Spinal Interneurons Research Articles

Neuroplasticity is the ability of the central nervous system (CNS) to adapt structurally and functionally in response to motor and sensory dysfunction caused by injury and disease. Spinal interneurons (INs) are key components of neuroplastic changes that ameliorate impaired motor function after CNS injury. A lateral spinal cord hemisection model exhibited spontaneous motor recovery of the hind limb on the affected side. Hence, neuroplastic changes within the spinal cord on the affected and/or unaffected side may occur during motor recovery following unilateral spinal cord injury (SCI). However, it remains unclear how the spinal neurons on the affected and unaffected side contribute to motor recovery in the ipsilesional hind limb following unilateral SCI. In this study, we aimed to explore whether the thoracic spinal neurons above the lesion were involved in the motor recovery of the ipsilesional hind limb in a unilateral SCI model. Following unilateral SCI, hind limb motor function on the ipsilateral side was initially impaired but showed spontaneous recovery in the behavioral tests, which was subsequently lost after ablation of thoracic spinal neurons in the ipsilesional spinal cord above the lesion. In contrast, changes in the ipsilesional hind limb motor function were not observed after ablation of the contralesional thoracic spinal neurons. These results suggest that thoracic spinal neurons on the ipsilesional side above the lesion are key components for hind limb motor recovery in a model of unilateral SCI.

The mammalian spinal locomotor network is composed of diverse populations of interneurons that collectively orchestrate and execute a range of locomotor behaviors. As the number of identified classes of spinal interneurons constituting the locomotor network continues to grow, it still remains unclear how the network’s collective activity corresponds to locomotor output on a step-by-step basis. To investigate this, we analyzed lumbar interneuron population recordings and multi-muscle electromyography from spinalized female cats performing air stepping and used artificial intelligence methods to uncover state space trajectories of spinal interneuron population activity on single step cycles and at millisecond timescales. Our analyses of interneuron population trajectories revealed that traversal of specific state space regions held millisecond-timescale correspondence to the timing adjustments of extensor-flexor alternation. Similarly, we found that small variations in the path of state space trajectories were tightly linked to single-step, microvolt-scale adjustments in the magnitude of muscle output. These results reveal a previously-unseen regional organization of spinal interneuron state space, which may serve as a unifying framework to study spinal network function across the diversity of behaviors the spinal cord is capable of producing.

Following traumatic cervical spinal cord injury (SCI), injury-induced functional changes within the perilesional forelimb circuits are an important cause of neurological dysfunction. K+/Cl− cotransporter 2 (KCC2) is a neuron-specific transmembrane protein essential for inhibitory neurotransmission. Reduced KCC2 expression post-SCI disrupts the excitatory/inhibitory ratio in spinal interneurons and blocks supraspinal neurotransmission. Recent advances in AAV9-based gene therapies present a promising approach to upregulate KCC2 and restore functional communication in the injured spinal circuits. This study aims to characterize the neurophysiological changes in a rodent model of bilateral contusion-compression cervical SCI and to assess the functional impact of KCC2 gene therapy in the injured spinal cord. We demonstrate that intrathecal AAV9 delivery of KCC2 enhances long-term forelimb and hindlimb motor recovery and improves neurophysiological outcomes following cervical SCI. This is accompanied by Luminex assay, transcriptional analysis, and immunohistochemical observations suggesting improvements in neuroanatomical preservation and neuroglial alterations in the perilesional circuits.

A major unresolved issue in managing severe pain is tolerance caused by repeated treatment of opioid analgesics. Here, it is demonstrated that tolerance-inducing treatment with morphine results in the persistent downregulation of transient receptor potential canonical 5 (TRPC5), impairing the Ca2+ homeostasis in GABAergic interneurons of the spinal dorsal horn (SDH) and consequently reducing GABA release. Spinal activation of TRPC5 by riluzole (RLZ) or lentiviral-mediated TRPC5 overexpression in GABAergic interneurons produces a long-lasting enhancement of morphine's analgesic effect. In contrast, pharmacological inhibition of TRPC5 and mice lacking TRPC5 accelerates the development of morphine tolerance. Mechanistically, it is found that transcriptional suppression of Trpc5 results from enhancer of zeste homolog 2 (EZH2)-mediated epigenetic modifications at the Trpc5 gene promoter. Morphine decreases the enrichment of RNA polymerase II at the Trpc5 promoter. Moreover, exposure to morphine increases EZH2 binding to the Trpc5 promoter, leading to the enrichment of histone H3 lysine-27 trimethylation (H3K27me3). Pharmacological blockade of EZH2 by EPZ6438 or genetic silencing in GABAergic interneurons reverses morphine tolerance. Thus, it is proposed that the clinical translation of these findings may help reduce the suffering of individuals with intractable pain.

