Knockdown of calpain1 in lumbar motoneurons reduces spasticity after spinal cord injury in adult rats.

Spasticity, affecting ∼75% of patients with spinal cord injury (SCI), leads to hyperreflexia, muscle spasms, and cocontractions of antagonist muscles, greatly affecting their quality of life. Spasticity primarily stems from the hyperexcitability of motoneurons below the lesion, driven by an upregulation of the persistent sodium current and a downregulation of chloride extrusion. This imbalance results from the post-SCI activation of calpain1, which cleaves Nav1.6 channels and KCC2 cotransporters. Our study was focused on mitigating spasticity by specifically targeting calpain1 in spinal motoneurons. We successfully transduced lumbar motoneurons in adult rats with SCI using intrathecal administration of adeno-associated virus vector serotype 6, carrying a shRNA sequence against calpain1. This approach significantly reduced calpain1 expression in transduced motoneurons, leading to a noticeable decrease in spasticity symptoms, including hyperreflexia, muscle spasms, and cocontractions in hindlimb muscles, which are particularly evident in the second month post-SCI. In addition, this decrease, which prevented the escalation of spasticity to a severe grade, paralleled the restoration of KCC2 levels in transduced motoneurons, suggesting a reduced proteolytic activity of calpain1. These findings demonstrate that inhibiting calpain1 in motoneurons is a promising strategy for alleviating spasticity in SCI patients.

Segmental organization of vestibulospinal inputs to spinal interneurons mediating crossed activation of thoracolumbar motoneurons in the neonatal mouse.

Vestibulospinal pathways activate contralateral motoneurons (MNs) in the thoracolumbar spinal cord of the neonatal mouse exclusively via axons descending ipsilaterally from the vestibular nuclei via the lateral vestibulospinal tract (LVST; Kasumacic et al., 2010). Here we investigate how transmission from the LVST to contralateral MNs is mediated by descending commissural interneurons (dCINs) in different spinal segments. We test the polysynaptic nature of this crossed projection by assessing LVST-mediated ventral root (VR) response latencies, manipulating synaptic responses pharmacologically, and tracing the pathway transynaptically from hindlimb extensor muscles using rabies virus (RV). Longer response latencies in contralateral than ipsilateral VRs, near-complete abolition of LVST-mediated calcium responses in contralateral MNs by mephenesin, and the absence of transsynaptic RV labeling of contralateral LVST neurons within a monosynaptic time window all indicate an overwhelmingly polysynaptic pathway from the LVST to contralateral MNs. Optical recording of synaptically mediated calcium responses identifies LVST-responsive ipsilateral dCINs that exhibit segmental differences in proportion and dorsoventral distribution. In contrast to thoracic and lower lumbar segments, in which most dCINs are LVST responsive, upper lumbar segments stand out because they contain a much smaller and more ventrally restricted subpopulation of LVST-responsive dCINs. A large proportion of these upper lumbar LVST-responsive dCINs project to contralateral L5, which contains many of the hindlimb extensor MNs activated by the LVST. A selective channeling of LVST inputs through segmentally and dorsoventrally restricted subsets of dCINs provides a mechanism for targeting vestibulospinal signals differentially to contralateral trunk and hindlimb MNs in the mammalian spinal cord.

Activation of 5-HT2A receptors upregulates the function of the neuronal K-Cl cotransporter KCC2.

In healthy adults, activation of γ-aminobutyric acid (GABA)(A) and glycine receptors inhibits neurons as a result of low intracellular chloride concentration ([Cl(-)](i)), which is maintained by the potassium-chloride cotransporter KCC2. A reduction of KCC2 expression or function is implicated in the pathogenesis of several neurological disorders, including spasticity and chronic pain following spinal cord injury (SCI). Given the critical role of KCC2 in regulating the strength and robustness of inhibition, identifying tools that may increase KCC2 function and, hence, restore endogenous inhibition in pathological conditions is of particular importance. We show that activation of 5-hydroxytryptamine (5-HT) type 2A receptors to serotonin hyperpolarizes the reversal potential of inhibitory postsynaptic potentials (IPSPs), E(IPSP), in spinal motoneurons, increases the cell membrane expression of KCC2 and both restores endogenous inhibition and reduces spasticity after SCI in rats. Up-regulation of KCC2 function by targeting 5-HT(2A) receptors, therefore, has therapeutic potential in the treatment of neurological disorders involving altered chloride homeostasis. However, these receptors have been implicated in several psychiatric disorders, and their effects on pain processing are controversial, highlighting the need to further investigate the potential systemic effects of specific 5-HT(2A)R agonists, such as (4-bromo-3,6-dimethoxybenzocyclobuten-1-yl)methylamine hydrobromide (TCB-2).

