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International audience; Neuronal rhythmogenesis in the spinal cord is correlated with variations in extracellular K + levels ([K + ] e). Astrocytes play important role in [K + ] e homeostasis and compute neuronal information. Yet it is unclear how neuronal oscillations are regulated by astrocytic K + homeostasis. Here we identify the astrocytic inward-rectifying K + channel Kir4.1 (a.k.a. Kcnj10) as a key molecular player for neuronal rhythmicity in the spinal central pattern generator (CPG). By combining two-photon calcium imaging with electrophysiology, immunohistochemistry and genetic tools, we report that astrocytes display Ca 2+ transients before and during oscillations of neighboring neurons. Inhibition of astrocytic Ca 2+-transients with BAPTA decreases the barium-sensitive Kir4.1 current responsible of K + clearance. Finally, we show in mice that Kir4.1 knockdown in astrocytes progressively prevents neuronal oscillations and alters the locomotor pattern resulting in lower motor performances in challenging tasks. These data identify astroglial Kir4.1 channels as key regulators of neuronal rhythmogenesis in the CPG driving locomotion.

SummaryPersistent sodium current (INaP) in the spinal locomotor network promotes two distinct nonlinear firing patterns: a self-sustained spiking triggered by a brief excitation in bistable motoneurons and bursting oscillations in interneurons of the central pattern generator (CPG). Here, we identified the NaV channels responsible forINaPand their role in motor behaviors. We report the axonalNav1.6as the main molecular player forINaPin lumbar motoneurons. The motoneuronal inhibition ofNav1.6, but not ofNav1.1, impairsINaP, bistability, postural tone and locomotor performance. In interneurons of the CPG region,Nav1.6withNav1.1equally mediateINaPand the inhibition of both channels is required to abolish oscillatory bursting activities and the locomotor rhythm. Overall,Nav1.6plays a significant role both in posture and locomotion by governingINaP-dependent bistability in motoneurons and working in tandem withNav1.1to provideINaP-dependent rhythmogenic properties of the CPG.

SummaryNeuronal rhythmogenesis in the spinal cord is correlated with variations in extracellular K+ levels ([K+]e). Astrocytes play important role in[K+]e homeostasis and compute neuronal information. Yet it is unclear how neuronal oscillations are regulated by astrocytic K+ homeostasis. Here we identify the astrocytic inward-rectifying K+ channel Kir4.1 (a.k.a. Kcnj10) as a key molecular player for neuronal rhythmicity in the spinal central pattern generator (CPG). By combining two-photon calcium imaging with electrophysiology, immunohistochemistry and genetic tools, we report that astrocytes display Ca2+ transients before and during oscillations of neighbouring neurons. Inhibition of astrocytic Ca2+ transients with BAPTA decreases the barium-sensitive Kir4.1 current responsible of K+ clearance. Finally, we show in mice that Kir4.1 knockdown in astrocytes progressively prevents neuronal oscillations and alters the locomotor pattern resulting in lower motor performances in challenging tasks. These data identify astroglial Kir4.1 channels as key regulators of neuronal rhythmogenesis in the CPG driving locomotion.Significance statementDespite decades of research, the cellular mechanisms responsible of the synchronized rhythmic oscillations driving locomotion remain elusive. To gain insight into the function of the spinal locomotor network, numerous studies have characterized diverse classes of locomotor-related neurons to determine their role in generating rhythmic movements during locomotion. In contrast, studies investigating non-neuronal components of the spinal cord are sparse. Our study represents a significant breakthrough by identifying astrocytic K+ uptake as a key regulator of neuronal rhythmicity synchronization and locomotor pattern at the cellular, microcircuit and system levels. These data provide mechanistic insights into the neuroglial dialogue at play during rhythmogenesis and point to a novel astroglial target for restoring normal neuronal network excitability in brain disorders and neurodegenerative diseases.

Bistable motoneurons of the spinal cord exhibit warmth-activated plateau potential driven by Na+ and triggered by a brief excitation. The thermoregulating molecular mechanisms of bistability and their role in motor functions remain unknown. Here, we identify thermosensitive Na+-permeable Trpm5 channels as the main molecular players for bistability in mouse motoneurons. Pharmacological, genetic or computational inhibition of Trpm5 occlude bistable-related properties (slow afterdepolarization, windup, plateau potentials) and reduce spinal locomotor outputs while central pattern generators for locomotion operate normally. At cellular level, Trpm5 is activated by a ryanodine-mediated Ca2+ release and turned off by Ca2+ reuptake through the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump. Mice in which Trpm5 is genetically silenced in most lumbar motoneurons develop hindlimb paresis and show difficulties in executing high-demanding locomotor tasks. Overall, by encoding bistability in motoneurons, Trpm5 appears indispensable for producing a postural tone in hindlimbs and amplifying the locomotor output., The authors show that Trpm5, but not Trpm4, is the main Na+ -permeant channel mediating the warmth-activated ICaN in lumbar motoneurons. Trpm5 is also critical in generating plateau potentials in bistable motoneurons that are essential for producing a postural tone in hindlimbs and amplifying the locomotor output.

Amyotrophic lateral sclerosis (ALS) is a devastating motoneuron (Mn) disease without effective cure currently available. Death of MNs in ALS is preceded by failure of neuromuscular junctions and axonal retraction. Neuregulin 1 (NRG1) is a neurotrophic factor highly expressed in MNs and neuromuscular junctions that support axonal and neuromuscular development and maintenance. NRG1 and its ErbB receptors are involved in ALS. Reduced NRG1 expression has been found in ALS patients and in the ALS SOD1(G93A) mouse model; however, the expression of the isoforms of NRG1 and its receptors is still controversial. Due to the reduced levels of NRG1 type III (NRG1-III) in the spinal cord of ALS patients, we used gene therapy based on intrathecal administration of adeno-associated virus to overexpress NRG1-III in SOD1(G93A) mice. The mice were evaluated from 9 to 16 weeks of age by electrophysiology and rotarod tests. At 16 weeks, samples were harvested for histological and molecular analyses. Our results indicate that overexpression of NRG1-III is able to preserve neuromuscular function of the hindlimbs, improve locomotor performance, increase the number of surviving MNs, and reduce glial reactivity in the treated female SOD1(G93A) mice. Furthermore, the NRG1-III/ErbB4 axis appears to regulate MN excitability by modulating the chloride transporter KCC2 and reduces the expression of the MN vulnerability marker MMP-9. However, NRG1-III did not have a significant effect on male mice, indicating relevant sex differences. These findings indicate that increasing NRG1-III at the spinal cord is a promising approach for promoting MN protection and functional improvement in ALS. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13311-019-00811-7) contains supplementary material, which is available to authorized users.

