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International audience; 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 reorganization 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) moto-neurons (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.

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.

International audience; 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.

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. J. Comp. Neurol. 519:3470–3487, 2011. © 2011 Wiley-Liss, Inc.

Serotonin (5-HT) plays a critical role in locomotor pattern generation by modulating the rhythm and the coordinations. Pet-1, a transcription factor selectively expressed in the raphe nuclei, controls the differentiation of 5-HT neurons. Surprisingly, inactivation of Pet-1 (Pet-1(-/-) mice) that causes a 70% reduction in the number of 5-HT-positive neurons in the raphe does not impair locomotion in adult mice. The goal of the present study was to investigate the operation of the locomotor central pattern generator (CPG) in neonatal Pet-1(-/-) mice. We first confirmed, by means of immunohistochemistry, that there is a marked reduction of 5-HT innervation in the lumbar spinal cord of Pet-1(-/-) mice. Fictive locomotion was induced in the in vitro neonatal mouse spinal cord preparation by bath application of N-methyl-d,l-Aspartate (NMA) alone or together with dopamine and 5-HT. A locomotor pattern characterized by left-right and flexor-extensor alternations was observed in both conditions. Increasing the concentration of 5-HT from 0.5 to 5 μm impaired the pattern in Pet-1(-/-) mice. We tested the role of endogenous 5-HT in the NMA-induced fictive locomotion. Application of 5-HT(2) or 5-HT(7) receptor antagonists affected the NMA-induced fictive locomotion in both heterozygous and homozygous mice although the effects were weaker in the latter strain. This may be, at least partly, explained by the reduced expression of 5-HT(2A) R as observed by means of immunohistochemistry. These results suggest that compensatory mechanisms take place in Pet-1(-/-) mice that make locomotion less dependent upon 5-HT.

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.

The classical GABA/glycine hyperpolarizing inhibition is not observed in the immature spinal cord. GABA A and glycine receptors are anions channels and the efficacy of inhibitory transmission in the spinal cord is largely determined by the gradient between intracellular and extracellular chloride concentrations. The concentration of intracellular chloride in neurons is mainly regulated by two cation–chloride cotransporters, the potassium–chloride cotransporter 2 (KCC2) and the sodium–potassium–chloride co-transporter 1 (NKCC1). In this study, we measured the reversal potential of IPSPs (E IPSP ) of lumbar motoneurons during the first postnatal week and we investigated the expression of KCC2 and NKCC1 in the ventral horn of the spinal cord from the embryonic day 17 to the postnatal day 20 in the rat. Our results suggest that the negative shift of E IPSP from above to below the resting membrane potential occurs during the first postnatal week when the expression of KCC2 increases significantly and the expression of NKCC1 decreases. KCC2 immunolabeling surrounded motoneurons, presumably in the plasma membrane and NKCC1 immunolabeling appeared outside this KCC2-labeled fine strip. Taken together, the present results indicate that maturation of chloride homeostasis is not completed at birth in the rat and that the upregulation of KCC2 plays a key role in the shift from depolarizing to hyperpolarizing IPSPs.

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.

Locomotor burst activity in the mature intact spinal cord alternates between the left and right sides of a segment through reciprocal inhibition. By contrast, all motor bursts are in phase in the fetus. The alternating pattern disappears after neonatal spinal cord transection which suppresses supraspinal influences upon the locomotor networks. These data reveal the plasticity of spinal cord locomotor networks. This review describes recent evidence suggesting that regulation of cation–chloride cotransporter expression and activity may underlie this plasticity. 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. This post-synaptic action depends on the intracellular concentration of chloride ions ([Cl − ] i ) which is regulated by a protein in the plasma membrane: the K + -Cl − cotransporter (KCC2) extruding both K + and Cl − ions. No or a reduced KCC2 expression leads to a depolarizing (excitatory) action of GABA and glycine. This latter situation is observed early during development and in several pathological conditions, such as epilepsy, neuronal injury and chronic pain.

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.

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.

