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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.

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.

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; 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.

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.

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.

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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.