Renshaw Cells Research Articles

Two inhibitory neuronal classes govern acquisition and recall of spinal sensorimotor adaptation.

Spinal circuits are central to movement adaptation, yet the mechanisms within the spinal cord responsible for acquiring and retaining behavior upon experience remain unclear. Using a simple conditioning paradigm, we found that dorsal inhibitory neurons are indispensable for adapting protective limb-withdrawal behavior by regulating the transmission of a specific set of somatosensory information to enhance the saliency of conditioning cues associated with limb position. By contrast, maintaining previously acquired motor adaptation required the ventral inhibitory Renshaw cells. Manipulating Renshaw cells does not affect the adaptation itself but flexibly alters the expression of adaptive behavior. These findings identify a circuit basis involving two distinct populations of spinal inhibitory neurons, which enables lasting sensorimotor adaptation independently from the brain.

Investigating the roles of reflexes and central pattern generators in the control and modulation of human locomotion using a physiologically plausible neuromechanical model

Objective. Studying the neural components regulating movement in human locomotion is obstructed by the inability to perform invasive experimental recording in the human neural circuits. Neuromechanical simulations can provide insights by modeling the locomotor circuits. Past neuromechanical models proposed control of locomotion either driven by central pattern generators (CPGs) with simple sensory commands or by a purely reflex-based network regulated by state-machine mechanisms, which activate and deactivate reflexes depending on the detected gait cycle phases. However, the physiological interpretation of these state machines remains unclear. Here, we present a physiologically plausible model to investigate spinal control and modulation of human locomotion. Approach. We propose a bio-inspired controller composed of two coupled CPGs that produce the rhythm and pattern, and a reflex-based network simulating low-level reflex pathways and Renshaw cells. This reflex network is based on leaky-integration neurons, and the whole system does not rely on changing reflex gains according to the gait cycle state. The musculoskeletal model is composed of a skeletal structure and nine muscles per leg generating movement in sagittal plane. Main results. Optimizing the open parameters for effort minimization and stability, human kinematics and muscle activation naturally emerged. Furthermore, when CPGs were not activated, periodic motion could not be achieved through optimization, suggesting the necessity of this component to generate rhythmic behavior without a state machine mechanism regulating reflex activation. The controller could reproduce ranges of speeds from 0.3 to 1.9 m s−1. The results showed that the net influence of feedback on motoneurons (MNs) during perturbed locomotion is predominantly inhibitory and that the CPGs provide the timing of MNs’ activation by exciting or inhibiting muscles in specific gait phases. Significance. The proposed bio-inspired controller could contribute to our understanding of locomotor circuits of the intact spinal cord and could be used to study neuromotor disorders.

Inhibitory interneurons show early dysfunction in a SOD1 mouse model of amyotrophic lateral sclerosis.

Few studies in amyotrophic lateral sclerosis (ALS) measure effects of the disease on inhibitory interneurons synapsing onto motoneurons (MNs). However, inhibitory interneurons could contribute to dysfunction, particularly if altered before MN neuropathology, and establish a long-term imbalance of inhibition/excitation. We directly assessed excitability and morphology of glycinergic (GlyT2 expressing) ventral lumbar interneurons from SOD1G93AGlyT2eGFP (SOD1) and wild-type GlyT2eGFP (WT) mice on postnatal days 6-10. Patch clamp revealed dampened excitability in SOD1 interneurons, including depolarized persistent inward currents (PICs), increased voltage and current threshold for firing action potentials, along with a marginal decrease in afterhyperpolarization duration. Primary neurites of ventral SOD1 inhibitory interneurons were larger in volume and surface area than WT. GlyT2 interneurons were then divided into threesubgroups based on location: (1) interneurons within 100μm of the ventral white matter, where Renshaw cells (RCs) are located, (2) interneurons interspersed with MNs in lamina IX, and (3) interneurons in the intermediate ventral area including laminae VII and VIII. Ventral interneurons in the RC area were the most profoundly affected, exhibiting more depolarized PICs and larger primary neurites. Interneurons in lamina IX had depolarized PIC onset. In lamina VII-VIII, interneurons were least affected. In summary, inhibitory interneurons show very early region-specific perturbations poised to impact excitatory/inhibitory balance of MNs, modify motor output and provide early biomarkers of ALS. Therapeutics like riluzole that universally reduce CNS excitability could exacerbate the inhibitory dysfunction described here. KEY POINTS: Spinal inhibitory interneurons could contribute to amyotrophic lateral sclerosis (ALS) pathology, but their excitability has never been directly measured. We studied the excitability and morphology of glycinergic interneurons in early postnatal transgenic mice (SOD1G93A GlyT2eGFP). Interneurons were less excitable and had marginally smaller somas but larger primary neurites in SOD1mice. GlyT2 interneurons were analysed according to their localization within the ventral spinal cord. Interestingly, the greatest differences were observed in the most ventrally located interneurons. We conclude that inhibitory interneurons show presymptomatic changes that may contribute to excitatory/inhibitory imbalance in ALS.