SummaryWe recently described new pathogenic variants in VRK1, in patients affected with distal Hereditary Motor Neuropathy associated with upper motor neurons signs. Specifically, we provided evidences that hiPSC-derived Motor Neurons (hiPSC-MN) from these patients display Cajal bodies (CBs) disassembly and defects in neurite outgrowth and branching.We here focused on the Axonal Initial Segment (AIS) and the related firing properties of hiPSC-MNs from these patients. We found that the patient’s Action Potential (AP) was smaller in amplitude, larger in duration, and displayed a more depolarized threshold while the firing patterns were not altered. These alterations were accompanied by a decrease in the AIS length measured in patients’ hiPSC-MNs. These data indicate that mutations in VRK1 impact the AP waveform and the AIS organization in MNs and may ultimately lead to the related motor neuron disease.HighlightshiPSC-MNs are functional and sustain firing patterns, typical of spinal MNshiPSC-MNs from patients with VRK1 mutations have altered Action PotentialAxonal Initial Segment is shorter in hiPSC-MNs from patients with mutated VRK1hiPSC-MNs are a useful platform to study Motor Inherited Peripheral NeuropathieseTOC BlurbIn human spinal Motor Neurons derived from induced Pluripotent Stem Cells from patients with VRK1 -related distal Hereditary Motor Neuropathy, Bos, Rihan et al. show that the mutations in VRK1 affect the electrical properties of these neurons: they display defects in the initiation of the Action Potential due to a shortening of the Axonal Initial Segment.

The central pattern generator (CPG) for locomotion is a set of pacemaker neurons endowed with inherent bursting driven by the persistent sodium current (INaP). How they proceed to regulate the locomotor rhythm remained unknown. Here, in neonatal rodents, we identified a persistent potassium current critical in regulating pacemakers and locomotion speed. This current recapitulates features of the M-current (IM): a subthreshold noninactivating outward current blocked by 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone dihydrochloride (XE991) and enhanced by N-(2-chloro-5-pyrimidinyl)-3,4-difluorobenzamide (ICA73). Immunostaining and mutant mice highlight an important role of Kv7.2-containing channels in mediating IM. Pharmacological modulation of IM regulates the emergence and the frequency regime of both pacemaker and CPG activities and controls the speed of locomotion. Computational models captured these results and showed how an interplay between IM and INaP endows the locomotor CPG with rhythmogenic properties. Overall, this study provides fundamental insights into how IM and INaP work in tandem to set the speed of locomotion.

Up-regulation of the persistent sodium current (INaP) and down-regulation of the potassium/chloride extruder KCC2 lead to spasticity after spinal cord injury (SCI). We here identified calpain as the driver of the up- and down-regulation of INaP and KCC2, respectively, in neonatal rat lumbar motoneurons. Few days after SCI, neonatal rats developed behavioral signs of spasticity with the emergence of both hyperreflexia and abnormal involuntary muscle contractions on hindlimbs. At the same time, in vitro isolated lumbar spinal cords became hyperreflexive and displayed numerous spontaneous motor outputs. Calpain-I expression paralleled with a proteolysis of voltage-gated sodium (Nav) channels and KCC2. Acute inhibition of calpains reduced this proteolysis, restored the motoneuronal expression of Nav and KCC2, normalized INaP and KCC2 function, and curtailed spasticity. In sum, by up- and down-regulating INaP and KCC2, the calpain-mediated proteolysis of Nav and KCC2 drives the hyperexcitability of motoneurons which leads to spasticity after SCI.

International audience; The locomotor network is a neural circuit capable of generating quite varied and complex patterns of motor activity. While neurotransmission and neuromodulation play a major role in fine-tuning motor function, neuron's firing properties driven by a palette of intrinsic ionic conductances and pump currents, play an equally important role. This review highlights the papers published over the past few years studying ionic conductances in spinal circuits governing locomotion. I discuss how this ensemble of currents underpins the operation of the locomotor network and summarize progress towards identifying the channels that mediate the ionic conductances.

In the context of the White Book of Physical and Rehabilitation Medicine (PRM), this paper deals with Research, the future of PRM. PRM students and specialists are mainly involved in biomedical research, investigating the biological processes, the causes of diseases, their medical diagnosis, the evaluation of their consequences on functioning, disability and health and the effects of health interventions at an individual and a societal level. Most of the current PRM research, often interdisciplinary, originates from applied research which, using existing knowledge, is directed towards specific goals. Translational medical research, research and development, implementation research and clinical impact research are in this field. PRM physicians, mainly master or PhD students, are nowadays increasing their participation in basic research and in pre- clinical trials. PRM physicians are involved in primary research, which is an original first hand research, but also in secondary research, which is the analysis and interpretation of primary research publications in a field, with a specific methodology. Secondary research remains an important activity of the UEMS PRM section and it will be the field of the new created Cochrane Rehabilitation. Secondary research with interest for persons with disabilities, will be developed world wide on the basis of evidence based medicine, with the participation of PRM physicians and of all other health and social professionals involved in rehabilitation. The development of research activities with interest for PRM in Europe is a challenge for the future, which has to be faced now. The European PRM schools, the European master and PhD program with their supporting research and clinical facilities, the European PRM organizations with their websites, the PRM scientific journals and European congresses are a strong basis to develop research activities, together with the development of Cochrane Rehabilitation field and of our cooperation with European high level research facilities, European and international scientific societies in different fields. PRM will be a leader in this field of research.

