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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.

Fast synaptic inhibition relies on tight regulation of intracellular Cl(-). Chloride dysregulation is implicated in several neurological and psychiatric disorders. Beyond mere disinhibition, the consequences of Cl(-) dysregulation are multifaceted and best understood in terms of a dynamical system involving complex interactions between multiple processes operating on many spatiotemporal scales. This dynamical perspective helps explain many unintuitive manifestations of Cl(-) dysregulation. Here we discuss how taking into account dynamical regulation of intracellular Cl(-) is important for understanding how synaptic inhibition fails, how to best detect that failure, why Cl(-) regulation is energetically so expensive, and the overall consequences for therapeutics.

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.

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

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.

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

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

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

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.

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.

International audience; 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 se-rotonin hyperpolarizes the reversal potential of inhibitory postsyn-aptic 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 ago-nists, such as (4-bromo-3,6-dimethoxybenzocyclobuten-1-yl)methyl-amine hydrobromide (TCB-2). T he neuron-specific K +-Cl-cotransporter KCC2 (encoded by the solute carrier family 12 member 5, Slc12a5) extrudes Cl-and is responsible for the low [Cl-] i in mature neurons (1-3), a prerequisite for hyperpolarizing inhibition mediated by GABA A receptors (GABA A Rs) and glycine receptors (GlyRs). The expression or the function of KCC2 is reduced in several neurological disorders (2, 4), and the resulting slight increase in [Cl-] i (depola-rizing shift of the chloride equilibrium potential, E Cl) dramatically compromises the inhibitory control of firing rate and excitatory inputs (5-7). Given the role of KCC2 in regulating the strength of inhibitory synaptic transmission, identifying tools that may increase KCC2 function and, hence, restore endogenous inhibition in pathological conditions is of particular importance. Spasticity is a disabling complication affecting individuals with spinal cord injury (SCI) and is characterized by a velocity-dependent increase in muscle tone resulting from hyperexcitable stretch reflexes, spasms, and hypersensitivity to normally innocuous sensory stimulations (8, 9). Down-regulation of KCC2 after SCI in rats is implicated in the development of spasticity (10) and chronic pain (11, 12). Notably, the expression of KCC2 in the motoneuron membrane is reduced, and, concomitantly, the density of cyto-plasmic clusters is higher, suggesting that the surface stability of the transporter is reduced in these pathological conditions (10). Mounting evidence indicates that phosphorylation of KCC2 in the C-terminal intracellular domain dynamically regulates its activity and surface expression (1). In particular, phosphorylation by protein kinase (PK)C, enhances KCC2 activity and reduces endo-cytosis (13). Interestingly, activation of 5-hydroxytryptamine type 2 receptors (5-HT 2 Rs) to serotonin stimulates PKC and strengthens the left-right alternation of motor bursts observed during loco-motion (14-16), which rely on reciprocal inhibition (17, 18). We hypothesized that 5-HT 2 R activity modulates KCC2 function and/ or expression. Our results indicate that the activation of the 5-HT 2A R subtype hyperpolarizes E IPSP via a PKC-dependent mechanism , increases KCC2 expression in the plasma membrane of motoneurons, and reduces SCI-induced spasticity. Results Negative Shift of E IPSP and Up-Regulation of KCC2. We first examined the effect of the 5-HT 2A/2B/2C R agonist (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI) (10 μM; Table S1) on E IPSP in control neonatal rats [postnatal day (P)5-P7]. DOI-hyperpolarized E IPSP within 10-20 min (Fig. 1 A and B). This effect was long-lasting (at least 2 h; Fig. 1B). E IPSP was significantly more hyperpolarized when motoneurons were recorded in the presence of DOI compared with control (8 mV; Fig. 1C, Left). There was a concomitant trend toward a depolarization of the resting membrane potential (V rest) by DOI (+2 mV; P > 0.05). As a result, the amplitude of hyperpolarizing IPSPs recorded at V rest increased significantly (Fig. 1C, Right). The next series of experiments was performed on animals that underwent a neonatal SCI. E IPSP was significantly more depolar-ized in those animals tested at P5-P7, compared with controls of the same age, as shown previously (19) (compare Fig. 1 C and D). Because of the increased sensitivity of neurons to 5-HT in those animals (15), DOI was tested at a lower concentration (1-1.5 μM) than in controls. DOI induced an ∼8-mV hyperpolarization of E IPSP (Fig. 1D, Left). As a result, E IPSP shifted from above to below V rest (Fig. 1D, Right). Another set of animals was treated chronically with DOI from P4 to P6-P7 [0.15 mg/kg, i.p. (15, 20) twice a day]. E IPSP was more hyperpolarized in those DOI-treated animals than in untreated transected animals (Fig. 1E). Values were similar to those measured in control animals. We performed subcellular fractionation of proteins from the lumbosacral spinal cord, followed by immunoblotting with a specific antibody against KCC2. The amount of KCC2 in the membrane fraction (KCC2Mb) was significantly increased after chronic DOI treatment, compared with NaCl-treated pups (Fig. 1F). There was a trend toward an increase in the amount of KCC2 in the cytosolic fraction. As a result, the ratio KCC2Mb/KCC2 cy-toplasm was nonsignificantly increased. We then analyzed the expression of KCC2 by immunohistochemistry. All of the analyses were performed on a homogeneous population of retrogradely labeled lumbar motoneurons [triceps surae (TS) muscles (ankle extensors); Fig. 1G]. GlyRs are colocalized with the anchoring protein gephyrin and can, therefore, be used to label the plasma Author contributions: R.B. and