Adult spinal Dmrt3 neurons receive direct somatosensory inputs from ipsi- and contralateral primary afferents and from brainstem motor nuclei.

In the spinal cord, sensory-motor circuits controlling motor activity are situated in the dorso-ventral interface. The neurons identified by the expression of the transcription factor Doublesex and mab-3 related transcription factor 3 (Dmrt3) have previously been associated with the coordination of locomotion in horses (Equus caballus, Linnaeus, 1758), mice (Mus musculus, Linnaeus, 1758), and zebrafish (Danio rerio, F. Hamilton, 1822). Based on earlier studies, we hypothesized that, in mice, these neurons may be positioned to receive sensory and central inputs to relay processed commands to motor neurons. Thus, we investigated the presynaptic inputs to spinal Dmrt3 neurons using monosynaptic retrograde replication-deficient rabies tracing. The analysis showed that lumbar Dmrt3 neurons receive inputs from intrasegmental neurons, and intersegmental neurons from the cervical, thoracic, and sacral segments. Some of these neurons belong to the excitatory V2a interneurons and to plausible Renshaw cells, defined by the expression of Chx10 and calbindin, respectively. We also found that proprioceptive primary sensory neurons of type Ia2, Ia3, and Ib, defined by the expression of calbindin, calretinin, and Brn3c, respectively, provide presynaptic inputs to spinal Dmrt3 neurons. In addition, we demonstrated that Dmrt3 neurons receive inputs from brain areas involved in motor regulation, including the red nucleus, primary sensory-motor cortex, and pontine nuclei. In conclusion, adult spinal Dmrt3 neurons receive inputs from motor-related brain areas as well as proprioceptive primary sensory neurons and have been shown to connect directly to motor neurons. Dmrt3 neurons are thus positioned to provide sensory-motor control and their connectivity is suggestive of the classical reflex pathways present in the spinal cord.

Transient increase in recurrent inhibition in amyotrophic lateral sclerosis as a putative protection from neurodegeneration.

Adaptive mechanisms in spinal circuits are likely involved in homeostatic responses to maintain motor output in amyotrophic lateral sclerosis. Given the role of Renshaw cells in regulating the motoneuron input/output gain, we investigated the modulation of heteronymous recurrent inhibition. Electrical stimulations were used to activate recurrent collaterals resulting in the Hoffmann reflex depression. Inhibitions from soleus motor axons to quadriceps motoneurons, and vice versa, were tested in 38 patients and matched group of 42 controls. Compared with controls, the mean depression of quadriceps reflex was larger in patients, while that of soleus was smaller, suggesting that heteronymous recurrent inhibition was enhanced in quadriceps but reduced in soleus. The modulation of recurrent inhibition was linked to the size of maximal direct motor response and lower limb dysfunctions, suggesting a significant relationship with the integrity of the target motoneuron pool and functional abilities. No significant link was found between the integrity of motor axons activating Renshaw cells and the level of inhibition. Enhanced inhibition was particularly observed in patients within the first year after symptom onset and with slow progression of lower limb dysfunctions. Normal or reduced inhibitions were mainly observed in patients with motor weakness first in lower limbs and greater dysfunctions in lower limbs. We provide the first evidence for enhanced recurrent inhibition and speculate that Renshaw cells might have transient protective role on motoneuron by counteracting hyperexcitability at early stages. Several mechanisms likely participate including cortical influence on Renshaw cell and reinnervation by slow motoneurons.

Synaptic Projections of Motoneurons Within the Spinal Cord.