Bradykinin (Bk) is a potent inflammatory mediator that causes hyperalgesia. The action of Bk on the sensory system is well documented but its effects on motoneurons, the final pathway of the motor system, are unknown. By a combination of patch-clamp recordings and two-photon calcium imaging, we found that Bk strongly sensitizes spinal motoneurons. Sensitization was characterized by an increased ability to generate self-sustained spiking in response to excitatory inputs. Our pharmacological study described a dual ionic mechanism to sensitize motoneurons, including inhibition of a barium-sensitive resting K+ conductance and activation of a nonselective cationic conductance primarily mediated by Na+. Examination of the upstream signaling pathways provided evidence for postsynaptic activation of B2 receptors, G protein activation of phospholipase C, InsP3 synthesis, and calmodulin activation. This study questions the influence of motoneurons in the assessment of hyperalgesia since the withdrawal motor reflex is commonly used as a surrogate pain model. DOI: http://dx.doi.org/10.7554/eLife.06195.001, eLife digest When we accidentally place our hand on a hot stove, we normally experience a painful sensation that starts with the sensory nerves under our skin. These nerves respond by transmitting electrical impulses to our brain, where the painful sensation is then processed. At the same time, these impulses are also transmitted to the motor nerves that control the muscles in our hand to trigger an immediate reflex to withdraw the hand from the hot stove. Pain therefore has a useful role as it can reduce how bad an injury is. People with a condition called hyperalgesia have an increased sensitivity to pain. This condition can result from a chemical called bradykinin ‘sensitizing’ the sensory nerves, causing them to transmit more electrical impulses in response to pain than normal. This makes the injury feel much more painful, and can make the pain last for longer than is beneficial. It was less clear whether bradykinin also affects motor nerves and so triggers a withdrawal reflex. By recording the electrical activity of motor nerve cells taken from the spinal cords of newborn rats, Bouhadfane et al. now show that these motor nerves become more active when exposed to bradykinin. Nerve cells generate electrical signals when ions—such as potassium, sodium, and calcium ions—move through channels in the membranes of the cell. Therefore, to investigate how bradykinin influences the electrical activity of motor nerves, Bouhadfane et al. exposed the cells to drugs that inhibit particular ion channels. This revealed that bradykinin sensitizes the motor nerves by blocking a type of potassium ion channel and activating another ion channel that mainly transports sodium ions. Furthermore, Bouhadfane et al. were able to identify the signaling pathways that allow bradykinin to affect the motor nerve cells. The study implies that the neuronal circuitry for pain does not rely exclusively on sensory nerve cells but should also integrate motor nerve cells. A future challenge remains in developing a protocol to resolve the contribution of motor nerve cells to hyperalgesia assessed by reflex withdrawal. DOI: http://dx.doi.org/10.7554/eLife.06195.002

Bicuculline is the most commonly used GABA(A) receptor antagonist to investigate the contribution of these receptors in motor control. However, this compound has been shown recently to potentiate the burst firing of neurons in various brain regions by blocking a calcium-activated potassium current underlying the spike after-hyperpolarization (AHP). This effect may distort our understanding of the role of GABA(A) receptors at the network level. In vitro brainstem-spinal cord preparations isolated from neonatal rats were used to compare the effects of bicuculline methiodide (bicuculline-M) and picrotoxin (PTX), another GABA(A) receptor antagonist, on the AHP of lumbar motoneurons as well as on spontaneous and locomotor-like motor activities. Intracellular recordings of lumbar motoneurons showed that bicuculline-M (20 microM) reduced the AHP to 57% of control whereas PTX (20-60 microM) had no significant effect. Bath-application of increasing concentrations of PTX caused an increase in spontaneous ventral root activity, which further increased significantly when bicuculline-M was added. The effects of both antagonists were tested on fictive locomotion. The left-right alternation was disrupted in the presence of bicuculline-M. A slow synchronous bursting activity of large amplitude also appeared in the presence of PTX. This slow rhythm was superimposed on a faster rhythm which still exhibited some degree of left-right alternation. These data demonstrate that bicuculline-M may not reveal accurately the contribution of GABA(A) receptors in motor control and the intrinsic properties of disinhibited networks.

The central pattern generators (CPGs) for locomotion, located in the lumbar spinal cord, are functional at birth in the rat. Their maturation occurs during the last few days preceding birth, a period during which the first projections from the brainstem start to reach the lumbar enlargement of the spinal cord. Locomotor burst activity in the mature intact spinal cord alternates between flexor and extensor motoneurons through reciprocal inhibition and between left and right sides through commisural inhibitory interneurons. By contrast, all motor bursts are in phase in the fetus. The alternating pattern disappears after neonatal spinal cord transection which suppresses supraspinal influences upon the locomotor networks. This article will review the role of serotonin (5-HT), in particular 5-HT2 receptors, in shaping the alternating pattern. For instance, pharmacological activation of these receptors restores the left-right alternation after injury. Experiments aimed at either reducing the endogenous level of serotonin in the spinal cord or blocking the activation of 5-HT2 receptors. We then describe recent evidence that the action of 5-HT2 receptors is mediated, at least in part, through a modulation of chloride homeostasis. The postsynaptic action of GABA and glycine depends on the intracellular concentration of chloride ions which is regulated by a protein in the plasma membrane, the K(+)-Cl(-) cotransporter (KCC2) extruding both K(+) and Cl(-) ions. Absence or reduction of KCC2 expression leads to a depolarizing action of GABA and glycine and a marked reduction in the strength of postsynaptic inhibition. This latter situation is observed early during development and in several pathological conditions, such as after spinal cord injury, thereby causing spasticity and chronic pain. It was recently shown that specific activation of 5-HT2A receptors is able to up-regulate KCC2, restore endogenous inhibition and reduce spasticity.

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.

