genetic approaches to directly address the function of mouse V1 neurons, a class of local circuit inhibitory interneurons that selectively express the transcription factor Engrailed1. Our results show that V1 neurons shape motor outputs during locomotion and are required for generating 'fast' motor bursting. These findings outline an important role for inhibition in regulating the frequency of the locomotor CPG rhythm, and also suggest that V1 neurons may have an evolutionarily conserved role in controlling the speed of vertebrate locomotor movements.

Mafa-dependent GABAergic activity promotes mouse neonatal apneas.

While apneas are associated with multiple pathological and fatal conditions, the underlying molecular mechanisms remain elusive. We report that a mutated form of the transcription factor Mafa (Mafa4A) that prevents phosphorylation of the Mafa protein leads to an abnormally high incidence of breath holding apneas and death in newborn Mafa4A/4A mutant mice. This apneic breathing is phenocopied by restricting the mutation to central GABAergic inhibitory neurons and by activation of inhibitory Mafa neurons while reversed by inhibiting GABAergic transmission centrally. We find that Mafa activates the Gad2 promoter in vitro and that this activation is enhanced by the mutation that likely results in increased inhibitory drives onto target neurons. We also find that Mafa inhibitory neurons are absent from respiratory, sensory (primary and secondary) and pontine structures but are present in the vicinity of the hypoglossal motor nucleus including premotor neurons that innervate the geniohyoid muscle, to control upper airway patency. Altogether, our data reveal a role for Mafa phosphorylation in regulation of GABAergic drives and suggest a mechanism whereby reduced premotor drives to upper airway muscles may cause apneic breathing at birth.

A Functional Topographic Map for Spinal Sensorimotor Reflexes.

Cutaneous somatosensory modalities play pivotal roles in generating a wide range of sensorimotor behaviors, including protective and corrective reflexes that dynamically adapt ongoing movement and posture. How interneurons (INs) in the dorsal horn encode these modalities and transform them into stimulus-appropriate motor behaviors is not known. Here, we use an intersectional genetic approach to functionally assess the contribution that eight classes of dorsal excitatory INs make to sensorimotor reflex responses. We demonstrate that the dorsal horn is organized into spatially restricted excitatory modules composed of molecularly heterogeneous cell types. Laminae I/II INs drive chemical itch-induced scratching, laminae II/III INs generate paw withdrawal movements, and laminae III/IV INs modulate dynamic corrective reflexes. These data reveal a key principle in spinal somatosensory processing, namely, sensorimotor reflexes are driven by the differential spatial recruitment of excitatory neurons.

Mechanical Allodynia Circuitry in the Dorsal Horn Is Defined by the Nature of the Injury.

The spinal dorsal horn is a major site for the induction and maintenance of mechanical allodynia, but the circuitry that underlies this clinically important form of pain remains unclear. The studies presented here provide strong evidence that the neural circuits conveying mechanical allodynia in the dorsal horn differ by the nature of the injury. Calretinin (CR) neurons in lamina II inner convey mechanical allodynia induced by inflammatory injuries, while protein kinase C gamma (PKCγ) neurons at the lamina II/III border convey mechanical allodynia induced by neuropathic injuries. Cholecystokinin (CCK) neurons located deeper within the dorsal horn (laminae III-IV) are important for both types of injuries. Interestingly, the Maf+ subset of CCK neurons is composed of transient vesicular glutamate transporter 3 (tVGLUT3) neurons, which convey primarily dynamic allodynia. Identification of an etiology-based circuitry for mechanical allodynia in the dorsal horn has important implications for the mechanistic and clinical understanding of this condition.

Selective and quickly reversible inactivation of mammalian neurons in vivo using the Drosophila allatostatin receptor.

Genetic strategies for perturbing activity of selected neurons hold great promise for understanding circuitry and behavior. Several such strategies exist, but there has been no direct demonstration of reversible inactivation of mammalian neurons in vivo. We previously reported quickly reversible inactivation of neurons in vitro using expression of the Drosophila allatostatin receptor (AlstR). Here, adeno-associated viral vectors are used to express AlstR in vivo in cortical and thalamic neurons of rats, ferrets, and monkeys. Application of the receptor's ligand, allatostatin (AL), leads to a dramatic reduction in neural activity, including responses of visual neurons to optimized visual stimuli. Additionally, AL eliminates activity in spinal cords of transgenic mice conditionally expressing AlstR. This reduction occurs selectively in AlstR-expressing neurons. Inactivation can be reversed within minutes upon washout of the ligand and is repeatable, demonstrating that the AlstR/AL system is effective for selective, quick, and reversible silencing of mammalian neurons in vivo.