International audience; In mature neurons, low intracellular chloride level required for inhibition is maintained by the potassium-chloride cotransporter, KCC2. Impairment of Cl-extrusion after KCC2 dysfunction has been involved in many central nervous system disorders, such as seizures, neuropathic pain, or spasticity, after a spinal cord injury (SCI). This makes KCC2 an appealing drug target for restoring Cl-homeostasis and inhibition in pathological conditions. In the present study, we screen the Prestwick Chemical Library Ò and identify conventional antipsychotics phenothiazine derivatives as enhancers of KCC2 activity. Among them, prochlorperazine hyperpolarizes the Cl-equilibrium potential in motoneurons of neonatal rats and restores the reciprocal inhibition post-SCI. The compound alleviates spasticity in chronic adult SCI rats with an efficacy equivalent to the antispastic agent, baclofen, and rescues the SCI-induced downregulation of KCC2 in moto-neurons below the lesion. These pre-clinical data support prochlorperazine for a new therapeutic indication in the treatment of spasticity post-SCI and neurological disorders involving a KCC2 dysfunction.

After a spinal cord injury (SCI), patients develop spasticity, a motor disorder characterized by hyperreflexia and stiffness of muscles. Spasticity results from alterations in motoneurons with an upregulation of their persistent sodium current (I

International audience; After a spinal cord injury (SCI), patients develop spasticity, a motor disorder characterized by hyperreflexia and stiffness of muscles. Spasticity results from alterations in motoneurons with an upregulation of their persistent sodium current (INaP), simultaneously with a disinhibition caused by a reduction of expression of chloride (Cl-) co-transporters KCC2. Until recently the origin of alterations was unknown. After reviewing pathophysiology of spasticity, the manuscript relates our recent work showing a tight relationship between the calpain-dependent proteolysis of voltage-gated sodium channels, the upregulation of INaP and spasticity following SCI. We also discuss KCC2 as a substrate of calpains which may contribute to the disinhibition of motoneurons below the lesion. This led us to consider the proteolytic cleavage of both sodium channels and KCC2 as the upstream mechanism contributing to the development of spasticity after SCI.; Suite à un traumatisme de la moelle épinière, la plupart des patients développent une exagération du tonus musculaire appelée spasticité, qui aboutit souvent à une incapacité motrice. Dans cette revue, nous résumerons les principaux mécanismes physiopathologiques de la spasticité qui découlent d’une lésion médullaire puis décrirons l’apport de nos récents travaux identifiant une protéase, la calpaïne, comme le promoteur de ces mécanismes physiopathologiques. Cette découverte ouvre de nouvelles pistes thérapeutiques dans le traitement de la spasticité.Suite à un traumatisme de la moelle épinière, la plupart des patients développent une exagération du tonus musculaire appelée spasticité, qui aboutit souvent à une incapacité motrice. Dans cette revue, nous résumerons les principaux mécanismes physiopathologiques de la spasticité qui découlent d’une lésion médullaire puis décrirons l’apport de nos récents travaux identifiant une protéase, la calpaïne, comme le promoteur de ces mécanismes physiopathologiques. Cette découverte ouvre de nouvelles pistes thérapeutiques dans le traitement de la spasticité.

Downregulation of the potassium chloride cotransporter type 2 (KCC2) after a spinal cord injury (SCI) disinhibits motoneurons and dorsal horn interneurons causing spasticity and neuropathic pain, respectively. We showed recently (Bos et al., 2013) that specific activation of 5-HT

The development and the ionic nature of bistable behavior in lumbar motoneurons were investigated in rats. One week after birth, almost all (∼80%) ankle extensor motoneurons recorded in whole-cell configuration displayed self-sustained spiking in response to a brief depolarization that emerged when the temperature was raised >30°C. The effect of L-type Ca2+channel blockers on self-sustained spiking was variable, whereas blockade of the persistent sodium current (INaP) abolished them. When hyperpolarized, bistable motoneurons displayed a characteristic slow afterdepolarization (sADP). The sADPs generated by repeated depolarizing pulses summed to promote a plateau potential. The sADP was tightly associated with the emergence of Ca2+spikes. Substitution of extracellular Na+or chelation of intracellular Ca2+abolished both sADP and the plateau potential without affecting Ca2+spikes. These data suggest a key role of a Ca2+-activated nonselective cation conductance (ICaN) in generating the plateau potential. In line with this, the blockade of ICaNby flufenamate abolished both sADP and plateau potentials. Furthermore, 2-aminoethoxydiphenyl borate (2-APB), a common activator of thermo-sensitive vanilloid transient receptor potential (TRPV) cation channels, promoted the sADP. Among TRPV channels, only the selective activation of TRPV2 channels by probenecid promoted the sADP to generate a plateau potential. To conclude, bistable behaviors are, to a large extent, determined by the interplay between three currents: L-type ICa, INaP, and a Na+-mediated ICaNflowing through putative TRPV2 channels.

Downregulation of the potassium chloride cotransporter type 2 (KCC2) after a spinal cord injury (SCI) disinhibits motoneurons and dorsal horn interneurons causing spasticity and neuropathic pain, respectively. We showed recently (Bos et al., 2013) that specific activation of 5-HT2A receptors by TCB-2 [(4-bromo-3,6-dimethoxybenzocyclobuten-1-yl)methylamine hydrobromide] upregulates KCC2 function, restores motoneuronal inhibition and reduces SCI-induced spasticity. Here, we tested the potential analgesic effect of TCB-2 on central (thoracic hemisection) and peripheral [spared nerve injury (SNI)] neuropathic pain. We found mechanical and thermal hyperalgesia reduced by an acute administration of TCB-2 in rats with SCI. This analgesic effect was associated with an increase in dorsal horn membrane KCC2 expression and was prevented by pharmacological blockade of KCC2 with an intrathecal injection of DIOA [(dihydroindenyl)oxy]alkanoic acid]. In contrast, the SNI-induced neuropathic pain was not attenuated by TCB-2 although there was a slight increase of membrane KCC2 expression in the dorsal horn ipsilateral to the lesion. Up-regulation of KCC2 function by targeting 5-HT2A receptors, therefore, has therapeutic potential in the treatment of neuropathic pain induced by SCI but not by SNI.

Locomotor disorders profoundly impact quality of life of patients with spinal cord injury. Understanding the neuronal networks responsible for locomotion remains a major challenge for neuroscientists and a fundamental prerequisite to overcome motor deficits. Although neuronal circuitry governing swimming activities in lower vertebrates has been studied in great details, determinants of walking activities in mammals remain elusive. The manuscript reviews some of the principles relevant to the functional organization of the mammalian locomotor network and mainly focuses on mechanisms involved in rhythmogenesis. Based on recent publications supplemented with new experimental data, the authors will specifically discuss a new working hypothesis in which pacemakers, cells characterized by inherent oscillatory properties, might be functionally integrated in the locomotor network in mammals.