The in vitro brain stem–spinal cord preparation of neonatal (0- to five-day-old) rats was used to establish whether pathways descending from the brain stem are capable of modulating synaptic transmission from primary afferents to lumbar motoneurons within the first few days after birth. We stimulated the ventral funiculus of the spinal cord at the cervical (C1–C2) level. Single-pulse stimulations evoked both excitatory and inhibitory postsynaptic potentials in ipsilateral lumbar (L2–L5) motoneurons which were recorded intracellularly. Twin-pulse stimulations evoked bursts of action potentials in ventral roots. The amplitude of the monosynaptic dorsal root-evoked excitatory postsynaptic potential decreased when a conditioning stimulation was applied to the ventral funiculus 50–300 ms prior to the stimulation of the ipsilateral dorsal root. A decreased input resistance of the motoneurons during the early part (25–100 ms after the artifact) of the ventral funiculus-evoked postsynaptic potentials could account, at least partly, for the decreased amplitude of the dorsal root-evoked response. However, the duration of the inhibition of the dorsal root-evoked excitatory postsynaptic potential was longer than that of the decrease in input resistance. Ventral funiculus stimulation evoked antidromic discharges in dorsal roots. Recordings of dorsal root potentials showed that these discharges were generated by the underlying afferent terminal depolarizations reaching firing threshold. The dorsal root discharge overlapped with most of the time-course of the ventral funiculus-evoked inhibition of the response to dorsal root stimulation, suggesting that part of this inhibition may be exerted at a presynaptic level. The number of antidromic action potentials evoked in dorsal roots by ventral funiculus stimulation increased significantly in saline solution with chloride concentration reduced to 50% of control. Bursts of action potentials disappeared when chloride was removed completely. Antidromic discharges were therefore due to chloride conductance. The number of action potentials evoked in ventral roots was increased in low-chloride saline solutions. Removing chloride from the bathing solution resulted in an unstable ventral root activity. Bath application of the GABA A receptor antagonist, bicuculline (5–10 μ M), blocked the ventral funiculus-evoked antidromic discharges in the dorsal roots. The increase in chloride conductance which generated the depolarizations underlying the dorsal root discharges was therefore mediated by an activation of GABA A receptors. In contrast, bursts of action potentials in the ventral roots were increased in both amplitude and duration under bicuculline. Our data demonstrate that pathways running in the ventral funiculus of the spinal cord exert a control on interneurons mediating presynaptic inhibition at birth.

This report describes the early motor behaviour in the neonatal rat in relation with the maturation of sensory and motor elements of the central nervous system (CNS). The role of vestibular information during the week before (E14-21) and the 2 weeks after (P0-15) birth will be considered. There is a rostro-caudal gradient in the maturation of posture and locomotion with a control of the head and forelimbs during the first postnatal week and then a sudden acceleration in the functional maturation of the hindlimb. At birth, the neonatal rat is blinded and deaf; despite the immaturity of the other sensory systems, the animal uses its olfactory system to find the mother nipple. Vestibular development takes place between E8 and P15. Most descending pathways from the brainstem start to reach the lumbar enlargement of the spinal cord a few days before birth (reticulo-, vestibulospinal pathways as well as the serotonergic and noradrenergic projections); their development is not completed until the end of the second postnatal week. At birth, in an in vitro preparation, a locomotor activity can be evoked by perfusing excitatory amino acids and serotonin over the lumbar region. The descending pathways which trigger the activity of the CPG are also partly functional. At the same age both air stepping and swimming can be induced. Complex locomotion such as walking, trotting and galloping start later because it requires the maturation of the vestibular system, descending pathways and postural reflex regulation. The period around birth is critical to properly define how the vestibular information is essential for the structuring of the motor behaviour. Different types of experiments (hypergravity, microgravity) are planned to test this hypothesis.

The present study was aimed at identifying spinal neurons ascending to the brainstem outside the dorsal columns in the lamprey. Two retrograde tracers (cobalt-lysine and horseradish peroxidase [HRP]) were injected in the brainstem or rostral spinal cord in vivo or in vitro. Labeled cells were distributed bilaterally with a contralateral dominance, along the whole rostrocaudal extent of the spinal cord. The density of cells markedly decreased rostrocaudally. Several classes of brainstem-projecting neurons were identified. Most cells with a short axon were small and formed columns, in the dorsolateral and ventrolateral gray matter, at the transition between the rhombencephalon and the spinal cord. Dorsal elongated cells were spindle shaped, located medially, in the first two spinal segments. Lateral elongated cells were medium to large size neurons, located in the intermediate and lateral gray matter, mainly contralateral to the injection site. Their axon emerging from the lateral part of the soma crossed the midline, ventral to the central canal. These cells were present throughout the rostral spinal cord. Cells were also labeled in the lateral white matter. Some of them had the typical dendritic arborizations of edge cells (intraspinal stretch receptor neurons) and were located in the most rostral segments, bilaterally. Other medium to large size neurons were identified dorsal and medial to most of the edge cells. We suggest that at least the group of lateral elongated cells exhibits rhythmic membrane potential oscillations during fictive locomotion. These cells may, together with the rostral edge cells, be responsible for the locomotor-related modulation of activity in reticulospinal and vestibulospinal neurons.

