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Occupational or environmental exposure to excessive Mn would cause manganism, which is resembled Parkinson disease. However, the mechanism underlying manganism is still unknown. It had been documented that astrocytes play important roles in physiological function in brain. Therefore, in the present study, the cultured astrocytes were exposed to 0, 125, 250, and 500 μM MnCl(2), and cell viability, lactate dehydrogenase (LDH) leakage, morphological change, cell cycle progression, and apoptosis were determined. In addition, 100 μM riluzole (a glutamatergic modulator) was pretreated for 6 h before no MnCl(2) exposure or 500 μM MnCl(2) exposure. The results showed that cell viability inhibited, LDH leakage elevated, morphology injured, G(0)/G(1) phase cell cycle arrested, and apoptosis rate increased in a concentration-dependent manner. Further investigation indicated that riluzole pretreatment reversed cytotoxicity, cell cycle aberration, and apoptosis on astrocytes caused by MnCl(2). These results suggested that MnCl(2) could cause cytotoxicity, cell cycle arrest, and apoptosis concentration-dependently; riluzole might antagonize Mn toxicity on astrocytes.

The neurotoxic effects of cerebrospinal fluid (CSF) from patients suffering amyotrophic lateral sclerosis (ALS), have been reported by various authors. However, variable results have been communicated and the mechanism of such neurotoxicity has been attributed to excess glutamate concentrations in ALS/CSF. We have studied here the properties of 14 CSFs from control patients and 29 CSFs from patients of ALS. We found that while ALS/CSF impairs the viability of rat brain cortical motoneurons maintained in primary cultures, this effect seemed to be exerted through a glutamate-independent mechanism. Resveratrol protected against such neurotoxic effects and antagonized the [Ca(+2)](c) elevation produced by ALS/CSF. However, riluzole did not afford protection and antagonized the resveratrol-elicited neuroprotective effects. We conclude that ALS/CSF elicited neurotoxicity on in vitro cultures of rat brain cortical motor neurons may become a sound microassay to test available novel multitargeted neuroprotective compounds with potential therapeutic application in ALS patients., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Paroxysmal extreme pain disorder (PEPD) and inherited erythromelalgia (IEM) are inherited pain syndromes arising from different sets of gain-of-function mutations in the sensory neuronal sodium channel isoform Nav1.7. Mutations associated with PEPD, but not IEM, result in destabilized inactivation of Nav1.7 and enhanced resurgent sodium currents. Resurgent currents arise after relief of ultra-fast open-channel block mediated by an endogenous blocking particle and are thought to influence neuronal excitability. As such, enhancement of resurgent currents may constitute a pathological mechanism contributing to sensory neuron hyperexcitability and pain hypersensitivity associated with PEPD. Furthermore, pain associated with PEPD, but not IEM, is alleviated by the sodium channel inhibitor carbamazepine. We speculated that selective attenuation of PEPD-enhanced resurgent currents might contribute to this therapeutic effect. Here we examined whether carbamazepine and two other sodium channel inhibitors, riluzole and anandamide, exhibit differential inhibition of resurgent currents. To gain further insight into the potential mechanism(s) of resurgent currents, we examined whether these inhibitors produced correlative changes in other properties of sodium channel inactivation. Using stably transfected human embryonic kidney 293 cells expressing wild-type Nav1.7 and the PEPD mutants T1464I and M1627K, we examined the effects of the three drugs on Navβ4 peptide-mediated resurgent currents. We observed a correlation between resurgent current inhibition and a drug-mediated increase in the rate of inactivation and inhibition of persistent sodium currents. Furthermore, although carbamazepine did not selectively target resurgent currents, anandamide strongly inhibited resurgent currents with minimal effects on the peak transient current amplitude, demonstrating that resurgent currents can be selectively targeted.

Valproate produces analgesia in animals and humans, however, its mechanisms of action are yet unknown. The present study examined effects of repeated administration of valproate on behavioral hypersensitivity and expression of glutamate transporter-1 (GLT-1) and glutamate-aspartate transporter (GLAST) in the spinal dorsal horn in rats after L5-L6 spinal nerve ligation (SNL). SNL significantly reduced mechanical withdrawal threshold and expression of GLT-1 and GLAST in the spinal dorsal horn. Repeated oral administration of valproate reduced hypersensitivity, restored down-regulated expression of GLT-1 and GLAST in the spinal dorsal horn, and enhanced analgesia from the glutamate transporter activator riluzole. This analgesia from valproate was blocked by the selective GLT-1 blocker dihydrokainic acid (DHK). These data suggest that valproate restores down-regulated expression of glutamate transporters in the spinal cord to presumably reduce glutamate signaling and to reduce hypersensitivity after nerve injury, and that combination of valproate with riluzole produces enhanced analgesia which relies on the spinal glutamate transporters., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Kainate-mediated excitotoxicity of organotypic spinal cord cultures is an in vitro model advantageous to investigate basic mechanisms of acute spinal injury and its pharmacological neuroprotection. Using such cultures, the putative neuroprotective agent riluzole applied at 5 μM (plasma therapeutic concentration) was studied for its ability to prevent neurotoxicity evoked by 1 h administration of kainate. We monitored real-time release of glutamate, release of lactate dehydrogenase (LDH) (cell damage marker), occurrence of cell pyknosis, the number of surviving neurons and motoneurons, and cell culture metabolic activity. Co-applied riluzole strongly blocked the kainate-evoked early rise in extracellular glutamate (via calcium dependent or independent processes) and suppressed LDH release (limited to <20% of total). Although there were no significant cell losses within the first h after kainate washout, pyknosis, fewer neurons and motoneurons were observed 24 h later. MTT assay demonstrated that surviving cells were metabolically competent. Co-application of kainate and tetrodotoxin also failed to protect spinal cord slices 24 h later. When riluzole application begun at kainate washout and continued for 24 h, significant neuroprotection was observed for neurons in the central and dorsal regions, while ventral horn cells (including motoneurons) were not protected. Our data suggest that riluzole neuroprotection against excitotoxicity was feasible, although it paradoxically required delayed drug administration, and was not extended to the ventral horn. We propose that riluzole was acting on yet-unidentified processes downstream of glutamate release and receptor activation. Deciphering their identity and role in cell death mechanisms may be an important goal to develop neuroprotection., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