Maturation of inhibitory postsynaptic transmission onto motoneurons in the rat occurs during the perinatal period, a time window during which pathways arising from the brainstem reach the lumbar enlargement of the spinal cord. There is a developmental switch in miniature IPSCs (mIPSCs) from predominantly long-duration GABAergic to short-duration glycinergic events. We investigated the effects of a complete neonatal [postnatal day 0 (P0)] spinal cord transection (SCT) on the expression of Glycine and GABAAreceptor subunits (GlyR and GABAAR subunits) in lumbar motoneurons. In control rats, the density of GlyR increased from P1 to P7 to reach a plateau, whereas that of GABAAR subunits dropped during the same period. In P7 animals with neonatal SCT (SCT-P7), the GlyR densities were unchanged compared with controls of the same age, while the developmental downregulation of GABAAR was prevented. Whole-cell patch-clamp recordings of mIPSCs performed in lumbar motoneurons at P7 revealed that the decay time constant of miniature IPSCs and the proportion of GABAergic events significantly increased after SCT. After daily injections of the 5-HT2R agonist DOI, GABAAR immunolabeling on SCT-P7 motoneurons dropped down to values reported in control-P7, while GlyR labeling remained stable. A SCT made at P5 significantly upregulated the expression of GABAAR 1 week later with little, if any, influence on GlyR. We conclude that the plasticity of GlyR is independent of supraspinal influences whereas that of GABAAR is markedly influenced by descending pathways, in particular serotoninergic projections.

A unilateral activation of the mesencephalic locomotor region (MLR) produces symmetrical bilateral locomotion in all vertebrate species tested to date. How this occurs remains unresolved. This study examined the possibility that the symmetry occurred at the level of the inputs from the MLR to reticulospinal (RS) cells. In lamprey semi-intact preparations, we recorded intracellular responses of pairs of large, homologous RS cells on both sides to stimulation of the MLR on one side. The synaptic responses on both sides were very similar in shape, amplitude, and threshold intensity. Increasing MLR stimulation intensity produced a symmetrical increase in the magnitude of the responses on both sides. Ca2+imaging confirmed the bilateral activation of smaller-sized RS cells as well. In a high-divalent cation solution, the synaptic responses of homologous RS cells persisted and exhibited a constant latency during high-frequency stimulation. Moreover, during gradual replacement of normal Ringer's solution with a Ca2+-free solution, the magnitude of responses showed a gradual reduction with a similar time course in the homologous RS cells. These results support the idea that the MLR projects monosynaptically to RS cells on both sides with symmetrical inputs. During locomotion of the semi-intact preparation, the discharge pattern was also very similar in homologous bilateral RS cells. Anatomical experiments confirmed the presence of MLR neurons projecting ipsilaterally to the reticular formation intermingled with neurons projecting contralaterally. We conclude that the bilaterally symmetrical MLR inputs to RS cells are likely contributors to generating symmetrical locomotor activity.

Rhythm generation in neuronal networks relies on synaptic interactions and pacemaker properties. Little is known about the contribution of the latter mechanisms to the integrated network activity underlying locomotion in mammals. We tested the hypothesis that the persistent sodium current (INaP) is critical in generating locomotion in neonatal rodents using both slice and isolated spinal cord preparations. After removing extracellular calcium, 75% of interneurons in the area of the central pattern generator (CPG) for locomotion exhibited bursting properties andINaPwas concomitantly upregulated. Putative CPG interneurons such as commissural and Hb9 interneurons also expressedINaP-dependent (riluzole-sensitive) bursting properties. Most bursting cells exhibited a pacemaker-like behavior (i.e., burst frequency increased with depolarizing currents). Veratridine upregulatedINaP, induced riluzole-sensitive bursting properties, and slowed down the locomotor rhythm. This study provides evidence thatINaPgenerates pacemaker activities in CPG interneurons and contributes to the regulation of the locomotor activity.