Perinatal cerebral hypoxia–ischemia (HI) can lead to severe neurodevelopmental disorders. Studies in humans and animal models mainly focused on cerebral outcomes, and little is known about the mechanisms that may affect the brainstem and the spinal cord. Dysfunctions of neuromodulatory systems, such as the serotonergic (5-HT) projections, critical for the development of neural networks, have been postulated to underlie behavioral and motor deficits, as well as metabolic changes. The aim of this study was to investigate brainstem and spinal cord functions by means of plethysmography and sensorimotor tests in a neonatal Rice–Vanucci model of HI in mice. We also evaluated bioaminergic contents in central regions dedicated to the motor control of autonomic functions. Mice with cerebral infarct expressed motor disturbances and had a lower body weight and a decreased respiratory frequency than SHAM, suggesting defects of brainstem neural network involved in the motor control of feeding, suckling, swallowing, and respiration. Moreover, our study revealed changes of monoamine and amino acid contents in the brainstem and the spinal cord of HI mice. Our results suggest that monoaminergic neuromodulation plays an important role in the physiopathology of HI brain injury that may represent a good therapeutic target.

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.

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.

Stimulation of trigeminal afferents has been reported to have powerful effects on the spinal cord in adult animals of several species. In the present study, the pathway transmitting these influences was investigated in the neonatal rat. Experiments were performed on in vitro brainstem/spinal cord preparations. Stimulation of the trigeminal nerve evoked bilateral polysynaptic discharges in lumbar ventral roots. Intracellular recordings from lumbar motoneurons showed mainly excitatory responses, although a few inhibitory responses were also observed. Experiments with perfusion of different parts of the preparation with general or selective synaptic blockers revealed a synaptic relay under GABAergic control in the brainstem, and at least one synapse in the cervical and in the thoracic spinal cord. The involvement of lumbar interneurons was established by perfusing the lumbar enlargement with saline containing either a high concentration of divalent ions or mephenesin in order to reduce transmission along polysynaptic pathways. The contribution of excitatory amino acid transmission was evaluated and was found to evoke mixed receptor responses. The course of the pathway was traced by using different lesions to the brainstem and spinal cord. The pathway was found to be ipsilateral in the brainstem and to become bilateral in the spinal cord. The results of the present study demonstrate that polysynaptic sensorimotor pathways are present at birth. The results are discussed in relation to the pontomedullary locomotor strip, which has been thought to share many features with the trigeminal system.

In vitro studies have repeatedly demonstrated that the neurotransmitters γ-aminobutyric acid (GABA) and glycine depolarize immature neurons in many areas of the CNS, including the spinal cord. This widely accepted phenomenon was recently challenged by experiments showing that the depolarizing action of GABA on neonatal hippocampus and neocortex in vitro was prevented by adding energy substrates (ES), such as the ketone body metabolite dl-β-hydroxybutyric acid (DL-BHB), lactate, or pyruvate to the artificial cerebrospinal fluid (ACSF). It was suggested that GABA-induced depolarizations in vitro might be an artifact due to inadequate energy supply when glucose is the sole energy source, consistent with the energy metabolism of neonatal rat brain being largely dependent on ESs other than glucose. Here we examined the effects of these ESs (DL-BHB, lactate, pyruvate) on inhibitory postsynaptic potentials (IPSPs) recorded from neonatal rat lumbar spinal cord motoneurons (MNs), in vitro. We report that supplementing the ACSF with physiologic concentrations of DL-BHB, lactate, or pyruvate does not alter the reversal potential of IPSPs ( EIPSP). Only high concentrations of pyruvate hyperpolarized EIPSP. In addition, the depolarizing action of GABA on primary afferent terminals was not affected by supplementing the ACSF with ES at physiologic concentrations. We conclude that depolarizing IPSPs in immature MNs and the primary afferent depolarizations are not caused by inadequate energy supply. Glucose at its standard concentration appears to be an adequate ES for the neonatal spinal cord in vitro.

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.

Lamprey spino-bulbar neurones modulate the activity of reticulospinal cells during locomotion. The aim of the present study was to investigate the effects of interrupting or increasing this feedback from the spinal cord on the fictive locomotor pattern. Double-bath experiments were performed on in vitro brain stem/spinal cord preparations. Fictive locomotion was induced by perfusing the spinal cord with 150 microM N-methyl-D-aspartate (NMDA). Blocking the synaptic transmission in the brain stem by exposing it to Mn2+ ions increased the locomotor rhythm. Conversely, stimulation of single reticulospinal neurones during the ipsilateral ventral root burst, when they were depolarized, increased the cycle duration by prolonging the ipsilateral motor burst. The spino-reticulo-spinal loop is an integral part of the locomotor network.

Lamprey reticulospinal neurons show phasic oscillations of their membrane potential during fictive locomotion. This modulation originates from the spinal cord locomotor networks. The aim of the present study was to elucidate the pattern of discharge of the spino-bulbar axons responsible for this modulation. Experiments were performed on in vitro brainstem/spinal cord preparations. Two baths were formed in the recording chamber. The caudal one was perfused with 150 μMN-methyl- d -aspartate (NMDA) solution to induce fictive locomotion. The rostral bath containing the brain and the first 3–5 segments of the spinal cord was exposed to a 0 Ca2+ + 2.6 mM Mn2+ solution to block synaptic transmission and therefore to abolish any rhythmic descending activity. Spinobulbar axons were recorded intracellularly at the level of the brain/spinal cord junction. They exhibited phasic discharges correlated with the ongoing motor activity in the caudal pool. Some discharged in phase with either the ipsilateral or the contralateral ventral root bursts, others with either of the transition phases between these two bursts. These spinal cells with ascending axons, running in the ventrolateral spinal cord, may be important for modulating the activity of supraspinal neurons to match the ongoing locomotor activity.