International audience; During brain development, there is a progressive reduction of intracellular chloride associated with a shift in GABA polarity: GABA depolarizes and occasionally excites immature neurons, subsequently hyperpolarizing them at later stages of development. This sequence, which has been observed in a wide range of animal species, brain structures and preparations, is thought to play an important role in activity-dependent formation and modulation of functional circuits. This sequence has also been considerably reinforced recently with new data pointing to an evolutionary preserved rule. In a recent ``Hypothesis and Theory Article,'' the excitatory action of GABA in early brain development is suggested to be ``an experimental artefact'' (Bregestovski and Bernard, 2012). The authors suggest that the excitatory action of GABA is due to an inadequate/insufficient energy supply in glucose-perfused slices and/or to the damage produced by the slicing procedure. However, these observations have been repeatedly contradicted by many groups and are inconsistent with a large body of evidence including the fact that the developmental shift is neither restricted to slices nor to rodents. We summarize the overwhelming evidence in support of both excitatory GABA during development, and the implications this has in developmental neurobiology.

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.

International audience; Hyperexcitability of spinal reflexes and reduced synaptic inhibition are commonly associated with spasticity after spinal cord injury (SCI). In adults, the activation of γ-aminobutyric acidA (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.

The inhibitory action of glycine and GABA in adult neurons consists of both shunting incoming excitations and moving the membrane potential away from the action potential (AP) threshold. By contrast, in immature neurons, inhibitory postsynaptic potentials (IPSPs) are depolarizing; it is generally accepted that, despite their depolarizing action, these IPSPs are inhibitory because of the shunting action of the Cl − conductance increase. Here we investigated the integration of depolarizing IPSPs (dIPSPs) with excitatory inputs in the neonatal rodent spinal cord by means of both intracellular recordings from lumbar motoneurons and a simulation using the compartment model program “Neuron.” We show that the ability of IPSPs to suppress suprathreshold excitatory events depends on E Cl and the location of inhibitory synapses. The depolarization outlasts the conductance changes and spreads electrotonically in the somatodendritic tree, whereas the shunting effect is restricted and local. As a consequence, dIPSPs facilitated AP generation by subthreshold excitatory events in the late phase of the response. The window of facilitation became wider as E Cl was more depolarized and started earlier as inhibitory synapses were moved away from the excitatory input. GAD65/67 immunohistochemistry demonstrated the existence of distal inhibitory synapses on motoneurons in the neonatal rodent spinal cord. This study demonstrates that small dIPSPs can either inhibit or facilitate excitatory inputs depending on timing and location. Our results raise the possibility that inhibitory synapses exert a facilitatory action on distant excitatory inputs and slight changes of E Cl may have important consequences for network processing.

The persistent sodium current ( INaP) is known to play a role in rhythm generation in different systems. Here, we investigated its contribution to locomotor pattern generation in the neonatal rat spinal cord. The locomotor network is mainly located in the ventromedial gray matter of upper lumbar segments. By means of whole cell recordings in slices, we characterized membrane and INaP biophysical properties of interneurons located in this area. Compared with motoneurons, interneurons were more excitable, because of higher input resistance and membrane time constant, and displayed lower firing frequency arising from broader spikes and longer AHPs. Ramp voltage-clamp protocols revealed a riluzole- or TTX-sensitive inward current, presumably INaP, three times smaller in interneurons than in motoneurons. However, in contrast to motoneurons, INaP mediated a prolonged plateau potential in interneurons after reducing K+ and Ca2+ currents. We further used in vitro isolated spinal cord preparations to investigate the contribution of INaP to locomotor pattern. Application of riluzole (10 μM) to the whole spinal cord or to the upper lumbar segments disturbed fictive locomotion, whereas application of riluzole over the caudal lumbar segments had no effect. The effects of riluzole appeared to arise from a specific blockade of INaP because action potential waveform, dorsal root–evoked potentials, and miniature excitatory postsynaptic currents were not affected. This study provides new functional features of ventromedial interneurons, with the first description of INaP-mediated plateau potentials, and new insights into the operation of the locomotor network with a critical implication of INaP in stabilizing the locomotor pattern.

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.

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.

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.