International audience; a r t i c l e s nature medicine advance online publication Spasticity, a common debilitating complication in people with spinal cord injury (SCI), is characterized by a velocity-dependent increase in the tonic stretch reflex and spasms 1. It is primarily attributed to a reduction in postsynaptic inhibition and an increase in the excit-ability of motoneurons below the lesion 2. Although disinhibition is related to a dysregulation of chloride homeostasis 3 , the mechanisms that cause motoneuron hyperexcitability are not yet fully understood. In the healthy spinal cord, the excitability of motoneurons is set by brainstem-derived serotonin (5-hydroxytryptamine (5-HT)). The activation of 5-HT type 2 receptors (5-HT 2) facilitates voltage-gated persistent calcium and sodium currents 4,5 (persistent inward currents, or PICs). These PICs considerably amplify the activity of brief synaptic excitatory inputs, which enables sustained muscle contractions 6. PICs are reduced early after SCI 7 as compared to those in healthy spinal cords, but slowly recover within weeks, leading to excessive motoneu-ron activity that is characterized by the plateau potentials associated with muscle spasms 8,9. The upregulation of Ca 2+ PICs in the chronic phase after the injury is due to the increased expression of 5-HT sub-type 2C receptors (5-HT 2C), which become constitutively active 10. However, a major question that remains is how the Na + PIC (I NaP)—a key conductance of the locomotor network 11–14 that drives plateau potentials in motoneurons 15 —is upregulated. In adult rats, spinal cord neurons express mRNA encoding five α-subunits of sodium channels (Nav1.1, Nav1.2, Nav1.3, Nav1.6 and Nav1.7) 16 , but the main α-subunits in spinal motoneurons are Nav1.1 and Nav1.6 (ref. 17). We demonstrate here that the upregulation of I NaP after SCI is accompanied by a proteolytic cleavage of the α-subunit of Nav channels. We further show that calpains, a family of intracellular calcium-dependent cysteine proteases 18 , are responsible for the cleavage of Nav1.6 channels. Our results open new therapeutic avenues, given that blocking either I NaP or the activity of calpain reduces spasticity. RESULTS Upregulation of Nav1.6 a-subunit expression after SCI We tested whether abnormal expression of Nav channels accounts for the upregulation of I NaP after SCI. To model SCI in rodents, we carried out a complete transection at the T8–T9 level in adult female rats to avoid regeneration of the supraspinal tracts. At 15 d, 30 d and 60 d after SCI or sham surgery, we performed immunohistochemistry in lumbar segments L4–L5 (caudal to the lesion) to analyze the expression of the two main Nav α-subunits that are present in motoneurons (Nav1.1 and Nav1.6 (ref. 17)). A pan-Nav antibody that recognizes all Nav1 isoforms strongly stained axon initial segments (AISs) of motoneurons in both sham-operated and SCI rats (Fig. 1a). Although the Nav1.1 α-subunit was hardly detectable (Supplementary Fig. 1a), Nav1.6-specific immunolabeling largely overlapped the pan-staining (Fig. 1a, middle and right). The intensity of Nav immunostaining revealed by both the pan-Nav and the Nav1.6 antibodies was higher in motoneurons after SCI than in sham-operated controls as early as 15 d after SCI (P < 0.001; Fig. 1a–c). Conversely, the Nav1.6-specific immunostaining in the AIS segments of Renshaw cells did not change after SCI (P > 0.05; Supplementary Fig. 1b,c). Calpain mediates proteolysis of Nav1.6 channels after SCI To confirm the changes in Nav expression after SCI, we performed western blots on the membrane fractions isolated from the lumbar Upregulation of the persistent sodium current (I NaP) in motoneurons contributes to the development of spasticity after spinal cord injury (SCI). We investigated the mechanisms that regulate I NaP and observed elevated expression of voltage-gated sodium (Nav) 1.6 channels in spinal lumbar motoneurons of adult rats with SCI. Furthermore, immunoblots revealed a proteolysis of Nav channels, and biochemical assays identified calpain as the main proteolytic factor. Calpain-dependent cleavage of Nav channels after neonatal SCI was associated with an upregulation of I NaP in motoneurons. Similarly, the calpain-dependent cleavage of Nav1.6 channels expressed in human embryonic kidney (HEK) 293 cells caused the upregulation of I NaP. The pharmacological inhibition of calpain activity by MDL28170 reduced the cleavage of Nav channels, I NaP in motoneurons and spasticity in rats with SCI. Similarly, the blockade of I NaP by riluzole alleviated spasticity. This study demonstrates that Nav channel expression in lumbar motoneurons is altered after SCI, and it shows a tight relationship between the calpain-dependent proteolysis of Nav1.6 channels, the upregulation of I NaP and spasticity.

In vertebrates, locomotion is associated with changes in respiratory activity, but the neural mechanisms by which this occurs remain unknown. We began examining this in lampreys using a semi-intact preparation of young adult Petromyzon marinus, in which respiratory and locomotor behaviors can be recorded simultaneously with the activity of the underlying neural control systems. Spontaneous fictive respiration was recorded with suction electrodes positioned over the glossopharyngeal or the rostral vagal motor nucleus. In this preparation, locomotor activity, characterized by symmetrical tail movements (electromyogram recordings), was evoked by mechanical stimulation of the skin. During locomotion, the mean respiratory frequency and the mean area of the motor bursts were significantly increased (81.6+/-28.6% and 62.8+/-25.4%, respectively; P0.05). The frequency returned to normal 92+/-51 s after the end of locomotion. There were fluctuations in the instantaneous respiratory and locomotor frequencies that were rhythmical but antiphasic for the two rhythmic activities. The changes in respiratory activity were also examined during bouts of locomotion occurring spontaneously, and it was found that a modification in respiratory activity preceded the onset of spontaneous locomotion by 3.5+/-2.6 s. This suggests that the early respiratory changes are anticipatory and are not caused by feedback generated by locomotion. The increase in respiratory frequency during locomotion induced by sensory stimulation persisted after removal of the mesencephalon. When both the mesencephalon and spinal cord were removed, resulting in the isolation of the rhombencephalon, changes in the respiratory activity were also present following skin stimulations that would have normally induced locomotion. Altogether, the results suggest that respiratory changes are programmed to adjust ventilation prior to motor activity, and that a central rhombencephalic mechanism is involved.

In the cochlea, K+ is essential for mechano-electrical transduction. Here, we explore cochlear structure and function in mice lacking K+ channels of the two-pore domain family. A profound deafness associated with a decrease in endocochlear potential is found in adult Kcnk5−/− mice. Hearing occurs around postnatal day 19 (P19), and completely disappears 2 days later. At P19, Kcnk5−/− mice have a normal endolymphatic [K+] but a partly lowered endocochlear potential. Using Lac-Z as a gene reporter, KCNK5 is mainly found in outer sulcus Claudius', Boettcher's and root cells. Low levels of expression are also seen in the spiral ganglion, Reissner's membrane and stria vascularis. Essential channels (KCNJ10 and KCNQ1) contributing to K+ secretion in stria vascularis have normal expression in Kcnk5−/− mice. Thus, KCNK5 channels are indispensable for the maintenance of hearing. Among several plausible mechanisms, we emphasize their role in K+ recycling along the outer sulcus lateral route., Potassium is necessary for the mechanical-electrical transduction needed for hearing. Here the authors study mice lacking the potassium channel KCNK5 and show that these channels are mostly expressed in the outer sulcus and are required for hearing, pointing to their essential role in potassium recycling.

There is increasing evidence that a subpopulation of neurons in the dorsal principal sensory trigeminal nucleus are not simple sensory relays to the thalamus but may form the core of the central pattern generating circuits responsible for mastication. In this paper, we used whole cell patch recordings in brain stem slices of young rats to show that these neurons have intrinsic bursting abilities that persist in absence of extracellular Ca2+. Application of different K+ channel blockers affected duration and firing rate of bursts, but left bursting ability intact. Bursting was voltage dependent and was abolished by low concentrations of Na+ channel blockers. The proportion of bursting neurons increased dramatically in the second postnatal week, in parallel with profound changes in several electrophysiological properties. This is the period in which masticatory movements appear and mature. Bursting was associated with the development of an afterdepolarization that depend on maturation of a persistent sodium conductance ( INaP). An interesting finding was that the occurrence of bursting and the magnitude of INaP were both modulated by the extracellular concentration of Ca2+. Lowering extracellular [Ca2+] increased both INaP and probability of bursting. We suggest that these mechanisms underlie burst generation in mastication and that similar processes may be found in other motor pattern generators.