Serotonergic projections from raphe nuclei arrive in the lumbar enlargement of the spinal cord during the late fetal period in the rat, a time window during which the locomotor-related left/right and flexor/extensor coordinations switch from synchrony to alternation. The goal of the present study was to investigate the role played by serotonin (5-HT) in modulating the left/right and flexor/extensor alternations. Fictive locomotion was induced by bath application of N-methyl-D,L-aspartate (NMA) in the in vitro neonatal rat spinal cord preparation. By means of cross-correlation analysis we demonstrate that 5-HT, when added to NMA, improves left/right and flexor/extensor (recorded from the 3rd and 5th lumbar ventral roots, respectively) alternations. This effect was partly reproduced by activation of 5-HT(2A/2C) receptors. We then tested the contribution of endogenous 5-HT to NMA-induced fictive locomotion. Reducing the functional importance of endogenous 5-HT, either by inhibiting its synthesis with daily injections of p-chloro-phenylalanine (PCPA), starting on the day of birth, or by application of ketanserin (a 5-HT(2) receptor antagonist) or SB269970 (a 5-HT(7) receptor antagonist), disorganized the NMA-induced locomotor pattern. This pattern was restored in PCPA-treated animals by adding 5-HT to the bath. Blocking 5-HT(7) receptors disorganized the locomotor-like rhythm even in the absence of electrical activity in the brain stem, suggesting that NMA applied to the spinal cord does not cause 5-HT release by activating a spino-raphe-spinal loop. These results demonstrate that 5-HT is critical in improving the locomotor-related alternations in the neonatal rat.

Motor behaviors of some species, such as the rat and the human baby, are quite immature at birth. Here we review recent data on some of the mechanisms underlying the postnatal maturation of posture in the rat, in particular the development of pathways descending from the brain stem and projecting onto the lumbar enlargement of the spinal cord. A short-lasting depletion in serotonin affects both posture and the excitability of motoneurons. Here we try to extrapolate to human development and suggest that the abnormalities in motor control observed in childhood—e.g, deficits in motor coordination—might have their roots in the prenatal period, in particular serotonin depletion due to exposure to several environmental and toxicological factors during pregnancy.

Locomotion is a type of motor behaviour that is produced by spinal neuronal networks associated with the different limbs. The rat in which development lasts three weeks in utero and continues during the first three post-natal weeks appears to be a very attractive model for the study of interlimb coordination maturational mechanisms. An early expression of rhythmic locomotor activity is observed at birth in vitro. Neuroactive substances like excitatory amino acid or amines (5-HT...) applied on the entire brainstem/spinal cord preparation induce a unique rhythmical activity over the cervical and the lumbar generators. This strict coordination results from a mutual interaction between generators since both bursting frequencies are altered significantly after functional isolation, with a slowing down of the isolated cervical network and a significant acceleration of the lumbar burst generator. However the rat does not walk at birth due to an absence of postural regulations. During the first two weeks, the neonatal rat is able to swim or to produce “air stepping” with an ipsilateral and a contralateral alternation between the different limbs. Adult walking occurs only during the third postnatal week. At least two mechanisms are able to control this early interlimb coordination . Numerous studies showed that 5-HT has ubiquitous topic and trophic effects on the early development of neurons and synapses. Studies on “PCPA” treated and “spinal” neonatal rats demonstrated strong deficits in locomotor movements. The second mechanism concerns the sensory afferents after birth; proprioceptive peripheral loops feed the central network and adapt its activation in a coordinated manner.

In both vertebrates and invertebrates, the elaboration of locomotion, and its neural control by the central nervous system, are extremely flexible. This is due not only to the network properties of relevant sets of central neurons, but also to the active participation of mutually co-operative central and peripheral loops of neural projections and activity. In this chapter, we describe experiments in which the above concepts have been advanced by comparing locomotor properties in the adult vs. neonatal rat preparation. Data obtained from the in vivo vs. in vitro preparation, and swimming vs. walking behavior, suggest that the locomotor pattern progressively exhibited after birth corresponds to successive steps in the maturation of locomotor networks. Our work emphasises that during the late pre- and early postnatal period, three distinct neural entities--segmental sensory input, descending pathways, and motoneurons--play a key role in the maturation of locomotion and its neural control. We propose that the neonatal rat preparation is an excellent model for studying the conversion from immature to adult locomotion. Some neural controls are more clearly demonstrable in the developing animal preparation than in the adult because the latter exhibits an array of complex and redundant adaptive mechanisms.