Recently, we reported the tetrodotoxin (TTX)- and dihydropyridine (DHP)-resistant (TDR) inward currents in neonatal mouse spinal neurons. In this study, we further characterized these currents in the presence of 1-5 μM TTX and 20-30 μM DHP (nifedipine, nimodipine, or isradipine). TDR inward currents were recorded by voltage ramp (persistent inward current, TDR-PIC) and step (TDR-I(p)) protocols. TDR-PIC and TDR-I(p) were found in 80.2% of recorded neurons (101/126) crossing laminae I to X from T12 to L6. TDR-PIC activated at -28.6 ± 13 mV with an amplitude of 80.6 ± 75 pA and time constant of 470.6 ± 240 ms (n = 75). TDR-I(p) had an amplitude of 151.2 ± 151 pA and a voltage threshold of -17.0 ± 9 mV (n = 54) with a wide range of kinetics parameters. The half-maximal activation was -21.5 ± 8 mV (-37 to -12 mV, n = 29) with a time constant of 5.2 ± 2 ms (1.2-11.2 ms, n = 19), whereas the half-maximal inactivation was -26.9 ± 9 mV (-39 to -18 mV, n = 14) with a time constant of 1.4 ± 0.4 s (0.5-2.2 s, n = 19). TDR-PIC and TDR-I(p) could be reduced by 60% in zero calcium and completely removed in zero sodium solutions, suggesting that they were mediated by sodium ions. Furthermore, the reversal potential of TDR-I(p) was estimated as 56.6 ± 3 mV (n = 10). TDR-PIC and TDR-I(p) persisted in 1-205 μM TTX, 20-100 μM DHP, 3-30 μM riluzole, 50-300 μM flufenamic acid, and 2-30 mM intracellular BAPTA. They also persisted with T-, N-, P/Q-, and R-type calcium channel blockers. In conclusion, we demonstrated novel TTX-, DHP-, and riluzole-resistant sodium channels in neonatal rodent spinal neurons. The unique pharmacological and electrophysiological properties would allow these channels to play a functional role in spinal motor system.

Vasomotion is linked to the rapid oscillations of intracellular calcium levels. In rat pulmonary artery, this activity can manifest as a slow periodic on-off pattern, the timing of which depends on the type and intensity of pharmacological stimuli employed. In this study, we have sought to characterize a slow-wave vasomotor activity pattern induced in isolated arterial ring preparations by simultaneous exposure to the α(1) -adrenoceptor agonist phenylephrine (1-10 nm) and the L channel agonist S(-)-Bay K 8644 (3-20 nm). Treated tissues responded with a stable on-off pattern of vasomotion persisting for >5 h at 5-6 cycles/h. In intact rings, this response was suppressed by methacholine and restored or enhanced by N(ω) -nitro-l-arginine methyl ester. Analogous inhibitory effects were obtained with high Mg(2+) , 8-Br-cGMP (but not 8-Br-cAMP), riluzole, ryanodine, chelerythrine, and fasudil. Pinacidil (30 nm) increased off-cycle length without change in slow-wave amplitude. Conversely, tetraethylammonium (1.0-3.0 mm) augmented the latter without affecting periodicity. Carbenoxolone (10 μm) abolished slow-wave activity, while raising basal tone and inducing random phasic activity. In endothelium-denuded rings, the threshold of agonist-induced slow-wave vasomotion was lowered and a similar inhibitory effect obtained with carbenoxolone. In conclusion, the slow-wave pattern of vasomotion described here is (i) subject to inhibitory modulation by endothelial NO and an array of voltage-gated and leak K conductances yet to be fully characterized; (ii) dependent on Ca(2+) from both extracellular and sarcoendoplasmatic sources; (iii) controlled by kinase (Rho and PKC)-mediated regulation of myosin light chain phosphatase; and (iv) synchronized via intermyocyte gap junctions., (© 2010 The Authors Fundamental and Clinical Pharmacology © 2010 Société Française de Pharmacologie et de Thérapeutique.)