Substance P initiates locomotion when injected in the brain stem of mammals. This study examined the possible role of this peptide on the supraspinal locomotor command system in lampreys. Substance P was bath applied or locally injected into an in vitro isolated brain stem, and the effects of the drug were examined on reticulospinal cells and on the occurrence of swimming in a semi-intact preparation. Bath applications of substance P induced sustained depolarizations occurring rhythmically in intracellularly recorded reticulospinal cells. Spiking activity was superimposed on the depolarizations and swimming was induced. The sustained depolarizations were abolished by tetrodotoxin, and substance P did not affect the membrane resistance of reticulospinal cells nor their firing properties, suggesting that it did not directly effect reticulospinal cells. To establish where the effects were exerted, successive lesions of the brain stem were made as well as local applications of the drug in the brain stem. Removing the mesencephalon abolished the sustained depolarizations, whereas large ejections of the drug in the mesencephalon excited reticulospinal cells and elicited bouts of swimming. More local injections into the mesencephalic locomotor region (MLR) also elicited swimming. After an injection of substance P, the current threshold needed to induce locomotion by MLR stimulation was decreased, and the size of the postsynaptic responses of reticulospinal cells to MLR stimulation was increased. Substance P also reduced the frequency of miniature spontaneous postsynaptic currents in reticulospinal cells. Taken together, these results suggest that substance P plays a neuromodulatory role on the brain stem locomotor networks of lampreys.

This study was carried out to identify lamprey neurones relaying trigeminal sensory inputs to reticulospinal cells. Double labeling with fluorescent tracers was used in vitro. Fluorescein-conjugated dextran amines were applied to the proximal stump of the cut trigeminal nerve on both sides, and Texas Red-conjugated dextran amines were injected unilaterally in the middle (MRRN) or the posterior (PRRN) rhombencephalic reticular nuclei. Texas Red retrogradely labeled cells were found ipsi- and contralateral to each injection. Any of these cells with the soma or at least a major dendrite among the fluorescein-labeled trigeminal afferent axons was considered a candidate relay cell. Of these two possibilities, only cells with their soma among the fluorescein-labeled trigeminal afferents were found. The candidate relay cells projecting to the MRRN were mostly clustered at the caudal vestibular nerve level within the trigeminal descending tract, whereas the majority of those projecting to the PRRN were located more caudally. The diameter of candidate relay cells ranged from 9.2 to 24.6 mum and 9.2 to 46.1 mum, after MRRN and PRRN injections, respectively. A possible relay function for these cells was tested with electrophysiological experiments. The intracellular responses to trigeminal nerve stimulation were recorded in reticulospinal cells under control conditions and after ejections of a combination of glutamate ionotropic receptor antagonists over the candidate relay cells in small areas along the sulcus limitans. The synaptic responses elicited in MRRN reticulospinal cells were maximally depressed when ejections were made at the level of the vestibular nerve, in accord with the anatomical data. The synaptic responses in PRRN reticulospinal cells showed maximal depression when ejections were made slightly more caudally. Altogether, these results suggest that cells located within the trigeminal descending tract and projecting to reticular nuclei are likely to be the sensory trigeminal relays to reticulospinal neurones in lampreys.

We investigated the influence of gravity on the maturation of electrical properties of lumbar motoneurons and the development of their inputs from ventral descending pathways, which are important for the control of posture and locomotion. Using electrophysiological approaches in the in vitro brain stem-spinal cord preparation of neonatal rats born and reared in hypergravity field we demonstrate that: (1) the postnatal development of descending inputs to lumbar enlargement was reduced in animals submitted to hypergravity; (2) similar developmental pattern of basic electrical properties observed between motoneurons of hypergravity and control animals could not account for the changes in descending inputs. We concluded that gravity was critical to shape development of the supraspinal afferents in the lumbar spinal cord throughout the postnatal period.

In lampreys as in other vertebrates, brainstem centres play a key role in the initiation and control of locomotion. One such centre, the mesencephalic locomotor region (MLR), was identified physiologically at the mesopontine border. Descending inputs from the MLR are relayed by reticulospinal neurons in the pons and medulla, but the mechanisms by which this is carried out remain unknown. Because previous studies in higher vertebrates and lampreys described cholinergic cells within the MLR region, we investigated the putative role of cholinergic agonists in the MLR-controlled locomotion. The local application of either acetylcholine or nicotine exerted a direct dose-dependent excitation on reticulospinal neurons as well as induced active or fictive locomotion. It also accelerated ongoing fictive locomotion. Choline acetyltransferase-immunoreactive cells were found in the region identified as the MLR of lampreys and nicotinic antagonists depressed, whereas physostigmine enhanced the compound EPSP evoked in reticulospinal neurons by electrical stimulation of this region. In addition, cholinergic inputs from the MLR to reticulospinal neurons were found to be monosynaptic. When the brainstem was perfused with d-tubocurarine, the induction of swimming by MLR stimulation was depressed, but not prevented, in a semi-intact preparation. Altogether, the results support the hypothesis that cholinergic inputs from the MLR to reticulospinal cells play a substantial role in the initiation and the control of locomotion.

The first postnatal week is a critical period for the development of posture in the rat. The use of ankle extensor muscles in postural reactions increases during this period. Changes in excitability of motoneurons are probably an important factor underlying this maturation. The aim of this study was to identify whether variations in the maturation exist between motor pools innervating antagonistic muscles. Intracellular recordings in the in vitro brain stem-spinal cord preparation of neonatal rats (from postnatal day 0-5) were used to examine the developmental changes in excitability of motoneurons innervating the ankle flexors (F-MNs) and the antigravity ankle extensors (E-MNs). No significant difference in resting potential, action potential threshold, input resistance or rheobase was observed at birth. The age-related increase in rheobase was more pronounced for F-MNs than for E-MNs. The development of discharge properties of E-MNs lagged behind that of F-MNs. More F-MNs than E-MNs were able to fire repetitively in response to current injection at birth. F-MNs discharged at a higher frequency than E-MNs at all ages. Differences in the duration of action potential afterhyperpolarization accounted, at least partly, for the differences in discharge frequency between E-MNs and F-MNs at birth, and for the age-related increase in firing rate. These results suggest that E-MNs are more immature at birth than F-MNs and that there is a differential development of motoneurons innervating antagonistic muscles. This may be a critical factor in the development of posture and locomotion.