The in vitro neonatal rat spinal cord preparation is the first mammalian nervous system isolated from the brainstem to the caudal end of the spinal cord. It permits the study of the cellular properties of mammalian locomotor networks and is unique in containing all the nervous structures related to locomotion. Although being a very immature system, this model has been considered as an adult preparation in which mammalian locomotor central pattern generators can be studied in detail. Nevertheless, one can also follow the development of locomotor functions during the perinatal period. Contrary to the adult, all neuroactive substances can directly reach the cellular structures in the brainstem-spinal cord preparation. When a neuroactive substance is applied to the bath, a single rhythmic activity is recorded along the cord. In fact, three rhythms can be isolated: one at the cervical level for the forelimbs, one at the lumbar level for the hind limbs and one in the sacrococcygeal region for the tail. Studies carried out on this preparation deal with three major areas: (1) relations between spontaneous activity and maturation of spinal network, (2) organisation of the different spinal networks, (3) key role of the descending pathways.

The central pattern generators (CPGs) for locomotion, located in the lumbar spinal cord, are functional at birth in the rat. Their maturation occurs during the last few days preceding birth, a period during which the first projections from the brainstem start to reach the lumbar enlargement of the spinal cord. The goal of the present study was to investigate the effect of suppressing inputs from supraspinal structures on the CPGs, shortly after their formation. The spinal cord was transected at the thoracic level at birth [postnatal day 0 (P0)]. We examined during the first postnatal week the capacity of the CPGs to produce rhythmic motor activity in two complementary experimental conditions. Left and right ankle extensor muscles were recordedin vivoduring airstepping, and lumbar ventral roots were recordedin vitroduring pharmacologically evoked fictive locomotion. Mechanical stimulation of the tail elicited long-lasting sequences of airstepping in the spinal neonates and only a few steps in sham-operated rats.In vitroexperiments made simultaneously on spinal and sham animals confirmed the increased excitability of the CPGs after spinalization. A left–right alternating locomotor pattern was observed at P1–P3. Both types of experiments showed that the pattern was disorganized at P6–P7, and that the left–right alternation was lost. Alternation was restored after the activation of serotonergic 5-HT2receptorsin vivo. These results suggest that descending pathways, in particular serotonergic projections, control the strength of reciprocal inhibition and therefore shape the locomotor pattern in the neonatal rat.

The adult pattern of locomotion is observed at the end of the second postnatal week in the rat. The in vitro spinal cord isolated from immature rats has served as a valuable preparation to study the mechanisms underlying the development of locomotion. Although the rat is unable to walk at birth, because of an immature posture, its spinal cord networks can generate at least two kinds of motor patterns in vitro. One activity is called 'fictive locomotion' because it shares several common features with locomotion observed in vivo. This fictive locomotor pattern is rarely observed spontaneously and its release requires either pharmacological or electrical stimulation of the spinal cord. A second endogenously generated activity observed in this preparation occurs spontaneously and exhibits phase relationships between motor outputs that are quite different from the fictive locomotor pattern. Here we review some of the developmental functions this spontaneous activity may subserve. It is likely a major trigger for the maturation of lumbar networks in the fetus, at a stage when inputs from both the periphery and supraspinal structures are weak. Pathways descending from the brainstem arrive in the lumbar enlargement during the last week in utero and the first two postnatal weeks. These pathways, through the neurotransmitters they contain, especially monoamines, are essential for the expression of some neuronal properties and may regulate several ongoing developmental processes.

Charcot–Marie–Tooth disease type 1A is the most frequent hereditary neuropathy affecting the peripheral nervous system. A partial duplication of chromosome 17 (17p11.2) involving the PMP22 gene is responsible for dysmyelination–demyelination processes leading to motor and sensory impairments. Murine models of this disease are now widely used to investigate the mechanisms occurring at the behavioural and physiological levels. In this study, adult transgenic mice (6 months old) having integrated 7 copies of the human PMP22 gene were used to compare the motor performance, evaluated by using a complex locomotor test (the rotarod test), with both the number of functional motoneurons innervating the soleus muscle and the level of myelination in the sciatic nerve. Two levels of motor deficits were detected and led us to divide the population into two subgroups. In both impaired groups, the level of motor deficit was strongly correlated with the number of functional motoneurons evaluated by retrograde labeling from the muscle, but not with the number of myelinated fibers or the thickness of the myelin sheath (g-ratio). It therefore appears that the number of motor units may be a key element in motor impairments observed in Charcot–Marie–Tooth disease type 1A disease. These findings may have implications for therapeutic procedures, which should focus on the survival of the motoneuronal pool and/or the maintenance of functional neuro-muscular connexions to reduce motor impairments in humans.

Serotonin (5-HT) plays an important role both in the development and in the recovery of locomotion after spinalization in vertebrates. We investigated the contribution of the serotonergic system to the maturation of the lumbar motoneurons and networks in the neonatal rat. A 5-HT synthesis inhibitor, p -chlorophenylalanine (PCPA), was administered daily from the first postnatal day (P0) onward. This protocol depleted serotonin in the spinal cord within 3–4 d, as demonstrated by immunohistochemistry. PCPA-treated rats exhibited postural changes characterized by lesser flexion at the knee and ankle levels and lesser extension of the hip. Posture was asymmetric, suggesting possible deficits in the interlimb coordination. Intracellular recordings were made at P3–5 from motoneurons innervating different hindlimb muscles, using the in vitro brainstem–spinal cord–nerve-attached preparation. In PCPA-treated rats, the conduction velocity of motoneurons was increased, and their excitability was decreased (because of higher rehobase and input conductance) compared with sham animals. In accordance with postural observations, changes were more pronounced in hip extensor/knee flexor than in ankle extensor motoneurons. The maturation of repetitive firing properties was stopped by PCPA treatment, although PCPA, applied in vitro , had no effect on membrane properties. The spontaneous endogenously generated activity, which is a characteristic of immature networks, was increased in PCPA-treated rats, suggesting that developing lumbar networks are sensitive to 5-HT levels. Serotonin may play a critical role during development in regulating the balance between the excitability of motoneurons and that of interneurons. Interneuronal excitability is crucial for the activity-dependent development of spinal cord networks.