Neocortical oscillations result from synchronized activity of a synaptically coupled network and can be strongly influenced by the intrinsic firing properties of individual neurons. As such, the intrinsic electroresponsive properties of individual neurons may have important implications for overall network function. Rhythmic intrinsic bursting (rIB) neurons are of particular interest, as they are poised to initiate and/or strongly influence network oscillations. Although neocortical rIB neurons have been recognized in multiple species, the current study is the first to identify and characterize rIB neurons in the human neocortex. Using whole-cell current-clamp recordings, rIB neurons (n = 12) are identified in human neocortical tissue resected from pediatric patients with intractable epilepsy. In contrast to human regular spiking neurons (n = 12), human rIB neurons exhibit rhythmic bursts of action potentials at frequencies of 0.1-4 Hz. These bursts persist after blockade of fast excitatory neurotransmission and voltage-gated calcium channels. However, bursting is eliminated by subsequent application of the persistent sodium current (I(NaP)) blocker, riluzole. In the presence of riluzole (either 10 or 20 μm), human rIB neurons no longer burst, but fire tonically like regular spiking neurons. These data demonstrate that I(NaP) plays a critical role in intrinsic oscillatory activity observed in rIB neurons in the human neocortex. It is hypothesized that aberrant changes in I(NaP) expression and/or function may ultimately contribute to neurological diseases that are linked to abnormal network activity, such as epilepsy., (© 2011 The Authors. European Journal of Neuroscience © 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

Aim: Earlier studies have shown that TREK-1 and TREK-2 (TREKs), members of the two-pore domain K(+) (K(2P)) channel family that are highly expressed under pathological conditions, are activated by neuroprotective agents. Baicalein and wogonin, oriental flavonoids originating from the root of the medicinal herb Scutellaria baicalensis, are known to have beneficial effects for neuroprotection. However, little is known about the effects of baicalein and wogonin on ion channels including TREKs. We investigated whether baicalein and wogonin modulate the TREK-2 channel, which has been less studied than TREK-1., Methods: Single-channel recordings were performed in COS-7 cells transfected with rat TREK-2 and analyzed baicalein- or wogonin-induced channel activity., Results: We found that baicalein and wogonin activated the TREK-2 current by increasing the opening frequency (channel activity: from 0.05 ± 0.01 to 0.17 ± 0.06 in baicalein treatment and from 0.03 ± 0.01 to 0.29 ± 0.09 in wogonin treatment, P < 0.05), while leaving the single-channel conductance and mean open time unchanged. Baicalein continuously activated TREK-2, whereas wogonin transiently activated TREK-2. Application of baicalein and wogonin activated TREK-2 in both cell attached and excised patches, suggesting that baicalein and wogonin may modulate TREK-2 either directly or indirectly with different mechanisms., Conclusion: These results suggest that baicalein- and wogonin-induced TREK-2 activation help set the resting membrane potential of cells exposed to pathological conditions and thus may give beneficial effects in neuroprotection., (© 2011 The Authors. Acta Physiologica © 2011 Scandinavian Physiological Society.)

The objectives of this study were to probe the contribution of spinal neuron persistent sodium conductances to reflex hyperexcitability in human chronic spinal cord injury. The intrinsic excitability of spinal neurons provides a novel target for medical intervention. Studies in animal models have shown that persistent inward currents, such as persistent sodium currents, profoundly influence neuronal excitability, and recovery of persistent inward currents in spinal neurons of animals with spinal cord injury routinely coincides with the appearance of spastic reflexes. Pharmacologically, this neuronal excitability can be decreased by agents that reduce persistent inward currents, such as the selective persistent sodium current inhibitor riluzole. We were able to recruit seven subjects with chronic incomplete spinal cord injury who were not concurrently taking antispasticity medications into the study. Reflex responses (flexion withdrawal and H-reflexes) and volitional strength (isometric maximum voluntary contractions) were tested at the ankle before and after placebo-controlled, double-blinded oral administration of riluzole (50 mg). Riluzole significantly decreased the peak ankle dorsiflexion torque component of the flexion withdrawal reflex. Peak maximum voluntary torque in both dorsiflexion and plantarflexion directions was not significantly changed. Average dorsiflexion torque sustained during the 5-s isometric maximum voluntary contraction, however, increased significantly. There was no effect, however, on the monosynaptic plantar and dorsiflexor H-reflex responses. Overall, these results demonstrate a contribution of persistent sodium conductances to polysynaptic reflex excitability in human chronic spinal cord injury without a significant role in maximum strength production. These results suggest that intrinsic spinal cellular excitability could be a target for managing chronic spinal cord injury hyperreflexia impairments without causing a significant loss in volitional strength.

In addition to a fast activating and immediately inactivating inward sodium current, many types of excitable cells possess a noninactivating or slowly inactivating component: the persistent sodium current (I(NaP)). The I(NaP) is found in normal primary sensory neurons where it is mediated by tetrodotoxin-sensitive sodium channels. The dorsal root ganglion (DRG) is the gateway for ectopic impulses that originate in pathological pain signals from the periphery. However, the role of I(NaP) in DRG neurons remains unclear, particularly in neuropathic pain states. Using in vivo recordings from single medium- and large-diameter fibers isolated from the compressed DRG in Sprague-Dawley rats, we show that local application of riluzole, which blocks the I(NaP), also inhibits the spontaneous activity of A-type DRG neurons in a dose-dependent manner. Significantly, riluzole also abolished subthreshold membrane potential oscillations (SMPOs), although DRG neurons still responded to intracellular current injection with a single full-sized spike. In addition, the I(NaP) was enhanced in medium- and large-sized neurons of the compressed DRG, while bath-applied riluzole significantly inhibited the I(NaP) without affecting the transient sodium current (I(NaT)). Taken together, these results demonstrate for the first time that the I(NaP) blocker riluzole selectively inhibits I(NaP) and thereby blocks SMPOs and the ectopic spontaneous activity of injured A-type DRG neurons. This suggests that the I(NaP) of DRG neurons is a potential target for treating neuropathic pain at the peripheral level.