The rat is quite immature at birth and a rapid maturation of motor behavior takes place during the first 2 postnatal weeks. Lumbar motoneurons undergo a rapid development during this period. The last week before birth represents the initial stages of motoneuron differentiation, including regulation of the number of cells and the arrival of segmental and first supraspinal afferents. At birth, motoneurons are electrically coupled and receive both appropriate and inappropriate connections from the periphery; the control from supraspinal structures is weak and exerted mainly through polysynaptic connections. During the 1st postnatal week, inappropriate sensori-motor contacts and electrical coupling disappear, the supraspinal control increases gradually and myelin formation is responsible for an increased conduction velocity in both descending and motor axons. Both N-methyl-D-aspartate (NMDA) and non-NMDA receptors are transiently overexpressed in the neonatal spinal cord. The contribution of non-NMDA receptors to excitatory amino acid transmission increases with age. Activation of γ-aminobutyric acid A and glycine receptors leads to membrane depolarization in embryonic motoneurons but to hyperpolarization in older motoneurons. The firing properties of motoneurons change with development: they are capable of more repetitive firing at the end of the 1st postnatal week than before birth. However, maturation does not proceed simultaneously in the motor pools innervating antagonistic muscles; for instance, the development of repetitive firing of ankle extensor motoneurons lags behind that of flexor motoneurons. The spontaneous embryonic and neonatal network-driven activity, detected at the levels of motoneurons and primary afferent terminals, may play a role in neuronal maturation and in the formation and refinement of sensorimotor connections.

The development of the postural control of hindlimbs was investigated during the first postnatal week in the rat. The whole body was tilted in a vertical plane with the nose up. The proportion of animals producing a complete extension of both hindlimbs increased with age until the end of the first postnatal week. Motor responses were evoked by the pitch tilt already at birth with a slight extension of the hips, the knees and the ankles remaining bent in most cases. The extension produced at the ankle level increased gradually during the first postnatal week. This was correlated with a change in the EMG activity recorded from the triceps surae muscles (ankle extensors) during this postural reaction. There was a gradual acquisition of a tonic pattern. Characteristics of EMG responses changed significantly with age demonstrating an important increase in the use of triceps surae muscles in this postural task. These data demonstrate that the first postnatal week is a critical period for the development of postural reactions in the hindlimbs. They also suggest the existence of a proximo-distal gradient in the maturation of postural control. The mechanisms responsible for this reflex and for the maturation of posture are discussed.

Presynaptic inhibition of primary afferents can be evoked from at least three sources in the adult animal: 1) by stimulation of several supraspinal structures; 2) by spinal reflex action from sensory inputs; or 3) by the activity of spinal locomotor networks. The depolarisation in the intraspinal afferent terminals which is due, at least partly, to the activation of GABA(A) receptors may be large enough to reach firing threshold and evoke action potentials that are antidromically conducted into peripheral nerves. Little is known about the development of presynaptic inhibition and its supraspinal control during ontogeny. This article, reviewing recent experiments performed on the in vitro brainstem/spinal cord preparation of the neonatal rat, demonstrates that a similar organisation is present, to some extent, in the new-born rat. A spontaneous activity consisting of antidromic discharges can be recorded from lumbar dorsal roots. The discharges are generated by the underlying afferent terminal depolarizations reaching firing threshold. The number of antidromic action potentials increases significantly in saline solution with chloride concentration reduced to 50% of control. Bath application of the GABA(A) receptor antagonist, bicuculline (5-10 microM) blocks the antidromic discharges almost completely. Dorsal root discharges are therefore triggered by chloride-dependent GABA(A) receptor-mediated mechanisms; 1) activation of descending pathways by stimulation delivered to the ventral funiculus (VF) of the spinal cord at the C1 level; 2) activation of sensory inputs by stimulation of a neighbouring dorsal root; or 3) pharmacological activation of the central pattern generators for locomotion evokes antidromic discharges in dorsal roots. VF stimulation also inhibited the response to dorsal root stimulation. The time course of this inhibition overlapped with that of the dorsal root discharge suggesting that part of the inhibition of the monosynaptic reflex may be exerted at a presynaptic level. The existence of GABA(A) receptor-independent mechanisms and the roles of the antidromic discharges in the neonatal rat are discussed.

The in vitro brainstem-spinal cord preparation of newborn rats (0 to six-days-old) was used to investigate the development of pathways descending ventrally from the brainstem, which are important for the control of posture and locomotion. The ventral funiculus of the spinal cord was stimulated at the cervical (C1) level. Responses were recorded at the lumbar level from either motoneurons or ventral roots using intracellular microelectrodes or suction electrodes, respectively. Responses consisted of a pure excitation lasting 15 ms, followed by mixed excitatory/inhibitory responses. The inhibition was, at least partly, mediated by glycine. Excitatory amino acid transmission appears to be responsible for the excitation. The characteristics of the ventral funiculus-evoked postsynaptic potentials and ventral root potentials changed significantly with age. Their latency decreased whereas the slope and the area, measured over the first 15 ms, increased. The increase of the ventral funiculus input to motoneurons was slightly more pronounced than that of the monosynaptic dorsal root-evoked potentials from day 0 to day 4. These data suggest a gradual arrival of ventral descending axons in the lumbar enlargement which may be responsible for the gradual acquisition of postural control that takes place during the first days after birth. This is a prerequisite for the development of the adult pattern of quadrupedal locomotion, with elevated trunk.

SUMMARY Changes in the extracellular ionic concentrations occur as a natural consequence of firing activity in large populations of neurons. The extent to which these changes alter the properties of individual neurons and the operation of neuronal networks remains unknown. Here, we show that the locomotorlike activity in the isolated neonatal rodent spinal cord reduces the extracellular calcium ([Ca 2+ ]o )t o 0.9 mM and increases the extracellular potassium ([K + ]o) to 6 mM. Such changes in [Ca 2+ ]o and [K + ]o trigger pacemaker activities in interneurons considered to be part of the locomotor network. Experimental data and a modeling study show that the emergence of pacemaker properties critically involves a [Ca 2+ ]o-dependent activation of the persistent sodium current (INaP). These results support a concept for locomotor rhythm generation in which INaP-dependent pacemaker properties in spinal interneurons are switched on and tuned by activity-dependent changes in [Ca 2+ ]o and [K + ]o.