A somaesthetic pathway to the magnocellular red nucleus (RNm) via relays other than cortico- or cerebello-rubral relays was previously found to exist in the cat. At the brainstem level, the ascending spinorubral fibres follow the medial lemniscus (LM). The present paper aims at describing in detail and evaluating the quantitative importance of the short-latency responses in RNm cells after microstimulation performed in the LM through a monopolar electrode. The RNm cells, tested intracellularly in cats anaesthetized with alpha-choralose and placed in a stereotaxic device, were identified by their antidromic activation to stimulation of the rubrospinal tract in the cervical cord. It was established that single-shock stimulation below 100 miroA current delivered to the LM induced short-lantency postsynaptic potentials (PSPs) in 87% of all the rubrospinal cells tested. The responses were indeed due to activation of LM fibres, as demonstrated by different tests: the location of the electrode tip in the LM was verified by recording, with the same electrode, the potentials evoked by stimulating the dorsal columns of the cord. The site was later confirmed histologically. The absence of stimulus spread from the LM to the underlying pyramidal tract was systematically checked by simultaneously recording the responses evoked in RNm cells and in the motor cortex. Monosynaptic excitatory responses (EPSPs) were evoked in RNm cells with a minimum stimulating current of less than 20 microA in the LM and a mean threshold of 42 microA. Disynaptic inhibitory potentials (IPSPs) were evoked in 23% of these cells with single-pulse stimulation. These latter responses showed a temporal facilitation with short trains of three pulses, which indicated that they were transmitted through inhibitory interneurones. Recordings were also performed from presumed LM fibre terminals running inside the RNm. The results therefore confirm the existence of strong lemniscal projections to RNm and demonstrate that they transmit both excitatory and inhibitory messages to rubrospinal cells. As the somaesthetic pathway to the RNm was previously found to come from the spinal cord, where it is located in the ventral portion, the present results also confirm that the LM is composed of fibres originating not only from neurones in the dorsal column nuclei, but also from cells placed at the segmental levels of the cord. The presumed sensorimotor function of this ascending pathway is discussed.

The activity of cells in the magnocellular red nucleus (RNm) was recorded extra- and intracellularly in the curarized thalamic cat performing fictive locomotion. The locomotor episodes were detected from the rhythmic activity recorded in the motor nerves of the contralateral hindlimb. It was confirmed that, during fictive locomotion, a large proportion of the rubrospinal cells (56% in our sample) exhibit a rhythmic pattern of activity which is synchronized with the efferent spinal motor nerve activity. On the basis of the intracellular recordings it was established that phases of intense synaptic activity with mixed excitatory postsynaptic potentials (EPSPs) and inhibitory postsynaptic potentials (IPSPs) are involved in this rhythmicity. After eliminating the cerebellar input to the RNm, it was observed that the cells still received intense excitatory and inhibitory inputs, resulting in a continuous modulation of their membrane potential, due to the occurrence of EPSPs and IPSPs. During fictive locomotor-like activity and after elimination of the cerebellar afferents to the RNm, it was observed that the spontaneous PSPs in RNm cells (in the case of 45% of the cells) were organized in repetitive subthreshold bursts occurring in phase relationships with the activity recorded in the motor nerves. Some extracellularly recorded cells (12%) showed a rhythmic firing pattern. It is generally recognized that, in the thalamic cat preparation, the locomotor pattern observed in efferent nerves originates from the central pattern generator (CPG) of the spinal cord. It therefore seems likely that the rhythmicity observed here in the RNm may originate from the spinal CPG and be transmitted through the spino-rubral pathway ascending in the ventral part of the cord. It is concluded that the spino-rubral pathway may transmit both somatosensory information and corollary discharges relating to the activity of the spinal CPG for locomotion.