Riluzole is approved for the treatment of amyotrophic lateral sclerosis (ALS); however, recent accumulating evidence suggests that riluzole is also effective for the treatment of psychiatric disorders, such as mood disorders. Plastic change in the brain induced by neurotrophic factors/growth factors is thought to be involved in the mechanism of antidepressants. This study investigated the mechanism of riluzole-induced glial cell line-derived neurotrophic factor (GDNF) production in rat C6 glioma cells (C6 cells), a model of astrocytes. The study investigated the phosphorylation of cAMP response element binding protein (CREB), an important transcriptional factor of the gdnf gene, and found that riluzole increased CREB phosphorylation in a time-dependent manner, peaking at 40min after treatment. The riluzole-induced CREB phosphorylation was completely blocked by a mitogen-activated protein kinase kinase (MEK) inhibitor (U0126). Riluzole increased extracellular signal-regulated kinase (ERK) activation prior to CREB phosphorylation. These results suggest that riluzole rapidly activates the MEK/ERK/CREB pathway. Furthermore, two types of fibroblast growth factor receptor (FGFR) tyrosine kinase inhibitors (SU5402 and PD173074) completely blocked riluzole-induced CREB phosphorylation. In addition, riluzole rapidly phosphorylated FGFR substrate 2α (FRS2α), a major adaptor protein of FGFR. These findings suggest that riluzole induces CREB phosphorylation through FGFR. In addition, PD173074 inhibited riluzole-induced GDNF production. In contrast, l-glutamate and a glutamate transporter inhibitor (t-PDC) did not yield any effects in either CREB phosphorylation or GDNF production. These findings suggest that riluzole rapidly activates a MEK/ERK/CREB pathway through FGFR in a glutamate transporter-independent manner, followed by GDNF expression in C6 cells., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Purpose: Brain metastases are a common preterminal event in patients with metastatic melanoma and require radiation therapy. Our group has previously shown that human GRM1 (hGRM1) expressing melanoma cells release excess extracellular glutamate and are growth inhibited by riluzole, an inhibitor of glutamate release. Riluzole-treated cells accumulate in G(2)/M phase of the cell cycle at 24 hours, and then undergo apoptotic cell death. We evaluated whether riluzole enhanced radiosensitivity in melanoma cells., Experimental Design: Clonogenic assays were performed to evaluate clonogenic survival after treatment in hGRM1 expressing and nonexpressing melanoma cells. Western immunoblots were performed to confirm apoptotic cell death. A xenograft mouse model was used to validate the in vitro experiments. Tumors harvested from the xenografts were fixed and stained for apoptosis and DNA damage markers., Results: In the hGRM1-positive cell lines C8161 and UACC903, riluzole enhanced the lethal effects of ionizing radiation; no difference was seen in the hGRM1-negative UACC930 cell line. C8161 cells treated with riluzole plus irradiation also showed the highest levels of the cleaved forms of PARP and caspase-3; excised C8161 xenografts showed the greatest number of apoptotic cells by immunohistochemistry (P < 0.001). On cell cycle analysis, a sequence-dependent enrichment in the G(2)/M phase was shown with the combination of riluzole and irradiation. Xenografts treated with riluzole and weekly radiation fractions showed significant growth inhibition and revealed markedly increased DNA damage., Conclusions: We have shown, in vitro and in vivo, that the combination of riluzole and ionizing radiation leads to greater cytotoxicity. These results have clinical implications for patients with brain metastases receiving whole brain radiation therapy.

Background: Spinal cord injury (SCI) is a devastating event often resulting in permanent neurologic deficit. Research has revealed an understanding of mechanisms that occur after the primary injury and contribute to functional loss. By targeting these secondary mechanisms of injury, clinicians may be able to offer improved recovery after SCI., Questions/purposes: In this review, we highlight advances in the field of SCI by framing three questions: (1) What is the preclinical evidence for the neuroprotective agent riluzole that has allowed this agent to move into clinical trials? (2) What is the preclinical evidence for Rho antagonists that have allowed this group of compounds to move into clinical trials? (3) What is the evidence for early surgical decompression after SCI?, Methods: We conducted a systematic review of MEDLINE and EMBASE-cited articles related to SCI to address these questions., Results: As a result of an improved understanding of the secondary mechanisms of SCI, specific clinical strategies have been established. We highlight three strategies that have made their way from bench to bedside: the sodium-glutamate antagonist riluzole, the Rho inhibitor Cethrin, and early surgical decompression. Each of these modalities is under clinical investigation. We highlight the fundamental science that led to this development., Conclusions: As our understanding of the fundamental mechanisms of SCI improves, we must keep abreast of these discoveries to translate them into therapies that will hopefully benefit patients. We summarize this process of bench to bedside with regard to SCI.

Excitotoxic damage to motoneurons is thought to be an important contribution to the pathogenesis of amyotrophic lateral sclerosis (ALS), a slowly developing degeneration of motoneurons that, in most cases of sporadic occurrence, is associated with impaired glial glutamate uptake. Riluzole is the only drug licensed for symptomatic ALS treatment and is proposed to delay disease progression. As riluzole is administered only after full ALS manifestation, it is unclear if its early use might actually prevent motoneuron damage. We explored this issue by using, as a simple in vitro model, hypoglossal motoneurons (a primary target of ALS) of the neonatal rat brainstem slice preparation exposed to excitotoxic stress due to glutamate uptake block by DL-threo-β-benzyloxyaspartate (TBOA). TBOA evoked sustained network bursting, early (1 h) enhancement of the S100B immunostaining of gray matter astrocytes, and activated the motoneuronal stress ATF-3 transcription factor; 4 h later, loss (30%) of motoneuron staining ensued and pyknosis appeared. Riluzole (5 μM; applied 15 min after TBOA) inhibited bursting, decreased the frequency of spontaneous glutamatergic events, reversed changes in S100B immunostaining and prevented late loss of motoneuron staining. These results show that excitotoxicity induced by glutamate uptake block developed slowly, and was sensed by glia and motoneurons with delayed cell death. Our data provide novel evidence for the neuroprotective action of riluzole on motoneurons and glia when applied early after an excitotoxic stimulus., (© 2011 The Authors. European Journal of Neuroscience © 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