Conservation of locomotion-induced oculomotor activity through evolution in mammals.

Efference copies are neural replicas of motor outputs used to anticipate the sensory consequences of a self-generated motor action or to coordinate neural networks involved in distinct motor behaviors.1 An established example of this motor-to-motor coupling is the efference copy of the propulsive motor command, which supplements classical visuo-vestibular reflexes to ensure gaze stabilization during amphibian larval locomotion.2 Such feedforward replica of spinal pattern-generating circuits produces a spino-extraocular motor coupled activity that evokes eye movements, spatiotemporally coordinated to tail undulation independently of any sensory signal.3,4 Exploiting the developmental stages of the frog,1 studies in metamorphing Xenopus demonstrated the persistence of this spino-extraocular motor command in adults and its developmental adaptation to tetrapodal locomotion.5,6 Here, we demonstrate for the first time the existence of a comparable locomotor-to-ocular motor coupling in the mouse. In neonates, ex vivo nerve recordings of brainstem-spinal cord preparations reveal a spino-extraocular motor coupled activity similar to the one described in Xenopus. In adult mice, trans-synaptic rabies virus injections in lateral rectus eye muscle label cervical spinal cord neurons closely connected to abducens motor neurons. Finally, treadmill-elicited locomotion in decerebrated preparations7 evokes rhythmic eye movements in synchrony with the limb gait pattern. Overall, our data are evidence for the conservation of locomotor-induced eye movements in vertebrate lineages. Thus, in mammals as in amphibians, CPG-efference copy feedforward signals might interact with sensory feedback to ensure efficient gaze control during locomotion.

Differential plasticity of the GABAergic and glycinergic synaptic transmission to rat lumbar motoneurons after spinal cord injury.

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 GABA(A) receptor subunits (GlyR and GABA(A)R subunits) in lumbar motoneurons. In control rats, the density of GlyR increased from P1 to P7 to reach a plateau, whereas that of GABA(A)R 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 GABA(A)R 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-HT(2)R agonist DOI, GABA(A)R 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 GABA(A)R 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 GABA(A)R is markedly influenced by descending pathways, in particular serotoninergic projections.

Differential effects of spinal cord transection on glycinergic and GABAergic synaptic signaling in sub-lesional lumbar motoneurons.

This review takes stock on the impact of complete spinal cord transection (SCT) on the plasticity of inhibitory synaptic transmission on sub-lesional lumbar motoneurons (MNs), differentiating between studies in neonate and adult rats. After neonatal SCT, normal maturational up-regulation of glycine receptors was observed. On the other hand, the developmental downregulation of the GABAA receptors, as well as the up-regulation of the co-transporter KCC2 were prevented, but not the normal decrease of NKCC1. In adult SCT rats, glycinergic synaptic transmission, which is the major contributor to spinal MNs inhibition in adulthood, had normal control levels 2 months post-injury. On the other hand, the GABAergic transmission was altered through an up-regulation of the pre-signaling levels and a down-regulation in the density of post synaptic receptors. KCC2 membrane expression was down-regulated at all post-injury times tested (24h to 4 months), thereby depolarizing the Cl- equilibrium potential and reducing the strength of postsynaptic inhibition. The preservation of glycinergic pre- and post signaling is probably a key factor in the success of locomotor rehabilitation programs in adult SCT rats. However, these data highlight the need to develop strategies to restore KCC2 levels in lumbar MNs, to stabilize the excitation/inhibition balance, which is essential to the effective control of skeletal muscle activity.

Alteration of glycinergic receptor expression in lumbar spinal motoneurons is involved in the mechanisms underlying spasticity after spinal cord injury.

