Rostro-caudal inhibition of hindlimb movements in the spinal cord of mice.
ABSTRACT: Inhibitory neurons in the adult mammalian spinal cord are known to locally modulate afferent feedback--from muscle proprioceptors and from skin receptors--to pattern motor activity for locomotion and postural control. Here, using optogenetic tools, we explored how the same population of inhibitory interneurons globally affects hindlimb movements in the spinal cord of both anesthetized and freely moving mice. Activation of inhibitory interneurons up to the middle/lower spinal cord i.e. T8-T9, were able to completely and globally suppress all ipsilateral hindlimb movements. Furthermore, the same population of interneurons--which inhibited movements--did not significantly change the sensory and proprioceptive information from the affected limbs to the cortex. These results suggest a rostro-caudal organization of inhibition in the spinal cord motor output without modulation of ascending sensory pathways.
Project description:Studies in chicks and mice have suggested that transcription factors mark functional subtypes of interneurons in the developing spinal cord. We used genetic, morphological, and physiological studies to test this proposed association in zebrafish. We found that Engrailed-1 expression uniquely marks a class of ascending interneurons, called circumferential ascending (CiA) interneurons, with ipsilateral axonal projections in both motor and sensory regions of spinal cord. These cells express the glycine transporter 2 gene and are the only known ipsilateral interneurons positive for this marker of inhibitory transmission. Patch recordings show that the CiA neurons are rhythmically active during swimming. Pairwise recordings from the CiA interneurons and postsynaptic cells reveal that the Engrailed-1 neurons produce monosynaptic, strychnine-sensitive inhibition of dorsal sensory interneurons and also inhibit more ventral neurons, including motoneurons and descending interneurons. We conclude that Engrailed-1 expression marks a class of inhibitory interneuron that seems to provide all of the ipsilateral glycinergic inhibition in the spinal cord of embryonic and larval fish. Individual Engrailed-1-positive cells are multifunctional, playing roles in both sensory gating and motor pattern generation. This primitive cell type may have given rise to several, more specialized glycinergic inhibitory interneurons in birds and mammals. Our data support the view that the subdivision of spinal cord into different regions by transcription factors defines a primitive functional organization of spinal interneurons that formed a developmental and evolutionary foundation on which more complex systems were built.
Project description:The spinal cord contains neural networks that enable regionally distinct motor outputs along the body axis. Nevertheless, it remains unclear how segment-specific motor computations are processed because the cardinal interneuron classes that control motor neurons appear uniform at each level of the spinal cord. V2a interneurons are essential to both forelimb and hindlimb movements, and here we identify two major types that emerge during development: type I neurons marked by high Chx10 form recurrent networks with neighboring spinal neurons and type II neurons that downregulate Chx10 and project to supraspinal structures. Types I and II V2a interneurons are arrayed in counter-gradients, and this network activates different patterns of motor output at cervical and lumbar levels. Single-cell RNA sequencing (RNA-seq) revealed type I and II V2a neurons are each comprised of multiple subtypes. Our findings uncover a molecular and anatomical organization of V2a interneurons reminiscent of the orderly way motor neurons are divided into columns and pools.
Project description:GABAergic interneurons are key elements in neural coding, but the mechanisms that assemble inhibitory circuits remain unclear. In the spinal cord, the transfer of sensory signals to motor neurons is filtered by GABAergic interneurons that act presynaptically to inhibit sensory transmitter release and postsynaptically to inhibit motor neuron excitability. We show here that the connectivity and synaptic differentiation of GABAergic interneurons that mediate presynaptic inhibition is directed by their sensory targets. In the absence of sensory terminals these GABAergic neurons shun other available targets, fail to undergo presynaptic differentiation, and withdraw axons from the ventral spinal cord. A sensory-specific source of brain derived neurotrophic factor induces synaptic expression of the GABA synthetic enzyme GAD65--a defining biochemical feature of this set of interneurons. The organization of a GABAergic circuit that mediates presynaptic inhibition in the mammalian CNS is therefore controlled by a stringent program of sensory recognition and signaling.
Project description:BACKGROUND: Rhythmic motor patterns for locomotion in vertebrates are generated in spinal cord neural networks known as spinal Central Pattern Generators (CPGs). A key element in pattern generation is the role of glycinergic synaptic transmission by interneurons that cross the cord midline and inhibit contralaterally-located excitatory neurons. The glycinergic inhibitory drive permits alternating and precisely timed motor output during locomotion such as walking or swimming. To understand better the evolution of this system we examined the physiology of the neural network controlling swimming in an invertebrate chordate relative of vertebrates, the ascidian larva Ciona intestinalis. RESULTS: A reduced preparation of the larva consisting of nerve cord and motor ganglion generates alternating swimming movements. Pharmacological and genetic manipulation of glycine receptors shows that they are implicated in the control of these locomotory movements. Morphological molecular techniques and heterologous expression experiments revealed that glycine receptors are inhibitory and are present on both motoneurones and locomotory muscle while putative glycinergic interneurons were identified in the nerve cord by labeling with an anti-glycine antibody. CONCLUSIONS: In Ciona intestinalis, glycine receptors, glycinergic transmission and putative glycinergic interneurons, have a key role in coordinating swimming movements through a simple CPG that is present in the motor ganglion and nerve cord. Thus, the strong association between glycine receptors and vertebrate locomotory networks may now be extended to include the phylum chordata. The results suggest that the basic network for 'spinal-like' locomotion is likely to have existed in the common ancestor of extant chordates some 650 M years ago.