Amyotrophic lateral sclerosis (ALS) is a devastating and fatal neurodegenerative disease of adults which preferentially attacks the neuromotor system. Riluzole has been used as the only approved treatment for amyotrophic lateral sclerosis since 1995, but its mechanism(s) of action in slowing the progression of this disease remain obscure. Searching PubMed for "riluzole" found 705 articles published between January 1996 and June 2009. A systematic review of this literature found that riluzole had a wide range of effects on factors influencing neural activity in general, and the neuromotor system in particular. These effects occurred over a large dose range (<1 μM to >1 mM). Reported neural effects of riluzole included (in approximate ascending order of dose range): inhibition of persistent Na(+) current = inhibition of repetitive firing < potentiation of calcium-dependent K(+) current < inhibition of neurotransmitter release < inhibition of fast Na(+) current < inhibition of voltage-gated Ca(2+) current = promotion of neuronal survival or growth factors < inhibition of voltage-gated K(+) current = modulation of two-pore K(+) current = modulation of ligand-gated neurotransmitter receptors = potentiation of glutamate transporters. Only the first four of these effects commonly occurred at clinically relevant concentrations of riluzole (plasma levels of 1-2 μM with three- to four-fold higher concentrations in brain tissue). Treatment of human ALS patients or transgenic rodent models of ALS with riluzole most commonly produced a modest but significant extension of lifespan. Riluzole treatment was well tolerated in humans and animals. In animals, despite in vitro evidence that riluzole may inhibit rhythmic motor behaviors, in vivo administration of riluzole produced relatively minor effects on normal respiration parameters, but inhibited hypoxia-induced gasping. This effect may have implications for the management of hypoventilation and sleep-disordered breathing during end-stage ALS in humans., (© 2010 Blackwell Publishing Ltd.)

Heat shock factor 1 (HSF1) mediates the cellular response to stress to increase the production of heat shock protein (HSP) chaperones for proper protein folding, trafficking, and degradation; failure of this homeostatic mechanism likely contributes to neurodegeneration. We show that the neuroprotective drug riluzole increased the amount of HSF1 in NG108-15 neuroprogenitor cells by slowing the specific turnover of HSF1 and supporting a more robust and sustained activation of HSF1. Using Hsp70-luciferase as a functional readout of the activity of HSF1, we show that riluzole amplified the heat shock induction of the reporter gene with an optimal increase at 1 μM. Immunocytochemical staining and Western blot quantitation of HSP70 in NG108-15 neuroprogenitor cells and embryonic spinal cord neurons provided corroborative evidence that riluzole amplified the HSF1-dependent regulation of HSP70 expression. Parallel studies on the GLT1 glutamate transporter showed that riluzole increased GLT1-reporter and GLT1 protein expression and that the increase was enhanced by heat shock and coincident with the increased expression of HSP70 and HSP90. This result is consistent with the anti-glutamatergic profile of riluzole and the presence of multiple heat shock elements on the GLT1 gene promoter, suggesting that riluzole may modulate GLT1 expression through HSF1. The increased HSP chaperones and GLT1 transporter blunted glutamate-induced and N-methyl D-aspartate receptor-mediated excitotoxic death. In summary, we show that riluzole increased the amount and activity of HSF1 to boost the expression of HSPs and GLT1 for neuroprotection under stress.

Background K2P channels play a key role in stabilizing the resting membrane potential, thereby modulating cell excitability in the central and peripheral somatic nervous system. Whole-cell experiments revealed a riluzole-activated current (I(RIL)), transported by potassium, in mouse superior cervical ganglion (mSCG) neurons. The activation of this current by riluzole, linoleic acid, membrane stretch, and internal acidification, its open rectification and insensitivity to most classic potassium channel blockers, indicated that I(RIL) flows through channels of the TREK [two-pore domain weak inwardly rectifying K channel (TWIK)-related K channel] subfamily. Whole-ganglia and single-cell reverse transcription-PCR demonstrated the presence of TREK-1, TREK-2, and TRAAK (TWIK-related arachidonic acid-activated K(+) channel) mRNA, and the expression of these three proteins was confirmed by immunocytochemistry in mSCG neurons. I(RIL) was enhanced by zinc, inhibited by barium and fluoxetine, but unaffected by quinine and ruthenium red, strongly suggesting that it was carried through TREK-1/2 channels. Consistently, a channel with properties identical with the heterologously expressed TREK-2 was recorded in most (75%) cell-attached patches. These results provide the first evidence for the expression of K2P channels in the mammalian autonomic nervous system, and they extend the impact of these channels to the entire nervous system.

Folate analogue inhibitors of Leishmania major pteridine reductase (PTR1) are potential antiparasitic drug candidates for combined therapy with dihydrofolate reductase (DHFR) inhibitors. To identify new molecules with specificity for PTR1, we carried out a virtual screening of the Available Chemicals Directory (ACD) database to select compounds that could interact with L. major PTR1 but not with human DHFR. Through two rounds of drug discovery, we successfully identified eighteen drug-like molecules with low micromolar affinities and high in vitro specificity profiles. Their efficacy against Leishmania species was studied in cultured cells of the promastigote stage, using the compounds both alone and in combination with 1 (pyrimethamine; 5-(4-chlorophenyl)-6-ethylpyrimidine-2,4-diamine). Six compounds showed efficacy only in combination. In toxicity tests against human fibroblasts, several compounds showed low toxicity. One compound, 5c (riluzole; 6-(trifluoromethoxy)-1,3-benzothiazol-2-ylamine), a known drug approved for CNS pathologies, was active in combination and is suitable for early preclinical evaluation of its potential for label extension as a PTR1 inhibitor and antiparasitic drug candidate.