Spasticity is a disabling motor disorder affecting 70% of people with brain and spinal cord injury. The rate-dependent depression (RDD) of the H reflex is the only electrophysiological measurement correlated with the degree of spasticity assessed clinically in spastic patients. Several lines of evidence suggest that the mechanism underlying the H reflex RDD depends on the strength of synaptic inhibition through GABAA (GABAAR) and glycine receptors (GlyR). In adult rats with spinal cord transection (SCT), we studied the time course of the expression of GABAAR and GlyR at the membrane of retrogradely identified Gastrocnemius and Tibialis anterior motoneurons (MNs) 3, 8 and 16 weeks after injury, and measured the RDD of the H reflex at similar post lesion times. Three weeks after SCT, a significant decrease in the expression of GABAA and GlyR was observed compared to intact rats, and the H-reflex RDD was much less pronounced than in controls. Eight weeks after SCT, GlyR values returned to normal. Simultaneously, we observed a tendency to recover normal RDD of the H reflex at higher frequencies. We tested whether an anti-inflammatory treatment using methylprednisolone performed immediately after SCT could prevent alterations in GABAA/glycine receptors and/or the development of spasticity observed 3 weeks after injury. This treatment restored control levels of GlyR but not the expression of GABAAR, and it completely prevented the attenuation of RDD. These data strongly suggest that alteration of glycinergic inhibition of lumbar MNs is involved in the mechanisms underlying spasticity after SCI.

GABA-glutamate supramammillary neurons control theta and gamma oscillations in the dentate gyrus during paradoxical (REM) sleep.

Several studies suggest that neurons from the lateral region of the SuM (SuML) innervating the dorsal dentate gyrus (DG) display a dual GABAergic and glutamatergic transmission and are specifically activated during paradoxical (REM) sleep (PS). The objective of the present study is to characterize the anatomical, neurochemical and electrophysiological properties of the SuML-DG projection neurons and to determine how they control DG oscillations and neuronal activation during PS and other vigilance states. For this purpose, we combine structural connectivity techniques using neurotropic viral vectors (rabies virus, AAV), neurochemical anatomy (immunohistochemistry, in situ hybridization) and imaging (light, electron and confocal microscopy) with in vitro (patch clamp) and in vivo (LFP, EEG) optogenetic and electrophysiological recordings performed in transgenic VGLUT2-cre male mice. At the cellular level, we show that the SuML-DG neurons co-release GABA and glutamate on dentate granule cells and increase the activity of a subset of DG granule cells. At the network level, we show that activation of the SuML-DG pathway increases theta power and frequency during PS as well as gamma power during PS and waking in the DG. At the behavioral level, we show that the activation of this pathway does not change animal behavior during PS, induces awakening during slow wave sleep and increases motor activity during waking. These results suggest that the SuML-DG pathway is capable of supporting the increase of theta and gamma power in the DG observed during PS and plays an important modulatory role of DG network activity during this state.

Prenatal activation of 5-HT2A receptor induces expression of 5-HT1B receptor in phrenic motoneurons and alters the organization of their premotor network in newborn mice.

In newborn mice of the control [C3H/HeJ (C3H)] and monoamine oxidase A-deficient (Tg8) strains, in which levels of endogenous serotonin (5-HT) were drastically increased, we investigated how 5-HT system dysregulation affected the maturation of phrenic motoneurons (PhMns), which innervate the diaphragm. First, using immunocytochemistry and confocal microscopy, we observed a 5-HT(2A) receptor (5-HT(2A)-R) expression in PhMns of both C3H and Tg8 neonates at the somatic and dendritic levels, whereas 5-HT(1B) receptor (5-HT(1B)-R) expression was observed only in Tg8 PhMns at the somatic level. We investigated the interactions between 5-HT(2A)-R and 5-HT(1B)-R during maturation by treating pregnant C3H mice with a 5-HT(2A)-R agonist (2,5-dimethoxy-4-iodoamphetamine hydrochloride). This pharmacological overactivation of 5-HT(2A)-R induced a somatic expression of 5-HT(1B)-R in PhMns of their progeny. Conversely, treatment of pregnant Tg8 mice with a 5-HT(2A)-R antagonist (ketanserin) decreased the 5-HT(1B)-R density in PhMns of their progeny. Second, using retrograde transneuronal tracing with rabies virus injected into the diaphragm of Tg8 and C3H neonates, we studied the organization of the premotor network driving PhMns. The interneuronal network monosynaptically connected to PhMns was much more extensive in Tg8 than in C3H neonates. However, treatment of pregnant C3H mice with 2,5-dimethoxy-4-iodoamphetamine hydrochloride switched the premotoneuronal network of their progeny from a C3H- to a Tg8-like pattern. These results show that a prenatal 5-HT excess affects, via the overactivation of 5-HT(2A)-R, the expression of 5-HT(1B)-R in PhMns and the organization of their premotor network.