Motoneurons have long been considered as the final common pathway of the nervous system, transmitting the neural impulses that are transduced into action.While many studies have focussed on the inputs that motoneurons receive from local circuits within the spinal cord and from other parts of the CNS, relatively few have investigated the targets of local axonal projections from motoneurons themselves, with the notable exception of those contacting Renshaw cells or other motoneurons.Recent research has not only characterised the detailed features of the excitatory connections between motoneurons and Renshaw cells but has also established that Renshaw cells are not the only target of motoneurons axons within the spinal cord. Motoneurons also form synaptic contacts with other motoneurons as well as with a subset of ventrally located V3 interneurons. These findings indicate that motoneurons cannot be simply viewed as the last relay station delivering the command drive to muscles, but perform an active role in the generation and modulation of motor patterns.

Genetic targeting of adult Renshaw cells using a Calbindin 1 destabilized Cre allele for intersection with Parvalbumin or Engrailed1

Renshaw cells (RCs) are one of the most studied spinal interneurons; however, their roles in motor control remain enigmatic in part due to the lack of experimental models to interfere with RC function, specifically in adults. To overcome this limitation, we leveraged the distinct temporal regulation of Calbindin (Calb1) expression in RCs to create genetic models for timed RC manipulation. We used a Calb1 allele expressing a destabilized Cre (dgCre) theoretically active only upon trimethoprim (TMP) administration. TMP timing and dose influenced RC targeting efficiency, which was highest within the first three postnatal weeks, but specificity was low with many other spinal neurons also targeted. In addition, dgCre showed TMP-independent activity resulting in spontaneous recombination events that accumulated with age. Combining Calb1-dgCre with Parvalbumin (Pvalb) or Engrailed1 (En1) Flpo alleles in dual conditional systems increased cellular and timing specificity. Under optimal conditions, Calb1-dgCre/Pvalb-Flpo mice targeted 90% of RCs and few dorsal horn neurons; Calb1-dgCre/En1-Flpo mice showed higher specificity, but only a maximum of 70% of RCs targeted. Both models targeted neurons throughout the brain. Restricted spinal expression was obtained by injecting intraspinally AAVs carrying dual conditional genes. These results describe the first models to genetically target RCs bypassing development.

Two opposite voltage-dependent currents control the unusual early development pattern of embryonic Renshaw cell electrical activity.

Renshaw cells (V1R) are excitable as soon as they reach their final location next to the spinal motoneurons and are functionally heterogeneous. Using multiple experimental approaches, in combination with biophysical modeling and dynamical systems theory, we analyzed, for the first time, the mechanisms underlying the electrophysiological properties of V1R during early embryonic development of the mouse spinal cord locomotor networks (E11.5-E16.5). We found that these interneurons are subdivided into several functional clusters from E11.5 and then display an unexpected transitory involution process during which they lose their ability to sustain tonic firing. We demonstrated that the essential factor controlling the diversity of the discharge pattern of embryonic V1R is the ratio of a persistent sodium conductance to a delayed rectifier potassium conductance. Taken together, our results reveal how a simple mechanism, based on the synergy of two voltage-dependent conductances that are ubiquitous in neurons, can produce functional diversity in embryonic V1R and control their early developmental trajectory.

Unveiling the neuromechanical mechanisms underlying the synergistic interactions in human sensorimotor system

Motor synergies are neural organizations of a set of redundant motor effectors that interact with one another to compensate for each other’s error and ensure the stabilization of a performance variable. Recent studies have demonstrated that central nervous system synergistically coordinates its numerous motor effectors through Bayesian multi-sensory integration. Deficiency in sensory synergy weakens the synergistic interaction between the motor effectors. Here, we scrutinize the neuromechanical mechanism underlying this phenomenon through spectral analysis and modeling. We validate our model-generated results using experimental data reported in the literature collected from participants performing a finger force production task with and without tactile feedback (manipulated through injection of anesthetic in fingers). Spectral analysis reveals that the error compensation feature of synergies occurs only at low frequencies. Modeling suggests that the neurophysiological structures involving short-latency back-coupling loops similar to the well-known Renshaw cells explain the deterioration of synergy due to sensory deprivation.

Spatial and Temporal Arrangement of Recurrent Inhibition in the Primate Upper Limb.