Growing evidence indicates that the glutamatergic neurotransmitter system is central to the neurobiology and treatment of depression. Riluzole, a drug currently used to slow the progression of amyotrophic lateral sclerosis (ALS), directly affects the glutamatergic system. In this study, we investigated the effects of riluzole in olfactory bulbectomy (OBX) rats, an animal model of depression. The olfactory bulbs in rats were removed by suction. The emotionality of rats was measured by scoring their responses to given stimuli, i.e., attack, startle, struggle, and fight responses. The OBX rats chronically treated with vehicle for 7 days at 14 days following surgery showed significant increases in emotionality responses. Single (1st day administration) and subchronic (7th day administration) riluzole treatment (1-10 mg/kg, po) significantly and dose-dependently reduced hyperemotional responses in OBX rats. Both single and subchronic riluzole treatment (10 mg/kg, po) had no significant effects on the emotional responses in sham operated rats. In addition, we demonstrated that single riluzole treatment (10 mg/kg, po) significantly decreased extracellular glutamate levels in medial prefrontal cortex of OBX rats by in vivo microdialysis. We provide the first experimental evidence that riluzole rapidly attenuated hyperemotional responses in OBX rats, an animal model of depression., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Neuronal apoptosis has been shown to be associated with the development of tolerance to morphine. In the present study, we investigated the effect of intracerebroventricular (icv) administration of an inhibitor of glutamate release, riluzole, on morphine-induced apoptosis in the rat cerebral cortex. Various groups of rats received either morphine (intraperitoneally, ip) and vehicle (icv) or morphine (ip) and different doses of riluzole (icv) once per day for 8 days. An in situ terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method was used as an apoptosis assay. Levels of the anti-apoptotic factors Bcl-2 and HSP70 and the pro-apoptotic agent caspase-3 were evaluated by immunoblotting. The glutamate concentration in the cerebral cortex was measured by high performance liquid chromatography (HPLC). The results showed that icv administration of riluzole decreased the number of apoptotic cells in the cerebral cortex compared with the control group, which was treated with morphine (ip) and 1% Tween 80 in 0.9% normal saline (icv). The levels of the anti-apoptotic proteins Bcl-2 and HSP70 were higher in the riluzole groups than in the control. Furthermore, co-administration of riluzole with morphine significantly decreased caspase-3 protein levels and glutamate content of the cerebral cortex compared with the control. In conclusion, we found that icv administration of riluzole attenuates morphine-induced apoptosis in the cerebral cortex after the development of morphine tolerance.

TREK-1 potassium channels are involved in a number of physiopathological processes such as neuroprotection, pain and depression. Molecules able to open or to block these channels can be clinically important. Having a cell model for screening such molecules is of particular interest. Here, we describe the development of the first available cell line that constituvely expresses the TREK-1 channel. The TREK-1 channel expressed by the h-TREK-1/HEK cell line has conserved all its modulation properties. It is opened by stretch, pH, polyunsaturated fatty acids and by the neuroprotective molecule, riluzole and it is blocked by spadin or fluoxetine. We also demonstrate that the h-TREK-1/HEK cell line is protected against ischemia by using the oxygen-glucose deprivation model.

Although adult motoneurons do not die if their axons are injured at some distance from the cell body, they are unable to survive injury caused by ventral root avulsion. Some of the injured motoneurons can be rescued if the ventral root is re-inserted into the spinal cord. Brachial plexus injuries that involve the complete or partial avulsion of one or more cervical ventral roots can be treated successfully only if satisfactory numbers of motoneurons remain alive following such an injury at the time of reconstructive surgery. Here we investigated the various strategies that could be used to rescue injured rat cervical motoneurons. The seventh cervical ventral root (C7) was avulsed and various therapeutic approaches were applied to induce motoneuronal survival and regeneration. Avulsion of the root without reimplantation resulted in very low numbers of surviving motoneurons (65 ± 8 SEM), while treatment of the injured motoneurons with riluzole resulted in high numbers of surviving motoneurons (637 ± 26 SEM). When the C7 ventral root was reimplanted or a peripheral nerve implant was used to guide the regenerating axons to a muscle, considerable numbers of motoneurons regenerated their axons (211 ± 15 SEM and 274 ± 28 SEM, respectively). Much greater numbers of axons regenerated when reimplantation was followed by riluzole treatment (573 ± 9 SEM). These results show that injured adult motoneurons can be rescued by riluzole treatment, even if they cannot regenerate their axons. Reinnervation of the peripheral targets can also be further improved with riluzole treatment.