Changes in innervation of lumbar motoneurons and organization of premotor network following training of transected adult rats.

Rats with complete spinal cord transection (SCT) can recover hindlimb locomotor function under strategies combining exercise training and 5-HT agonist treatment. This recovery is expected to result from structural and functional re-organization within the spinal cord below the lesion. To begin to understand the nature of this reorganization, we examined synaptic changes to identified gastrocnemius (GS) or tibialis anterior (TA) motoneurons (MNs) in SCT rats after a schedule of early exercise training and delayed 5-HT agonist treatment. In addition, we analyzed changes in distribution and number of lumbar interneurons (INs) presynaptic to GS MNs using retrograde transneuronal transport of rabies virus. In SCT-untrained rats, we found few changes in the density and size of inhibitory and excitatory inputs impinging on cell bodies of TA and GS MNs compared to intact rats, whereas there was a marked trend for a reduction in the number of premotor INs connected to GS MNs. In contrast, after training of SCT rats, a significant increase of the density of GABAergic and glycinergic axon terminals was observed on both GS and TA motoneuronal cell bodies, as well as of presynaptic P-boutons on VGLUT1 afferents. Despite these changes in innervation the number of premotor INs connected to GS MNs was similar to control values although some new connections to MNs were observed. These results suggest that adaptation of gait patterns in SCT-trained rats was accompanied by changes in the innervation of lumbar MNs while the distribution of the spinal premotor circuitry was relatively preserved.

Early movement restriction leads to enduring disorders in muscle and locomotion.

Motor control and body representation in the central nervous system (CNS) as well as musculoskeletal architecture and physiology are shaped during development by sensorimotor experience and feedback, but the emergence of locomotor disorders during maturation and their persistence over time remain a matter of debate in the absence of brain damage. By using transient immobilization of the hind limbs, we investigated the enduring impact of postnatal sensorimotor restriction (SMR) on gait and posture on treadmill, age-related changes in locomotion, musculoskeletal histopathology and Hoffmann reflex in adult rats without brain damage. SMR degrades most gait parameters and induces overextended knees and ankles, leading to digitigrade locomotion that resembles equinus. Based on variations in gait parameters, SMR appears to alter age-dependent plasticity of treadmill locomotion. SMR also leads to small but significantly decreased tibial bone length, chondromalacia, degenerative changes in the knee joint, gastrocnemius myofiber atrophy and muscle hyperreflexia, suggestive of spasticity. We showed that reduced and atypical patterns of motor outputs, and somatosensory inputs and feedback to the immature CNS, even in the absence of perinatal brain damage, play a pivotal role in the emergence of movement disorders and musculoskeletal pathologies, and in their persistence over time. Understanding how atypical sensorimotor development likely contributes to these degradations may guide effective rehabilitation treatments in children with either acquired (ie, with brain damage) or developmental (ie, without brain injury) motor disabilities.

Differential organization of gamma-aminobutyric acid type A and glycine receptors in the somatic and dendritic compartments of rat abducens motoneurons.

Premotor inhibitory neurons responsible for the decrease in the firing discharge during fast or slow eye movements selectively target the cell bodies and the dendrites of abducens motoneurons. Gamma-aminobutyric acid (GABA) and glycine, the main inhibitory synaptic neurotransmitters in the central nervous system, act via glycine and GABAA receptors, assembled from various types of subunits, which determine the kinetics of the currents mediated. Therefore, our hypothesis was that the expression of the inhibitory receptors on the somatic and the dendritic compartments, involved in different functions, may differ. In this study, we compared the subcellular patterns of expression of the main GABAA receptor subunits (GABAARalpha1, alpha2, alpha3, alpha5), glycine receptors (GlyRalpha1), and gephyrin in the somatic and dendritic compartments of rat abducens motoneurons, using double or triple immunocytochemical experiments with confocal microscopy. Significant differences exist in the patterns of organization and the synaptic expression of the GlyR and GABAAR subunits in the cell bodies and dendrites of abducens motoneurons. In the somata, only the GABAARalpha1 subunit was expressed, whereas both GABAARalpha1 and GABAARalpha3 were present in the dendrites. The GlyRalpha1 to GABAARalpha1 density ratio was reversed in the somatic and dendritic compartments (0.9 vs. 2.3). A quantitative electron microscopy study showed that the modes whereby gephyrin reaches its postsynaptic inhibitory synaptic target differ between the somata and the dendrites. Therefore, our results support the idea that a structure-function adaptation occurs at the single-neuron level.

