Riluzole treatment modulates KCC2 and EAAT-2 receptor expression and Ca2+ accumulation following ventral root avulsion injury.

Avulsion injury results in motoneuron death due to the increased excitotoxicity developing in the affected spinal segments. This study focused on possible short and long term molecular and receptor expression alterations which are thought to be linked to the excitotoxic events in the ventral horn with or without the anti-excitotoxic riluzole treatment. In our experimental model the left lumbar 4 and 5 (L4, 5) ventral roots of the spinal cord were avulsed. Treated animals received riluzole for 2 weeks. Riluzole is a compound that acts to block voltage-activated Na+ and Ca2+ channels. In control animals the L4, 5 ventral roots were avulsed without riluzole treatment. Expression of astrocytic EAAT-2 and that of KCC2 in motoneurons on the affected side of the L4 spinal segment were detected after the injury by confocal and dSTORM imaging, intracellular Ca2+ levels in motoneurons were quantified by electron microscopy. The KCC2 labeling in the lateral and ventrolateral parts of the L4 ventral horn was weaker compared with the medial part of L4 ventral horn in both groups. Riluzole treatment dramatically enhanced motoneuron survival but was not able to prevent the down-regulation of KCC2 expression in injured motoneurons. In contrast, riluzole successfully obviated the increase of intracellular calcium level and the decrease of EAAT-2 expression in astrocytes compared with untreated injured animals. We conclude that KCC2 may not be an essential component for survival of injured motoneurons and riluzole is able to modulate the intracellular level of calcium and expression of EAAT-2.

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

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

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

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

Prochlorperazine Increases KCC2 Function and Reduces Spasticity after Spinal Cord Injury.

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® 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 motoneurons 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.

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

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

Cleavage of Na(+) channels by calpain increases persistent Na(+) current and promotes spasticity after spinal cord injury.

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.

Chloride Regulation: A Dynamic Equilibrium Crucial for Synaptic Inhibition.

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.

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

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

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

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

Contribution of the potassium-chloride cotransporter KCC2 to the strength of inhibition in the neonatal rodent spinal cord in vitro.

In healthy mature motoneurons (MNs), KCC2 cotransporters maintain the intracellular chloride concentration at low levels, a prerequisite for postsynaptic inhibition mediated by GABA and glycine. KCC2 expression in lumbar MNs is reduced after spinal cord injury (SCI) resulting in a depolarizing shift of the chloride equilibrium potential. Despite modeling studies indicating that such a downregulation of KCC2 function would reduce the strength of postsynaptic inhibition, physiological evidence is still lacking. The present study aimed at investigating the functional impact of a modification of KCC2 function. We focused on a well characterized disynaptic inhibitory pathway responsible for reciprocal inhibition between antagonistic muscles. We performed in vitro extracellular recordings on spinal cords isolated from rodents at the end of the first postnatal week. Genetic reduction of KCC2 expression, pharmacological blockade of KCC2, as well as SCI-induced downregulation of KCC2 all resulted in a reduction of the strength of reciprocal inhibition. We then tried to restore endogenous inhibition after SCI by means of zinc ions that have been shown to boost KCC2 function in other models. Zinc chloride indeed hyperpolarized the chloride equilibrium potential in MNs and increased reciprocal inhibition after neonatal SCI. This study demonstrates that the level of KCC2 function sets the strength of postsynaptic inhibition and suggests that the downregulation of KCC2 after SCI likely contributes to the high occurrence of flexor-extensor cocontractions in SCI patients.

Sensitization of neonatal rat lumbar motoneuron by the inflammatory pain mediator bradykinin.

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.

Serotonergic modulation of post-synaptic inhibition and locomotor alternating pattern in the spinal cord.

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.

Deficits of brainstem and spinal cord functions after neonatal hypoxia-ischemia in mice.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: Our results suggest that monoaminergic neuromodulation plays an important role in the physiopathology of HI brain injury that may represent a good therapeutic target.

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

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

Sodium-mediated plateau potentials in lumbar motoneurons of neonatal rats.

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 Ca(2+) channel blockers on self-sustained spiking was variable, whereas blockade of the persistent sodium current (I(NaP)) 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 Ca(2+) spikes. Substitution of extracellular Na(+) or chelation of intracellular Ca(2+) abolished both sADP and the plateau potential without affecting Ca(2+) spikes. These data suggest a key role of a Ca(2+)-activated nonselective cation conductance ((CaN)) in generating the plateau potential. In line with this, the blockade of (CaN) by 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 I(Ca), I(NaP), and a Na(+)-mediated I(CaN) flowing through putative TRPV2 channels.

Activity-dependent changes in extracellular Ca2+ and K+ reveal pacemakers in the spinal locomotor-related network.

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 locomotor-like activity in the isolated neonatal rodent spinal cord reduces the extracellular calcium ([Ca(2+)]o) to 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.

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

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

Refuting the challenges of the developmental shift of polarity of GABA actions: GABA more exciting than ever!

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.

Glucose is an adequate energy substrate for the depolarizing action of GABA and glycine in the neonatal rat spinal cord in vitro.

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-ß-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 (E(IPSP)). Only high concentrations of pyruvate hyperpolarized E(IPSP). 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.

Combinational spinal GAD65 gene delivery and systemic GABA-mimetic treatment for modulation of spasticity.

BACKGROUND: Loss of GABA-mediated pre-synaptic inhibition after spinal injury plays a key role in the progressive increase in spinal reflexes and the appearance of spasticity. Clinical studies show that the use of baclofen (GABA(B) receptor agonist), while effective in modulating spasticity is associated with major side effects such as general sedation and progressive tolerance development. The goal of the present study was to assess if a combined therapy composed of spinal segment-specific upregulation of GAD65 (glutamate decarboxylase) gene once combined with systemic treatment with tiagabine (GABA uptake inhibitor) will lead to an antispasticity effect and whether such an effect will only be present in GAD65 gene over-expressing spinal segments. METHODS/PRINCIPAL FINDINGS: Adult Sprague-Dawley (SD) rats were exposed to transient spinal ischemia (10 min) to induce muscle spasticity. Animals then received lumbar injection of HIV1-CMV-GAD65 lentivirus (LVs) targeting ventral α-motoneuronal pools. At 2-3 weeks after lentivirus delivery animals were treated systemically with tiagabine (4, 10, 20 or 40 mg/kg or vehicle) and the degree of spasticity response measured. In a separate experiment the expression of GAD65 gene after spinal parenchymal delivery of GAD65-lentivirus in naive minipigs was studied. Spastic SD rats receiving spinal injections of the GAD65 gene and treated with systemic tiagabine showed potent and tiagabine-dose-dependent alleviation of spasticity. Neither treatment alone (i.e., GAD65-LVs injection only or tiagabine treatment only) had any significant antispasticity effect nor had any detectable side effect. Measured antispasticity effect correlated with increase in spinal parenchymal GABA synthesis and was restricted to spinal segments overexpressing GAD65 gene. CONCLUSIONS/SIGNIFICANCE: These data show that treatment with orally bioavailable GABA-mimetic drugs if combined with spinal-segment-specific GAD65 gene overexpression can represent a novel and highly effective anti-spasticity treatment which is associated with minimal side effects and is restricted to GAD65-gene over-expressing spinal segments.