Background: The potential of the cardiac persistent sodium current as a target for protection of the myocardium from ischaemia and reperfusion injury is gaining increasing interest. We have investigated the anti-ischaemic and antiarrhythmic effects of riluzole, a selective INaP blocker, in an open chest pig model of infarction., Methods and Principal Findings: The left anterior descending coronary artery (LAD) was ligated in 27 anesthetised pigs (landrace or large white, either sex, 20-35 kg) which had received riluzole (8 mg/kg IP; n = 6), lidocaine (2.5-12 mg/kg bolus plus 0.05-0.24 mg/kg/min; n = 11) or vehicle (n = 10) 50 min prior. Arrhythmias could be delineated into phase 1a (0 to 20 min), phase 1b (20 to 50 min) and phase 2 (from 50 min to termination at 180 min) and were classified as premature ventricular contractions (PVCs), non-sustained ventricular tachycardia (VT) or ventricular fibrillation (VF) (spontaneously reverting within 15 s) or sustained VT or VF (ie. requiring cardioversion at 15 s). Riluzole reduced the average number of all arrhythmias in Phase 2 (PVCs from 484+/-119 to 32+/-13; non sustained arrhythmias from 8.9+/-4.4 to 0.7+/-0.5; sustained arrhythmias from 3.9+/-2.2 to 0.5+/-0.4); lidocaine reduced the average number of non-sustained and sustained arrhythmias (to 0.4+/-0.3 and 0.4+/-0.3 respectively) but not PVCs (to 390+/-234). Riluzole and lidocaine reduced the average number of sustained arrhythmias in phase 1b (from 1.8+/-0.4 to 0.17+/-0.13 (p<0.02) and to 0.55+/-0.26 (p = ns) respectively). Neither lidocaine or riluzole changed the ECG intervals: there was no statistical significance between groups at time zero (just before ligation) for any ECG measure. During the course of the 3 hour period of the ischaemia R-R, and P-R intervals shortened slightly in control and riluzole groups (not significantly different from each other) but not in the lidocaine group (significantly different from control). QRS and QTc did not change appreciably in any group Riluzole reduced the degree of histopathological tissue damage across the infarct zone considerably more than did lidocaine., Conclusions: At the doses used, riluzole was at least as effective as lidocaine at reducing the number of episodes of ischaemic VT or VF in pigs, and much more effective at reducing the number of PVCs. We propose that this is related to the ability of riluzole to block cardiac persistent sodium current.

The respiratory rhythm and motor pattern are hypothesized to be generated by a brain stem respiratory network with a rhythmogenic core consisting of neural populations interacting within and between the pre-Bötzinger (pre-BötC) and Bötzinger (BötC) complexes and controlled by drives from other brain stem compartments. Our previous large-scale computational model reproduced the behavior of this network under many different conditions but did not consider neural oscillations that were proposed to emerge within the retrotrapezoid nucleus/parafacial respiratory group (RTN/pFRG) and drive preinspiratory (or late-expiratory, late-E) discharges in the abdominal motor output. Here we extend the analysis of our previously published data and consider new data on the generation of abdominal late-E activity as the basis for extending our computational model. The extended model incorporates an additional late-E population in RTN/pFRG, representing a source of late-E oscillatory activity. In the proposed model, under normal metabolic conditions, this RTN/pFRG oscillator is inhibited by BötC/pre-BötC circuits, and the late-E oscillations can be released by either hypercapnia-evoked activation of RTN/pFRG or by hypoxia-dependent suppression of RTN/pFRG inhibition by BötC/pre-BötC. The proposed interactions between BötC/pre-BötC and RTN/pFRG allow the model to reproduce several experimentally observed behaviors, including quantal acceleration of abdominal late-E oscillations with progressive hypercapnia and quantal slowing of phrenic activity with progressive suppression of pre-BötC excitability, as well as to predict a release of late-E oscillations by disinhibition of RTN/pFRG under normal conditions. The extended model proposes mechanistic explanations for the emergence of RTN/pFRG oscillations and their interaction with the brain stem respiratory network.

The rhythmic and coordinated activation of axial muscles that underlie trunk movements during locomotion are generated by specialized networks in the spinal cord. The operation of these networks has been extensively investigated in limbless swimming vertebrates. But little is known about the architecture and functioning of the axial locomotor networks in limbed vertebrates. We investigated the rhythm-generating capacity of the axial segmental networks in the salamander (Pleurodeles waltlii). We recorded ventral root activity from hemisegments and segments that were surgically isolated from the mid-trunk cord and chemically activated with bath-applied N-methyl-d-aspartate (NMDA). We provide evidence that the rhythmogenic capacity of the axial network is distributed along the mid-trunk spinal cord without an excitability gradient. We demonstrate that the burst generation in a hemisegment depends on glutamatergic excitatory interactions. Reciprocal glycinergic inhibition between opposite hemisegments ensures left-right alternation and lowers the rhythm frequency in segments. Our results further suggest that persistent sodium current contributes to the rhythmic regenerating process both in hemisegments and segments. Burst termination in hemisegments is not achieved through the activation of apamine-sensitive Ca(2+)-activated K(+) channels and burst termination in segments relies on crossed glycinergic inhibition. Together our results indicate that the basic design of the salamander axial network is similar to most of axial networks investigated in other vertebrates, albeit with some significant differences in the cellular mechanism that underlies segmental bursting. This finding supports the view of a phylogenetic conservation of basic building blocks of the axial locomotor network among the vertebrates.