Spinal motor neuron plasticity after hindlimb unloading in aged mice and its modulation by exercise with TRPM8-mediated cutaneous stimulation.

Propriospinal interneurons in the spinal cord integrate multiple modalities of supraspinal and sensory inputs to modulate motor activity and facilitate complex motor behaviors, such as locomotion, skilled reaching, or grasping. The important ability of modulating motor activity in response to changes in the environment is partly mediated by a population of spinal interneurons marked by the expression Isl1, called dI3 neurons. These dI3 neurons are located throughout the cervical and lumbar spinal cord, receive cutaneous and proprioceptive feedback, and project to motoneurons. Previous work has demonstrated that dI3s are implicated in cutaneous-evoked reflexes and play a role in behaviors such as locomotion and grip strength, as well as motor recovery after spinal cord injury; however, it is unclear how different dI3 populations are connected to motor networks across the spinal cord to facilitate these diverse and complex functions. Through optogenetic activation of individual dI3 subpopulations located in different segments of the spinal cord, we mapped the functional connectivity of dI3 premotor circuits across the lumbar and cervical enlargements. We demonstrate that individual dI3 subpopulations have unique connectivity patterns and together form short and long propriospinal circuits that are either ipsilateral or commissural. Our findings suggest that dI3 subpopulations modulate the activity of distinct motor pools to differentially modulate complex motor functions such as grasping or locomotion.

Using DeepLabCut-Live to probe state dependent neural circuits of behavior with closed-loop optogenetic stimulation.

Recent advances in potential mechanisms of epidural spinal cord stimulation for movement disorders.

Spinal V3 interneurons are glutamatergic neurons that are distributed among the dorsal, intermediate, and ventral spinal cord. They are involved in broad neural circuit connections in the central nervous system. Functionally, they play important roles in locomotion, such as the maintenance of robust and balanced gaits during walking. More importantly, after spinal cord injury, these neurons maintain their excitability and facilitate proprioceptive sensory transmission to motor neurons, which are crucial for the initiation of complex coordinated reciprocal and rhythmic activities that resemble locomotion. Thus, spinal V3 interneurons appear to be good candidates for the restoration of locomotion after spinal cord injury. Nevertheless, therapeutic strategies targeting spinal V3 interneurons for spinal cord injury are scarce. In this review, we summarize the functional roles of spinal V3 interneurons in locomotion across uninjured and injured states and come up with possible strategies targeting them to restore locomotor function after spinal cord injury. Currently, an increasing number of studies are dedicated to identifying spinal V3 interneurons and their roles in motor, sensory, and autonomic nervous system functions. However, there are still many unclear questions regarding the molecular and functional characteristics of V3 interneurons in the spinal cord, as well as their potential therapeutic effects after spinal cord injury. Future research should prioritize the in-depth characterization of this specific neuronal subpopulation based on its sensorimotor features to further enhance spinal cord repair and functional recovery.

Targeted neuromodulation of spinal interneurons enhances breathing in chronic spinal cord injury.

End-organs such as the bladder rely on a delicate balance between internal urgency and voluntary restraint. However, the specific spinal circuits coordinating these functions remain poorly defined. Here, we identify a genetically defined population of lumbosacral spinal interneurons marked byTrpc4expression that gate bladder sensory input and regulate micturition reflexes. Single-cell transcriptomics and in situ physiology reveal molecularly distinctTrpc4⁺ subtypes. Functional manipulation reveals thatTrpc4⁺ neurons are essential for coordinating bladder-sphincter activity and gating visceral pain. Their ablation leads to bladder hypersensitivity and voiding dysfunction, while targeted activation reverses these maladaptive states. Circuit tracing reveals convergence of primary afferent and descending brainstem inputs ontoTrpc4⁺ neurons. These findings establishTrpc4⁺ interneurons are critical for bladder sensory-motor integration and extend classical spinal gating models to encompass visceral pain and organ reflex control.