Project description:Spasticity, a common complication after spinal cord injury (SCI), is frequently accompanied by chronic pain. The physiological origin of this pain (critical to its treatment) remains unknown, although spastic motor dysfunction has been related to the hyperexcitability of motoneurons and to changes in spinal sensory processing. Here we show that the pain mechanism involves changes in sensory circuits of the dorsal horn (DH) where nociceptive inputs integrate for pain processing. Spasticity is associated with the DH hyperexcitability resulting from an increase in excitation and disinhibition occurring in two respective types of sensory interneurons. In the tonic-firing inhibitory lamina II interneurons, glutamatergic drive was reduced while glycinergic inhibition was potentiated. In contrast, excitatory drive was boosted to the adapting-firing excitatory lamina II interneurons while GABAergic and glycinergic inhibition were reduced. Thus, increased activity of excitatory DH interneurons coupled with the reduced excitability of inhibitory DH interneurons post-SCI could provide a neurophysiological mechanism of central sensitization and chronic pain associated with spasticity.
Project description:The spinal cord contains neural networks that enable regionally-distinct motor outputs along the body axis. Nevertheless, it remains unclear how segment-specific motor computations are processed because the cardinal interneuron classes that control motor neurons appear uniform at each level of the spinal cord. V2a interneurons are essential to both fore and hindlimb movements and here we identified two major types that emerge during development: Type-I neurons marked by high Chx10 form recurrent networks with neighboring spinal neurons, and Type-II that downregulate Chx10 and project to supraspinal structures. Type-I and -II V2a interneurons are arrayed in counter-gradients and this network activates different patterns of motor output at cervical and lumbar levels. Single cell RNA-seq revealed Type-I and -II V2a neurons are each comprised of multiple subtypes. Our findings uncover a molecular and anatomical organization of V2a interneurons reminiscent of the orderly way motor neurons are divided into columns and pools. Overall design: Single-cell RNA-Seq (scRNA-Seq) conducted on cervical and lumbar spinal V2a interneurons from 2 P0 neonates. Cells were sorted via FACS and pooled as a single sample on the 10x Chromium Controller.
Project description:Little is known about the organizational and functional connectivity of the corticospinal (CS) circuits that are essential for voluntary movement. Here, we map the connectivity between CS neurons in the forelimb motor and sensory cortices and various spinal interneurons, demonstrating that distinct CS-interneuron circuits control specific aspects of skilled movements. CS fibers originating in the mouse motor cortex directly synapse onto premotor interneurons, including those expressing Chx10. Lesions of the motor cortex or silencing of spinal Chx10+ interneurons produces deficits in skilled reaching. In contrast, CS neurons in the sensory cortex do not synapse directly onto premotor interneurons, and they preferentially connect to Vglut3+ spinal interneurons. Lesions to the sensory cortex or inhibition of Vglut3+ interneurons cause deficits in food pellet release movements in goal-oriented tasks. These findings reveal that CS neurons in the motor and sensory cortices differentially control skilled movements through distinct CS-spinal interneuron circuits.
Project description:The neurotransmitter systems mediating spinal locomotion in response to epidural spinal cord stimulation (ES) have not been identified. Here, we examine the role of the serotonergic system in regulating locomotor behavior of decerebrated cats during ES at L4-L5. ES elicited coordinated, weight-bearing, hindlimb stepping with plantar foot placement on a moving treadmill belt. Ketanserin [a 5-hydroxytryptamine (serotonin) (5-HT)(2/7) receptor antagonist] depressed this locomotor activity: only weak rhythmic movements without plantar foot placement and depressed EMG activity were observed. Cyproheptadine, a nonselective 5-HT blocker, prevented facilitation of stepping by epidural stimulation. These data demonstrate an important role of the serotonergic system in facilitating locomotion in the presence of epidural stimulation. In the presence of ketanserin, passive movements of one forelimb in a step-like manner immediately induced stepping of both hindlimbs with EMG patterns similar to those observed with ES without ketanserin. Thus, a non-5-HT-dependent spinal circuitry projecting from the cervical to the lumbar region of the spinal cord can facilitate stepping. The specific neurotransmitters responsible for this forelimb-facilitated stepping of the hindlimbs are unknown. These data suggest that a 5-HT(2/7) receptor-dependent pathway that processes hindlimb locomotor-like proprioception to facilitate hindlimb stepping can be complemented with proprioceptive afferents from the forelimbs via a non-5-HT(2/7) receptor neurotransmitter system. Thus, different neurotransmitter receptor systems can be used to mediate the same type of sensory event, i.e., locomotor-like proprioception to facilitate the same motor task, i.e., hindlimb stepping.
Project description: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.
Project description:Muscle-specific populations of proprioceptive sensory neurons form selective connections with spinal motor neurons, implying the existence of molecular distinctions between proprioceptor subpopulations. Here, we compare the gene expression profiles of proprioceptors that supply an antagonistic muscle pair functioning at a single hindlimb joint. Overall design: TA and GS muscles in a Pv::YFP reporter line were injected at p0 with ctb-555; retrogradely labeled proprioceptors were manually isolated at p1 and pooled to form 25-30 neuron samples.