Although it has been known for a long time that in awake cats, natural stimulation of the skin induces short latency responses in rubrospinal cells, the pathway possibly involved has been identified only recently (Padel et al. 1988). This tract, which was described in acute, chloralose anaesthetized cats, ascends in the ventromedial spinal cord and is activated via collaterals of primary afferent fibres running in the dorsal columns of the spinal cord. The present study demonstrates that this newly described spino-rubral tract is able to send detailed somaesthetic information to the red nucleus. After lesions leaving intact only the spino-rubral pathway, excitatory and inhibitory responses to natural peripheral stimulations were recorded in identified rubral efferent cells. The most effective stimuli were touching the skin, passive joint rotation and hair displacement. Each cell was found to possess a particular receptive field. These fields which could be ipsi-, contra-, or bi-lateral were generally located on a single limb, although they could include two or more limbs, or even exceptionally the whole body with or without preferential zones. The topographic organization of receptive fields was arranged somatotopically in the red nucleus and overlapped the motor representation. The somaesthetic inputs transmitted through the spino-rubral pathway to the red nucleus are very similar to those previously observed in the intact cat, which supports the idea that this pathway may play a functional role in motor control. The spino-rubro-spinal loop may provide a fast adaptation of the descending motor command, thus producing a fine and harmonious tuning between the changing surroundings and the animal's movements.

In awake chronically implanted cat, the cells in the red nucleus (RN) can be either activated or inhibited by natural stimulation on periphery. The effective stimuli are touching the fur, rotating the joints and tapping the muscles. A somaesthetic map has been constructed with the face area dorsally, the forelimb more ventrally and the hindlimb lateroventrally in the RN. In acute preparations, after ablation of the motor cortex and the cerebellum and section of the dorsal columns of the spinal cord at cervical level, the RN cells were still reacting to natural stimulation of the skin or electrical stimulation of peripheral nerves. The course of the somaesthetic pathway was systematically mapped by microstimulation of the spinal cord. It was shown that it follows the primary afferents which enter the dorsal columns, where they give off collaterals which relay at segment levels. After decussation the fibres ascend the ventromedial quadrant of the cord. A large portion of the fibres relay a second time in the medulla. The existence of such a pathway can account for the somaesthetic responses recorded in the RN in awake cats.

The spinal interneurons in newborn rodents, when synaptically isolated by removing the extracellular calcium ([Ca2+]o), demonstrate intrinsic rhythmic bursting activity that can be suppressed by riluzole, a blocker of the persistent sodium current (INaP) [2]. This finding led to the suggestion that lowering of [Ca2+]o may enhance INaP by shifting its activation threshold toward more negative voltages, and raised the question of functional relevance of this finding to generation of locomotor rhythm. To assess this issue, a series of experiments was performed in vitro using the isolated spinal cord preparation from the neonatal rat with measurements of [Ca2+]o and extracellular potassium concentration ([K+]o) during pharmacologically induced fictive locomotion. We demonstrated that with the onset of fictive locomotion, [Ca2+]o reduced from 1.2 up to 0.9 mM whereas [K+]o increased from 4 up to 6 mM. At the same time, a special study performed on the isolated genetically identified Hb9 excitatory interneurons showed that, at [Ca2+]o= 1 mM and [K+]o=5 mM, 12% of Hb9 cells expressed intrinsic INaP-dependent bursting, and at the concentrations typical for fictive locomotion ([Ca2+]o= 0.9 mM and [K+]o=6 mM), as many as 50% of identified Hb9 interneurons expressed INaP-dependent bursting. Importantly, the threshold of [Ca2+]o to generate bursting decreased as [K+]o increased. The analysis of Hb9 neuron behavior during slow ramp increase of voltage revealed that lowering [Ca2+]o from 1.2 to 0.9 mM induced a negative shift (~ -3 mV) in the INaP half-activation voltage (V1/2NaP). In contrast, V1/2NaP was not changed when [K+]o increased from 4 to 6 mM. To theoretically investigate the effect of changing [Ca2+]o and [K+]o on the Hb9’s pacemaker properties and firing behavior, we developed a single-compartment computational model of Hb9 neuron. In this model, we explicitly simulated a negative shift of V1/2NaP occurring with the reduction of [Ca2+]o.. At [K+]o=6 mM, our model exhibited tonic activity at V1/2NaP = –50 mV (Fig. ​(Fig.1A,1A, left). The rhythmic bursting emerged at V1/2NaP = –51 mV, and further shifting V1/2NaP to the left produced stable bursting (Fig. ​(Fig.1A,1A, right). In turn, an increase in [K+]o reduced the potassium reversal potential and hence all voltage-gated potassium currents (IK), which provided an additional augmentation of INaP-dependent bursting [1]. To study a synergistic effect of [Ca2+]o and [K+]oon the emergence of bursting activity, we modeled a population of 50 uncoupled neurons with randomly distributed parameters (see Fig. ​Fig.1B).1B). Our simulations have shown that shifting V1/2NaP towards more negative values induced by reducing [Ca2+]o may play a major role in emergence of bursting activity in the population of spinal interneurons. We have also demonstrated that accumulation of [K+]ocan facilitate the emergence of INaP-dependent bursting via the reduction of IK. Figure 1 A. Switching from tonic to bursting activity in the modeled neuron by shifting V1/2NaP to more negative value. B. The synergistic effect of [Ca2+]o and [K+]ochanges on emergence of bursting activity in the modeled population of 50 uncoupled cells. In summary we suggest that co-regulation of INaP and IK by the corresponding changes in [Ca2+]o and [K+]o may convert activity of spinal interneurons from asynchronous/tonic to the synchronized bursting. This activity-dependent switching in firing behavior may represent a fundamental mechanism for locomotor rhythm generation in the spinal cord.