Renshaw cells mediate recurrent inhibition between motoneurons within the spinal cord. The function of this circuit is not clear; we previously suggested based on computational modeling that it may cancel oscillations in muscle activity around 10 Hz, thereby reducing physiological tremor. Such tremor is especially problematic for dexterous hand movements, yet knowledge of recurrent inhibitory function is sparse for the control of the primate upper limb, where no direct measurements have been made to date. In this study, we made intracellular penetrations into 89 motoneurons in the cervical enlargement of four terminally anesthetized female macaque monkeys, and recorded recurrent IPSPs in response to antidromic stimulation of motor axons. Recurrent inhibition was strongest to motoneurons innervating shoulder muscles and elbow extensors, weak to wrist and digit extensors, and almost absent to the intrinsic muscles of the hand. Recurrent inhibitory connections often spanned joints, for example from motoneurons innervating wrist and digit muscles to those controlling the shoulder and elbow. Wrist and digit flexor motoneurons sometimes inhibited the corresponding extensors, and vice versa. This complex connectivity presumably reflects the flexible usage of the primate upper limb. Using trains of stimuli to motor nerves timed as a Poisson process and coherence analysis, we also examined the temporal properties of recurrent inhibition. The recurrent feedback loop effectively carried frequencies up to 100 Hz, with a coherence peak around 20 Hz. The coherence phase validated predictions from our previous computational model, supporting the idea that recurrent inhibition may function to reduce tremor.SIGNIFICANCE STATEMENT We present the first direct measurements of recurrent inhibition in primate upper limb motoneurons, revealing that it is more flexibly organized than previous observations in cat. Recurrent inhibitory connections were relatively common between motoneurons controlling muscles that act at different joints, and between flexors and extensors. As in the cat, connections were minimal for motoneurons innervating the most distal intrinsic hand muscles. Empirical data are consistent with previous modeling: temporal properties of the recurrent inhibitory feedback loop are compatible with a role in reducing physiological tremor by suppressing oscillations around 10 Hz.

Comparative Study between Magnesium Sulfate and Pethidine for Controlling Shivering after Spinal Anesthesia

Abstract Background Shivering is an involuntary muscular activity. Increased muscle tone during shivering is due to temperature-induced changes in neuronal activity in the reticular formation. Synchronization of motor neurons during shivering may be mediated by recurrent inhibition through renshaw cells. Aim of the Work To verify the efficacy of magnesium sulfate for controlling post spinal shivering, to compare the efficacy of magnesium sulfate and pethidine for controlling post spinal shivering and to detect the side effects of both magnesium sulfate and pethidine after their use for controlling post spinal shivering. Patients and Methods This prospective study was conducted at El-Matarya Teaching Hospital from 2018 till 2019. After obtaining approval from the Research Ethical Committee of Ain Shams University, informed patient consent was obtained before the procedure. After giving the spinal anesthesia, only patients who developed post-spinal shivering were followed for the study. 60 patients with post-spinal shivering were included with the following criteria: Results Regarding age, weight, height and duration of surgery; there were no statistically significant differences between the two studied groups. Comparison of the two studied groups revealed no statistically significant changes at all times of measurement. Conclusion Magnesium sulfate in a dose of 30 mg/kg IV infusion in 100 ml normal saline over 10 min is effective for control of post spinal shivering. Pethidine in a dose of 0.5 mg/kg IV bolus is effective for control of post spinal shivering.

Recurrent inhibition during Jendrassik maneuver

Objective - Conflicting theoretical models exist regarding the mechanism related to the ability of the Jendrassik maneuver to reinforce reflex parameters. Our objective was to investigate if vigorous handgrip would induce changes in recurrent inhibition of soleus motoneurons. Method - Soleus H reflex was evoked by stimulating the tibial nerve at rest and during bilateral vigorous handgrip, alternating single (H1) and paired stimulation (H2). At paired stimulation we used interstimulus intervals of 10, 15, 20 and 25 ms and supramaximal test stimulus. H1- and H2-wave amplitudes were expressed as percentage of maximal M-wave amplitude. Conditioned H2 wave maximal (H2max) and minimal (H2) amplitudes evoked at rest and expressed as a percentage of the unconditioned H1max amplitude were compared with the corresponding values obtained during handgrip by means of paired Student test and Bonferroni correction. Subjects - At the study participated 28 healthy volunteers. Results - The H1max/Mmax × 100 values obtained during handgrip (37.5±10.1) were significantly higher than those obtained at rest (27.1±7.4). The H2max/H1max × 100-va-lues obtained at paired stimulation were significantly higher during handgrip than at rest, while no significant diffe-rence was found between the H2/H1max × 100-values obtained during handgrip and at rest respectively. Discussion - The H2max/H1max is determined by both the excitability of the motoneurons and the recurrent inhibition elicited by the conditioning stimulus, while H2/H1max indicates only the level of recurrent inhibition. According to our results the Renshaw cells retain their inhibitory effect on the soleus alpha motoneurons during remote muscle contraction. Conclusion - Soleus H reflex enhancement during Jendrassik maneuver is not due to decrease of recurrent inhibition.