GABA and glycine are classically called "inhibitory" amino acids, despite the fact that their action can rapidly switch from inhibition to excitation and vice versa. The postsynaptic action depends on the intracellular concentration of chloride ions ([Cl(-)](i)), which is regulated by proteins in the plasma membrane: the K(+)-Cl(-) cotransporter KCC2 and the Na(+)-K(+)-Cl(-) cotransporter NKCC1, which extrude and intrude Cl(-) ions, respectively. A high [Cl(-)](i) leads to a depolarizing (excitatory) action of GABA and glycine, as observed in mature dorsal root ganglion neurons and in motoneurons both early during development and in several pathological conditions, such as following spinal cord injury. Here, we review some recent data regarding chloride homeostasis in the spinal cord and its contribution to network operation involved in locomotion.

International audience; GABA and glycine are classically called "inhibitory" amino acids, despite the fact that their action can rapidly switch from inhibition to excitation and vice versa. The postsynaptic action depends on the intracellular concentration of chloride ions ([Cl À ] i), which is regulated by proteins in the plasma membrane: the K þ-Cl À cotransporter KCC2 and the Na þ-K þ-Cl À cotransporter NKCC1, which extrude and intrude Cl À ions, respectively. A high [Cl À ] i leads to a depolarizing (excitatory) action of GABA and glycine, as observed in mature dorsal root ganglion neurons and in motoneurons both early during development and in several pathological conditions, such as following spinal cord injury. Here, we review some recent data regarding chloride homeostasis in the spinal cord and its contribution to network operation involved in locomotion.

International audience; Patterned, spontaneous activity plays a critical role in the development of neuronal networks. A robust spontaneous activity is observed in vitro in spinal cord preparations isolated from immature rats. The rhythmic ventral root discharges rely mainly on the depolarizing/ excitatory action of GABA and glycine early during development, whereas at later stages glutamate drive is primarily responsible for the rhythmic activity and GABA/glycine are thought to play an inhibitory role. However, rhythmic discharges mediated by the activation of GABA A receptors are recorded from dorsal roots (DRs). In the present study, we used the in vitro spinal cord preparation of neonatal rats to identify the relationship between discharges that are conducted antidromically along DRs and the spontaneous activity recorded from lumbar motoneurons. We show that discharges in DRs precede those in ventral roots and that primary afferent depolarizations (PADs) start earlier than EPSPs in motoneurons. EPSP-triggered averaging revealed that the action potentials propagate not only antidromically in the DR but also centrally and trigger EPSPs in motoneurons. Potentiating GABAergic antidromic discharges by diazepam increased the EPSPs recorded from motoneurons; conversely, blocking DR bursts markedly reduced these EPSPs. High intracellular concentrations of chloride are maintained in primary afferent terminals by the sodium-potassium-chloride cotransporter NKCC1. Blocking these cotransporters by bumetanide decreased both dorsal and ventral root discharges. We conclude that primary afferent fibers act as excitatory interneurons and that GABA, through PADs reaching firing threshold, is still playing a key role in promoting spontaneous activity in neonates.

The spinal circuitry underlying the generation of basic locomotor synergies has been described in substantial detail in lampreys and the cellular mechanisms have been identified. The initiation of locomotion, on the other hand, relies on supraspinal networks and the cellular mechanisms involved are only beginning to be understood. This review examines some of the findings relative to the neural mechanisms involved in the initiation of locomotion of lampreys. Locomotion can be elicited by sensory stimulation or by internal cues associated with fundamental needs of the animal such as food seeking, exploration, and mating. We have described mechanisms by which escape swimming is elicited in lampreys in response to mechanical skin stimulation. A rather simple neural connectivity is involved, including sensory and relay neurons, as well as the brainstem rhombencephalic reticulospinal cells, which act as command neurons. We have shown that reticulospinal cells have intrinsic membrane properties that allow them to transform a short duration sensory input into a long-lasting excitatory command that activates the spinal locomotor networks. These mechanisms constitute an important feature for the activation of escape swimming. Other sensory inputs can also elicit locomotion in lampreys. For instance, we have recently shown that olfactory signals evoke sustained depolarizations in reticulospinal neurons and chemical activation of the olfactory bulbs with local injections of glutamate induces fictive locomotion. The mechanisms by which internal cues initiate locomotion are less understood. Our research has focused on one particular locomotor center in the brainstem, the mesencephalic locomotor region (MLR). The MLR is believed to channel inputs from many brain regions to generate goal-directed locomotion. It activates reticulospinal cells to elicit locomotor output in a graded fashion contrary to escape locomotor bouts, which are all-or-none. MLR inputs to reticulospinal cells use both glutamatergic and cholinergic transmission; nicotinic receptors on reticulospinal cells are involved. MLR excitatory inputs to reticulospinal cells in the middle (MRRN) are larger than those in the posterior rhombencephalic reticular nucleus (PRRN). Moreover at low stimulation strength, reticulospinal cells in the MRRN are activated first, whereas those in the PRRN require stronger stimulation strengths. The output from the MLR on one side activates reticulospinal neurons on both sides in a highly symmetrical fashion. This could account for the symmetrical bilateral locomotor output evoked during unilateral stimulation of the MLR in all animal species tested to date. Interestingly, muscarinic receptor activation reduces sensory inputs to reticulospinal neurons and, under natural conditions, the activation of MLR cholinergic neurons will likely reduce sensory inflow. Moreover, exposing the brainstem to muscarinic agonists generates sustained recurring depolarizations in reticulospinal neurons through pre-reticular effects. Cells in the caudal half of the rhombencephalon appear to be involved and we propose that the activation of these muscarinoceptive cells could provide additional excitation to reticulospinal cells when the MLR is activated under natural conditions. One important question relates to sources of inputs to the MLR. We found that substance P excites the MLR, whereas GABA inputs tonically maintain the MLR inhibited and removal of this inhibition initiates locomotion. Other locomotor centers exist such as a region in the ventral thalamus projecting directly to reticulospinal cells. This region, referred to as the diencephalic locomotor region, receives inputs from several areas in the forebrain and is likely important for goal-directed locomotion. In summary, this review focuses on the most recent findings relative to initiation of lamprey locomotion in response to sensory and internal cues in lampreys.