Alloknesis refers to itch caused by normally non itch-inducing stimuli, particularly light mechanical stimuli, such as contacts with clothes or other human bodies. This symptom occurs in patients suffering from chronic itch. While it has been mainly described in patients with atopic dermatitis, it is probably present in numerous other conditions and it could induce a severe burden. Until now, it is mainly diagnosed using Von Frey filaments and validated questionnaires are lacking. Alloknesis differs from mechanical pruritus in that it is linked to sensitization to pruritus and therefore occurs in pathological conditions, whereas mechanical pruritus (triggered by the presence of insects on the skin, for example) is a physiological phenomenon. While the role of central sensitization to pruritus in alloknesis is still poorly understood, the role of peripheral sensitization is becoming clearer. Interactions between low-threshold mechanoreceptors (LTMRs) and spinal interneurons are especially involved. Both the mechanical labelled pathway and the polymodal pathway have been shown to contribute to mechanical alloknesis. The mechanical labelled pathway comprises dedicated primary sensory neurons, spinal interneurons, and projection neurons that are functionally distinct from those involved in chemical itch. The polymodal pathway relies on a subset of primary sensory neurons traditionally associated with chemical itch, which can also transduce light mechanical stimuli through the activation of the mechanosensitive ion channel PIEZO1. Both converge onto the gastrin-releasing peptide (GRP) - GRP receptor (GRPR) chemical itch pathway in the spinal cord. Alloknesis is largely unknown to healthcare professionals and even more so to patients, and is not actively investigated. The objective of reducing alloknesis should be considered a therapeutic goal. To date, it has not been investigated in clinical trials. A novel research domain is emerging concerning this symptom, which exerts a substantial impact on the daily lives of numerous patients.

Background: The red nucleus (RN) is a phylogenetically conserved structure within the midbrain that is traditionally associated with general motor coordination; however, its specific role in controlling directional movement remains poorly understood. Methods: This study systematically investigates the function and mechanism of RN neurons in directional movement by combining stereotactic brain injections, fiber photometry recordings, multi-unit in vivo electrophysiological recordings, optogenetic manipulation, and anterograde trans-synaptic tracing. Results: We analyzed mice performing standardized T-maze turning tasks and revealed that anatomically distinct RN neuronal ensembles exhibit direction-selective activity patterns. These neurons demonstrate preferential activation during ipsilateral turning movements, with activity onset consistently occurring after movement initiation. We establish a causal relationship between RN neuronal activity and directional motor control: selective activation of RN glutamatergic neurons facilitates ipsilateral turning, whereas temporally precise inhibition significantly impairs the execution of these movements. Anterograde trans-synaptic tracing using H129 reveals that RN neurons selectively project to spinal interneuron populations responsible for ipsilateral flexion and coordinated limb movements. Conclusions: These findings offer a framework for understanding asymmetric motor control in the brain. This work redefines the RN as a specialized hub within midbrain networks that mediate lateralized movements and offers new avenues for neuromodulatory treatments for neurodegenerative and post-injury motor disorders.

EZH2-mediated suppression of TIMP1 in spinal GABAergic interneurons drives microglial activation via MMP-9-TLR2/4-NLRP3 signaling in neuropathic pain.

To date, precise restoration of proper connections between posttrauma axons and neurons following spinal cord injury (SCI) remains a substantial challenge. Here, we developed glutamate-linked upconversion nanoparticles (Glu-UCNP) to facilitate optogenetic control of axonal sprouting in SCI mice. After being specifically uptaken by the postsynaptic interneurons innervated by corticospinal tract (CST) axons, Glu-UCNP not only serves as internal light transducers that convert near-infrared light to visible light but also acts as nanobeacons that guide axonal sprouting toward postsynaptic neurons of glutamatergic synapses. This in situ optogenetic modulation successfully demonstrated the restoration of spinal motor circuits by rebuilding functional connections between CST axons and postsynaptic interneurons. It was corroborated by live-cell recording, immunofluorescence staining, in vivo Ca2+ imaging, and pellet-reaching tests. Transcriptome sequencing further elucidated the molecular network changes underlying this optogenetic modulation. These findings highlight the potential therapeutic applications of optogenetic modulation in the reassembly of neural circuits after SCI.