Disturbed sensorimotor and electrophysiological patterns in lead intoxicated rats during development are restored by curcumin I.

Lead poisoning is one of the most significant health problem of environmental origin. It is known to cause different damages in the central and peripheral nervous system which could be represented by several neurophysiological and behavioral symptoms. In this study we firstly investigated the effect of lead prenatal exposure in rats to (3g/L), from neonatal to young age, on the motor/sensory performances, excitability of the spinal cord and gaits during development. Then we evaluated neuroprotective effects of curcumin I (Cur I) against lead neurotoxicity, by means of grasping and cliff avoidance tests to reveal the impairment of the sensorimotor functions in neonatal rats exposed prenatally to lead. In addition, extracellular recordings of motor output in spinal cord revealed an hyper-excitability of spinal networks in lead treated rats. The frequency of induced fictive locomotion was also increased in treated rats. At the young age, rats exhibited an impaired locomotor gait. All those abnormalities were attenuated by Cur I treatment at a dose of 16g/kg. Based on our finding, Cur I has shown features of a potent chemical compound able to restore the neuronal and the relative locomotor behaviors disturbances induced by lead intoxication. Therefore, this chemical can be recommended as a new therapeutic trial against lead induced neurotoxicity.

Early postnatal maturation in vestibulospinal pathways involved in neck and forelimb motor control.

To assess the organization and functional development of vestibulospinal inputs to cervical motoneurons (MNs), we have used electrophysiology (ventral root and electromyographic [EMG] recording), calcium imaging, trans-synaptic rabies virus (RV) and conventional retrograde tracing and immunohistochemistry in the neonatal mouse. By stimulating the VIIIth nerve electrically while recording synaptically mediated calcium responses in MNs, we characterized the inputs from the three vestibulospinal tracts, the separate ipsilateral and contralateral medial vestibulospinal tracts (iMVST/cMVST) and the lateral vestibulospinal tract (LVST), to MNs in the medial and lateral motor columns (MMC and LMC) of cervical segments. We found that ipsilateral inputs from the iMVST and LVST were differentially distributed to the MMC and LMC in the different segments, and that all contralateral inputs to MMC and LMC MNs in each segment derive from the cMVST. Using trans-synaptic RV retrograde tracing as well as pharmacological manipulation of VIIIth nerve-elicited synaptic responses, we found that a substantial proportion of inputs to both neck and forelimb extensor MNs was mediated monosynaptically, but that polysynaptic inputs were also significant. By recording EMG responses evoked by natural stimulation of the vestibular apparatus, we found that vestibular-mediated motor output to the neck and forelimb musculature became more robust during the first 10 postnatal days, concurrently with a decrease in the latency of MN discharge evoked by VIIIth nerve electrical stimulation. Together, these results provide insight into the complexity of vestibulospinal connectivity in the cervical spinal cord and a cogent demonstration of the functional maturation that vestibulospinal connections undergo postnatally. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1061-1077, 2016.

Reorganization of supramammillary-hippocampal pathways in the rat pilocarpine model of temporal lobe epilepsy: evidence for axon terminal sprouting.

In mesial temporal lobe epilepsy (MTLE), spontaneous seizures likely originate from a multi-structural epileptogenic zone, including several regions of the limbic system connected to the hippocampal formation. In this study, we investigate the structural connectivity between the supramammillary nucleus (SuM) and the dentate gyrus (DG) in the model of MTLE induced by pilocarpine in the rat. This hypothalamic nucleus, which provides major extracortical projections to the hippocampal formation, plays a key role in the regulation of several hippocampus-dependent activities, including theta rhythms, memory function and emotional behavior, such as stress and anxiety, functions that are known to be altered in MTLE. Our findings demonstrate a marked reorganization of DG afferents originating from the SuM in pilocarpine-treated rats. This reorganization, which starts during the latent period, is massive when animals become epileptic and continue to evolve during epilepsy. It is characterized by an aberrant distribution and an increased number of axon terminals from neurons of both lateral and medial regions of the SuM, invading the entire inner molecular layer of the DG. This reorganization, which reflects an axon terminal sprouting from SuM neurons, could contribute to trigger spontaneous seizures within an altered hippocampal intrinsic circuitry.

Prenatal exposure to fenugreek impairs sensorimotor development and the operation of spinal cord networks in mice.