Supraspinal Control of Recurrent Inhibition during Anisometric Contractions.

Increase in recurrent inhibition was observed during eccentric compared with isometric and concentric maximal voluntary contractions but the neural mechanisms involved in this specific control of the Renshaw cell activity are unknown. This study was designed to investigate the supraspinal control of the recurrent inhibition during anisometric contractions of the plantar flexor muscles. To that purpose, the paired Hoffmann-reflex (H-reflex) technique permitted to assess changes in homonymous recurrent pathway by comparing the modulations of test and conditioning H-reflexes (H' and H1, respectively) in the soleus (SOL) muscle during maximal and submaximal isometric, concentric and eccentric contractions. Submaximal contraction intensity was set at 50% of the SOL electromyographic activity recorded during maximal isometric contraction. Fourteen volunteer subjects participated in an experimental session designed to assess the activity of the recurrent inhibition pathway. The results indicate that the amplitude of H1 normalized to the maximal M-wave were similar (P > 0.05) regardless of the muscle contraction type and intensity. Whatever the contraction intensity, the ratio between H' and H1 amplitudes was significantly decreased (P < 0.05) during eccentric compared with isometric and concentric contractions. Furthermore, this ratio was significantly smaller (P < 0.05) during submaximal compared with maximal contractions whatever the muscle contraction type. Together, the current results confirm the supraspinal control of the Renshaw cell activity when muscle contraction intensity is modulated and show that this control remains similar for isometric, concentric and eccentric contractions. Data further suggest that recurrent inhibition pathway may serve as variable gain regulator at motoneuronal level to improve resolution in the control of motor output for the SOL during eccentric contractions.

Motor units as tools to evaluate profile of human Renshaw inhibition.

To uncover the synaptic profile of Renshaw inhibition on motoneurons, we stimulated thick motor axons and recorded from voluntarily-activated motor units. Stimuli generated a direct motor response on the whole muscle and an inhibitory response in active motor units. We have estimated the profile of Renshaw inhibition indirectly using the response of motor unit discharge rates to the stimulus. We have put forward a method of extrapolation that may be used to determine genuine synaptic potentials as they develop on motoneurons. These optimized techniques can be used in research and in clinics to fully appreciate Renshaw cell function in various neurological disorders. Although Renshaw inhibition (RI) has been extensively studied for decades, its precise role in motor control is yet to be discovered. One of the main handicaps is a lack of reliable methods for studying RI in conscious human subjects. We stimulated the lowest electrical threshold motor axons (thickest axons) in the tibial nerve and analysed the stimulus-correlated changes in discharge of voluntarily recruited low-threshold single motor units (SMUs) from the soleus muscle. In total, 54 distinct SMUs from 12 subjects were analysed. Stimuli that generated only the direct motor response (M-only) on surface electromyography induced an inhibitory response in the low-threshold SMUs. Because the properties of RI had to be estimated indirectly using the background discharge rate of SMUs, its profile varied with the discharge rate of the SMU. The duration of RI was found to be inversely proportional to the discharge rate of SMUs. Using this important finding, we have developed a method of extrapolation for estimating RI as it develops on motoneurons in the spinal cord. The frequency methods indicated that the duration of RI was between 30 and 40ms depending on the background firing rate of the units, and the extrapolation indicated that RI on silent motoneurons was ∼55ms. The present study establishes a novel methodology for studying RI in human subjects and hence may serve as a tool for improving our understanding of the involvement of RI in human motor control.

Electrophysiological properties of Ia excitation and recurrent inhibition in cat abdominal motoneurons.