The proposed mechanisms of spinal cord stimulation (SCS) follow the polarization of dorsal column axons; however, the development of subparesthesia SCS has encouraged the consideration of different targets. Given their relative proximity to the stimulation electrodes and their role in pain processing (eg, synaptic processing and gate control theory), spinal cord dorsal horn interneurons may be attractive stimulation targets. We developed a computational modeling pipeline termed "quasiuniform-mirror assumption" and applied it to predict polarization of dorsal horn interneuron cell types (islet type, central type, stellate/radial, vertical-like) to SCS. The quasiuniform-mirror assumption allows the prediction of the peak and directional axes of dendrite polarization for each cell type and location in the dorsal horn, in addition to the impact of the stimulation pulse width and electrode configuration. For long pulses, the peak polarization per milliampere of SCS with a spaced bipolar configuration was islet type 3.5mV, central type 1.3mV, stellate/radial 1.4mV, and vertical-like 1.6mV. For stellate/radial, the peak dendrite polarization was dorsal-ventral, and for islet-type, the peak dendrite polarization was in the rostral-caudal axis. For islet type and central type cells, peak dendrite polarization was between stimulation electrodes, whereas for stellate/radial and vertical-like cells, peak dendrite polarization was under the stimulation electrodes. The impact of the pulse width depends on the membrane time constants. Assuming a 1-millisecond time constant, for a 1-millisecond or 100-μs pulse width, the peak dendrite polarization decreases (from direct current values) by approximately 33% and approximately 88%, respectively. Increasing the interelectrode distance beyond approximately 3 cm did not significantly increase the peak polarization but expanded the region of interneuron polarization. Predicted maximum polarization of islet-cells in the superficial dorsal horn at locations between electrodes is 4.6mV for 2 mA, 1-millisecond pulse SCS. A polarization of a few millivolts is sufficient to modulate synaptic processing through subthreshold mechanisms. Our simulations provide support for SCS approaches optimized to modulate the dendrites of dorsal horn neurons.

ABSTRACTLocomotion is a vital motor function for any living being. In vertebrates, a basic locomotor pattern is controlled by the spinal locomotor network (SLN). Although SLN has been extensively studied, due to technical difficulties, most data were obtained during fictive locomotion, and data about the activity of spinal neurons during locomotion with intact sensory feedback from limbs are extremely limited. Here, we overcame the technical problems and recorded the activity of putative spinal interneurons from spinal segments L4–L6 during treadmill forward locomotion evoked by stimulation of the mesencephalic locomotor region in the decerebrate cat. We found that neurons were activated and inactivated preferably within one of the four phase ranges presumably related to preparation for the limb lift‐off, the limb lift‐off, transition from the limb flexion to limb extension during swing, and the limb touch‐down. We analyzed the activity phases of recorded interneurons by using a new method that took into account the previously ignored information about the stability of neuronal modulation in the sequential locomotor cycles. We suggested that neurons with stable modulation (i.e., small dispersion of their activity phase in sequential cycles) represent the core of SLN. Our analysis revealed groups of neurons active approximately out of phase and presumably contributing to the control of vertical (VC) and horizontal (HC) components of the step. We found that most VC‐ and HC‐related neurons were located in the intermediate and dorsal/ventral parts of the grey matter, respectively. Our experimental data can be used as a benchmark for computational models of locomotor neuronal networks.