Fenugreek is a medicinal plant whose seeds are widely used in traditional medicine, mainly for its laxative, galactagogue and antidiabetic effects. However, consumption of fenugreek seeds during pregnancy has been associated with a range of congenital malformations, including hydrocephalus, anencephaly and spina bifida in humans. The present study was conducted to evaluate the effects of prenatal treatment of fenugreek seeds on the development of sensorimotor functions from birth to young adults. Pregnant mice were treated by gavage with 1 g/kg/day of lyophilized fenugreek seeds aqueous extract (FSAE) or distilled water during the gestational period. Behavioral tests revealed in prenatally treated mice a significant delay in righting, cliff avoidance, negative geotaxis responses and the swimming development. In addition, extracellular recording of motor output in spinal cord isolated from neonatal mice showed that the frequency of spontaneous activity and fictive locomotion was reduced in FSAE-exposed mice. On the other hand, the cross-correlation coefficient in control mice was significantly more negative than in treated animals indicating that alternating patterns are deteriorated in FSAE-treated animals. At advanced age, prenatally treated mice displayed altered locomotor coordination in the rotarod test and also changes in static and dynamic parameters assessed by the CatWalk automated gait analysis system. We conclude that FSAE impairs sensorimotor and coordination functions not only in neonates but also in adult mice. Moreover, spinal neuronal networks are less excitable in prenatally FSAE-exposed mice suggesting that modifications within the central nervous system are responsible, at least in part, for the motor impairments.

Characterization of last-order premotor interneurons by transneuronal tracing with rabies virus in the neonatal mouse spinal cord.

We characterized the interneurons involved in the control of ankle extensor (triceps surae [TS] muscles) motoneurons (MNs) in the lumbar enlargement of mouse neonates by retrograde transneuronal tracing using rabies virus (RV). Examination of the kinetics of retrograde transneuronal transfer at sequential intervals post inoculation enabled us to determine the time window during which only the first-order interneurons, i.e., interneurons likely monosynaptically connected to MNs (last-order interneurons [loINs]) were RV-infected. The infection of the network resulted exclusively from a retrograde transport of RV along the motor pathway. About 80% of the loINs were observed ipsilaterally to the injection. They were distributed all along the lumbar enlargement, but the majority was observed in L4 and L5 segments where TS MNs were localized. Most loINs were distributed in laminae V-VII, whereas the most superficial laminae were devoid of RV infection. Contralaterally, commissural loINs were found essentially in lamina VIII of all lumbar segments. Groups of loINs were characterized by their chemical phenotypes using dual immunolabeling. Glycinergic neurons connected to TS MNs represented 50% of loINs ipsilaterally and 10% contralaterally. As expected, the ipsilateral glycinergic loINs included Renshaw cells, the most ventral neurons expressing calbindin. We also demonstrated a direct connection between a group of cholinergic interneurons observed ipsilaterally in L3 and the rostral part of L4, and TS MNs. To conclude, transneuronal tracing with RV, combined with an immunohistochemical detection of neuronal determinants, allows a very specific mapping of motor networks involved in the control of single muscles.

Chapter 1--importance of chloride homeostasis in the operation of rhythmic motor networks.

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.

Down-regulation of the potassium-chloride cotransporter KCC2 contributes to spasticity after spinal cord injury.

Hyperexcitability of spinal reflexes and reduced synaptic inhibition are commonly associated with spasticity after spinal cord injury (SCI). In adults, the activation of gamma-aminobutyric acid(A) (GABAA) and glycine receptors inhibits neurons as a result of low intracellular chloride (Cl-) concentration, which is maintained by the potassium-chloride cotransporter KCC2 (encoded by Slc12a5). We show that KCC2 is downregulated after SCI in rats, particularly in motoneuron membranes, thereby depolarizing the Cl- equilibrium potential and reducing the strength of postsynaptic inhibition. Blocking KCC2 in intact rats reduces the rate-dependent depression (RDD) of the Hoffmann reflex, as is observed in spasticity. RDD is also decreased in KCC2-deficient mice and in intact rats after intrathecal brain-derived neurotrophic factor (BDNF) injection, which downregulates KCC2. The early decrease in KCC2 after SCI is prevented by sequestering BDNF at the time of SCI. Conversely, after SCI, BDNF upregulates KCC2 and restores RDD. Our results open new perspectives for the development of therapeutic strategies to alleviate spasticity.