Ia excitation and recurrent inhibition are basic neuronal circuits in motor control in hind limb. Renshaw cells receive synaptic inputs from axon collaterals of motoneurons and inhibit motoneurons and Ia inhibitory interneurons. It is important to know properties of Ia excitation and recurrent inhibition of trunk muscle such as abdominal muscles. The abdominal muscles have many roles and change those roles for different kind of functions. Intracellular recordings were obtained from the abdominal motoneurons of the upper lumbar segments in cats anesthetized. First, dorsal roots were left intact, and sensory and motor axons were electrically stimulated. Ia excitatory post-synaptic potentials were elicited in five of eight motoneurons at same segment stimulated. Second, dorsal roots were sectioned, and motor axons were electrically stimulated. Recurrent inhibitory post-synaptic potentials were elicited in one of 11 abdominal motoneurons. Renshaw cells extracellularly fired high-frequency bursts at short latency and at same segment stimulated.

Subtype Diversification and Synaptic Specificity of Stem Cell-Derived Spinal Interneurons

Neuronal diversification is a fundamental step in the construction of functional neural circuits, but how neurons generated from single progenitor domains acquire diverse subtype identities remains poorly understood.Here we developed an embryonic stem cell (ESC)-based system to model subtype diversification of V1 interneurons, a class of spinal neurons comprising four clades collectively containing dozens of molecularly distinct neuronal subtypes. We demonstrate that V1 subtype diversity can be modified by extrinsic signals. Inhibition of Notch and activation of retinoid signaling results in a switch to MafA clade identity and enriches differentiation of Renshaw cells, a specialized MafA subtype that mediates recurrent inhibition of spinal motor neurons. We show that Renshaw cells are intrinsically programmed to migrate to species-specific laminae upon transplantation and to form subtype-specific synapses with motor neurons. Our results demonstrate that stem cell-derived neuronal subtypes can be used to investigatemechanisms underlying neuronal subtype specification and circuit assembly.

Expressions of VGLUT1/2 in the inspiratory interneurons and GAD65/67 in the inspiratory Renshaw cells in the neonatal rat upper thoracic spinal cord

Although the inspiratory spinal interneurons are thought to provide a major fraction of the excitatory synaptic potentials to the inspiratory intercostal motoneurons, this has not been confirmed. To clarify whether some inspiratory spinal interneurons are glutamatergic, we obtained whole-cell recordings from the ventromedial area of the third thoracic segments in an isolated brainstem-spinal cord preparation from neonatal rat, and the recorded cells were filled with Lucifer Yellow for later visualization. We then examined the existence of mRNA of vesicular glutamate transporters 1 and/or 2 (VGLUT1/2) by performing in situ hybridization. To discriminate the interneurons from motoneurons, we electrically stimulated the third thoracic ventral root on the recorded side, and the results verified that the antidromic spike or excitatory postsynaptic potential was not evoked. In cases in which the ventral root stimulation evoked depolarizing postsynaptic potentials, we examined the existence of glutamic acid decarboxylase 65 and/or 67 (GAD65/67) mRNA using a mixed probe to verify whether the cell was truly a Renshaw cell. The long diameter of the recorded interneurons was 22 ± 8 μm; the short diameter was 13 ± 4 μm. The interneurons' input resistance was 598 ± 274 MΩ. The Renshaw cells had similar sizes and input resistance. Six of 11 interneurons expressed VGLUT1/2, and four of five Renshaw cells expressed GAD65/67. Our findings suggest that approximately one-half of the inspiratory interneurons in the ventromedial area of the neonatal rat thoracic spinal cord are glutamatergic, and these interneurons might enhance the inspiratory intercostal motor activity.

Persistent Sodium Current Drives Excitability of Immature Renshaw Cells in Early Embryonic Spinal Networks.