The central nervous system controls movement by combining neuromotor modules, known as muscle synergies. Previous studies suggest that spinal premotor interneurons (PreM-INs) contribute to the encoding of stable muscle synergies for voluntary movement. But descending and sensory inputs also influence motor outputs through the spinal interneuronal network, which may be configured by its inputs to encode different sets of muscle synergies depending on the network state, thereby recruiting different selections of synergies. Here we tested this possibility of state-dependent synergy encoding by examining the muscle synergies represented by the same upstream spinal interneurons under different activity states induced by various optogenetic stimulation patterns. Lumbosacral spinal units and electromyographic (EMG) activities of hindlimb muscles were simultaneously recorded from anaesthetized Thy1-ChR2 mice as the spinal cord was stimulated by one or two optic fibres at different intensities. The synergy encoded by each unit was revealed as a 'muscle field' derived from spike-triggered averages of EMG, whereas the entire muscle synergy set was factorized from the EMG. We found that although the muscle synergy set remained stable across stimulation conditions, the muscle fields of the same units were matched to different synergies within the set in different states. Thus the interneurons may flexibly adjust their connectivity with the motoneurons of the muscles as descending and sensory afferents impose different states on the spinal network. State-dependent encoding of muscle synergies may allow different synergies to be selected for producing stable movement in an ever-changing workspace environment. KEY POINTS: Muscle synergies for locomotion can be represented by spinal interneurons, as revealed by the interneurons' muscle fields derived from spike-triggered averages of EMG. The muscle field of a single spinal interneuron may vary under different stimulation conditions, as demonstrated by optogenetic stimulation. Encoding of muscle synergies is dependent on the state of spinal activities, thus facilitating the selection of appropriate synergies in different dynamic environments.

Idiopathic scoliosis (IS) is the most common form of spinal deformity with unclear pathogenesis. In this study, we first reanalyzed the loci associated with IS, drawing upon previous studies. Subsequently, we mapped these loci to candidate genes using either location-based or function-based strategies. To further substantiate our findings, we verified the enrichment of variants within these candidate genes across several large IS cohorts encompassing Chinese, East Asian, and European populations. Consequently, we identified variants in the EPHA4 gene as compelling candidates for IS. To confirm their pathogenicity, we generated zebrafish mutants of epha4a. Remarkably, the zebrafish epha4a mutants exhibited pronounced scoliosis during later stages of development, effectively recapitulating the IS phenotype. We observed that the epha4a mutants displayed defects in left-right coordination during locomotion, which arose from disorganized neural activation in these mutants. Our subsequent experiments indicated that the disruption of the central pattern generator (CPG) network, characterized by abnormal axon guidance of spinal cord interneurons, contributed to the disorganization observed in the mutants. Moreover, when knocked down efnb3b, the ligand for Epha4a, we observed similar CPG defects and disrupted left-right locomotion. These findings suggested that ephrin B3-Epha4 signaling is vital for the proper functioning of CPGs, and defects in this pathway could lead to scoliosis in zebrafish. Furthermore, we identified two cases of IS in NGEF, a downstream molecule in the EPHA4 pathway. Collectively, our data provide compelling evidence that neural patterning impairments and disruptions in CPGs may underlie the pathogenesis of IS.

Idiopathic scoliosis (IS) is the most common form of spinal deformity with unclear pathogenesis. In this study, we first reanalyzed the loci associated with IS, drawing upon previous studies. Subsequently, we mapped these loci to candidate genes using either location-based or function-based strategies. To further substantiate our findings, we verified the enrichment of variants within these candidate genes across several large IS cohorts encompassing Chinese, East Asian, and European populations. Consequently, we identified variants in the EPHA4 gene as compelling candidates for IS. To confirm their pathogenicity, we generated zebrafish mutants of epha4a. Remarkably, the zebrafish epha4a mutants exhibited pronounced scoliosis during later stages of development, effectively recapitulating the IS phenotype. We observed that the epha4a mutants displayed defects in left-right coordination during locomotion, which arose from disorganized neural activation in these mutants. Our subsequent experiments indicated that the disruption of the central pattern generator (CPG) network, characterized by abnormal axon guidance of spinal cord interneurons, contributed to the disorganization observed in the mutants. Moreover, when knocked down efnb3b, the ligand for Epha4a, we observed similar CPG defects and disrupted left-right locomotion. These findings suggested that ephrin B3-Epha4 signaling is vital for the proper functioning of CPGs, and defects in this pathway could lead to scoliosis in zebrafish. Furthermore, we identified two cases of IS in NGEF, a downstream molecule in the EPHA4 pathway. Collectively, our data provide compelling evidence that neural patterning impairments and disruptions in CPGs may underlie the pathogenesis of IS.