Spontaneous network activity (SNA) emerges in the spinal cord (SC) before the formation of peripheral sensory inputs and central descending inputs. SNA is characterized by recurrent giant depolarizing potentials (GDPs). Because GDPs in motoneurons (MNs) are mainly evoked by prolonged release of GABA, they likely necessitate sustained firing of interneurons. To address this issue we analyzed, as a model, embryonic Renshaw cell (V1R) activity at the onset of SNA (E12.5) in the embryonic mouse SC (both sexes). V1R are one of the interneurons known to contact MNs, which are generated early in the embryonic SC. Here, we show that V1R already produce GABA in E12.5 embryo, and that V1R make synaptic-like contacts with MNs and have putative extrasynaptic release sites, while paracrine release of GABA occurs at this developmental stage. In addition, we discovered that V1R are spontaneously active during SNA and can already generate several intrinsic activity patterns including repetitive-spiking and sodium-dependent plateau potential that rely on the presence of persistent sodium currents (INap). This is the first demonstration that INap is present in the embryonic SC and that this current can control intrinsic activation properties of newborn interneurons in the SC of mammalian embryos. Finally, we found that 5 μm riluzole, which is known to block INaP, altered SNA by reducing episode duration and increasing inter-episode interval. Because SNA is essential for neuronal maturation, axon pathfinding, and synaptogenesis, the presence of INaP in embryonic SC neurons may play a role in the early development of mammalian locomotor networks.SIGNIFICANCE STATEMENT The developing spinal cord (SC) exhibits spontaneous network activity (SNA) involved in the building of nascent locomotor circuits in the embryo. Many studies suggest that SNA depends on the rhythmic release of GABA, yet intracellular recordings of GABAergic neurons have never been performed at the onset of SNA in the SC. We first discovered that embryonic Renshaw cells (V1R) are GABAergic at E12.5 and spontaneously active during SNA. We uncover a new role for persistent sodium currents (INaP) in driving plateau potential in V1R and in SNA patterning in the embryonic SC. Our study thus sheds light on a role for INaP in the excitability of V1R and the developing SC.

Stoichiometry of the Heteromeric Nicotinic Receptors of the Renshaw Cell.

Neuronal nicotinic acetylcholine receptors (nAChRs) are pentamers built from a variety of subunits. Some are homomeric assemblies of α subunits, others heteromeric assemblies of α and β subunits which can adopt two stoichiometries (2α:3β or 3α:2β). There is evidence for the presence of heteromeric nAChRs with the two stoichiometries in the CNS, but it has not yet been possible to identify them at a given synapse. The 2α:3β receptors are highly sensitive to agonists, whereas the 3α:2β stoichiometric variants, initially described as low sensitivity receptors, are indeed activated by low and high concentrations of ACh. We have taken advantage of the discovery that two compounds (NS9283 and Zn) potentiate selectively the 3α:2β nAChRs to establish (in mice of either sex) the presence of these variants at the motoneuron-Renshaw cell (MN-RC) synapse. NS9283 prolonged the decay of the two-component EPSC mediated by heteromeric nAChRs. NS9283 and Zn also prolonged spontaneous EPSCs involving heteromeric nAChRs, and one could rule out prolongations resulting from AChE inhibition by NS9283. These results establish the presence of 3α:2β nAChRs at the MN-RC synapse. At the functional level, we had previously explained the duality of the EPSC by assuming that high ACh concentrations in the synaptic cleft account for the fast component and that spillover of ACh accounts for the slow component. The dual ACh sensitivity of 3α:2β nAChRs now allows to attribute to these receptors both components of the EPSC.SIGNIFICANCE STATEMENT Heteromeric nicotinic receptors assemble α and β subunits in pentameric structures, which can adopt two stoichiometries: 3α:2β or 2α:3β. Both stoichiometric variants are present in the CNS, but they have never been located and characterized functionally at the level of an identified synapse. Our data indicate that 3α:2β receptors are present at the spinal cord synapses between motoneurons and Renshaw cells, where their dual mode of activation (by high concentrations of ACh for synaptic receptors, by low concentrations of ACh for extrasynaptic receptors) likely accounts for the biphasic character of the synaptic current. More generally, 3α:2β nicotinic receptors appear unique by their capacity to operate both in the cleft of classical synapses and at extrasynaptic locations.

Recurrent excitation between motoneurones propagates across segments and is purely glutamatergic.

Spinal motoneurones (Mns) constitute the final output for the execution of motor tasks. In addition to innervating muscles, Mns project excitatory collateral connections to Renshaw cells (RCs) and other Mns, but the latter have received little attention. We show that Mns receive strong synaptic input from other Mns throughout development and into maturity, with fast-type Mns systematically receiving greater recurrent excitation than slow-type Mns. Optical recordings show that activation of Mns in one spinal segment can propagate to adjacent segments even in the presence of intact recurrent inhibition. While it is known that transmission at the neuromuscular junction is purely cholinergic and RCs are excited through both acetylcholine and glutamate receptors, here we show that neurotransmission between Mns is purely glutamatergic, indicating that synaptic transmission systems are differentiated at different postsynaptic targets of Mns.