Objective: To observe the expression of Nav1.1 and the effect of electroacupuncture therapy (ET) on it after acute cerebral ischemia., Methods: Focal acute cerebral ischemic model was established by occluding right middle cerebral artery. One hundred and forty-five male adult Sprague-Dawley rats were randomly divided into four groups: sham operation group, middle cerebral artery occlusion (MCAO) group, ET group and riluzole group. Double immunofluorescence and real-time PCR were used to observe Nav1.1 expression, and TTC staining was used to detect infarct volume at 6 hours, 1 day, 2 days, 3 days and 7 days after ischemia., Results: The expression of Nav1.1 in sham operation group had no change. After ischemia, the expression of Nav1.1 was up-regulated evidently at 6 hours compared with sham operation group, down-regulated at 1 day and up-regulated again from 2 to 7 days after ischemia; the expression at 7 days was lower than that at 6 hours. In ET group and riluzole group, Nav1.1 expression were down-regulated compared with that in MCAO group after ischemia, the infarct volume was smaller than that in MCAO group at the same time point, and the difference is significant (p<0.05); however, the difference between ET group and riluzole group is not significant (p>0.05)., Conclusion: ET and riluzole could regulate the expression of Nav1.1 after ischemia, decrease the infarction volume and reduce cerebral ischemic injury. One of the important mechanisms for ET's cerebral protective function may be that it could regulate the expression of Nav1.1 after ischemia.

We have suggested that in the lamprey, a medullary region called the paratrigeminal respiratory group (pTRG), is essential for respiratory rhythm generation and could correspond to the pre-Bötzinger complex (pre-BötC), the hypothesized kernel of the inspiratory rhythm-generating network in mammals. The present study was performed on in vitro brainstem preparations of adult lampreys to investigate whether some functional characteristics of the respiratory network are retained throughout evolution and to get further insights into the recent debated hypotheses on respiratory rhythmogenesis in mammals, such as for instance the "group-pacemaker" hypothesis. Thus, we tried to ascertain the presence and role of neurokinins (NKs) and burst-generating ion currents, such as the persistent Na(+) current (I(NaP)) and the Ca(2+)-activated non-specific cation current (I(CAN)), described in the pre-Bötzinger complex. Respiratory activity was monitored as vagal motor output. Substance P (SP) as well as NK1, NK2 and NK3 receptor agonists (400-800 nM) applied to the bath induced marked increases in respiratory frequency. Microinjections (0.5-1 nl) of SP as well as the other NK receptor agonists (1 microM) into the pTRG increased the frequency and amplitude of vagal bursts. Riluzole (RIL) and flufenamic acid (FFA) were used to block I(NaP) and I(CAN), respectively. Bath application of either RIL or FFA (20-50 microM) depressed, but did not suppress respiratory activity. Coapplication of RIL and FFA at 50 microM abolished the respiratory rhythm that, however, was restarted by SP microinjected into the pTRG. The results show that NKs may have a modulatory role in the lamprey respiratory network through an action on the pTRG and that I(NaP) and I(CAN) may contribute to vagal burst generation. We suggest that the "group-pacemaker" hypothesis is tenable for the lamprey respiratory rhythm generation since respiratory activity is abolished by blocking both I(NaP) and I(CAN), but is restored by enhancing network excitability., (Copyright (c) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

The goal of this study was to examine the effects of metabotropic glutamate receptor-1 (GRM1) blockade on melanoma anchorage-independent growth and invasion. We performed colony and invasion assays using GRM1-expressing melanoma lines and the GRM1-negative UACC930 line. Using the glutamate-release inhibitor Riluzole or the non-competitive GRM1 antagonist BAY 36-7620 we were able to induce considerable inhibition of colony formation and invasion in GRM1-expressing melanoma lines. Neither pharmacological agent induced significant reduction in colony formation or invasion in the GRM1-negative melanoma line, UACC930. Additionally we assessed the efficacy of these inhibitors to inhibit the growth of fresh melanoma tumor samples cultured on a 74-mum nylon mesh. Both Riluzole and BAY 36-7620 significantly inhibited tumor cell growth into the interstitial spaces of the mesh. When repeated with normal mole samples both inhibitors were much less effective in preventing the outgrowth of cells. These experiments show that a specific antagonist of GRM1 (BAY 36-7620) or an inhibitor of glutamate release (Riluzole) can significantly suppress melanoma migration, invasion and colony formation as well as inhibit the proliferation of fresh melanoma cells. These findings, added to our previous work, strengthen the case that GRM1 is a valid therapeutic target in patients with melanoma.

The antiglutamatergic drug riluzole (Rilutek) is presently being used off label in the treatment of psychiatric conditions in adult patients and, increasingly, in children. This article briefly reviews the pharmacology of this drug and its current investigative and clinical uses and adverse effects. It also reports on our experience to date in the study of the drug in children, with emphasis on adverse effects noted so far in these younger patients.

Opiates are the most effective drugs for pain relief. However, the repeated use of opiates induces tolerance to their analgesic effects. It has been shown that this morphine-induced tolerance is associated with apoptosis in the central nervous system. The aim of this study is to evaluate the effects of intracerebroventricular (i.c.v.) administration of riluzole, an anti-glutamatergic drug, on morphine-induced apoptosis in the lumbar region of the rat spinal cord. Animals were given daily injections of morphine and vehicle, morphine and riluzole, or riluzole alone. Nociception was assessed using a hot plate apparatus, and apoptosis was assessed using the in situ terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method. The levels of anti-apoptotic factors Bcl-2 and HSP 70 and the pro-apoptotic agent caspase-3 were evaluated using immunoblotting. The glutamate concentration in the lumbar spinal cord was measured with high performance liquid chromatography (HPLC). The results indicate that the i.c.v. administration of riluzole attenuated morphine tolerance and reduced the number of TUNEL positive cells. Immunoblotting revealed that the levels of the selected anti-apoptotic agents were greater in the treatment groups compared to the controls. Furthermore, the results demonstrated that the administration of riluzole can attenuate the morphine-induced elevation of glutamate in the lumbar spinal cord. In conclusion, i.c.v. administration of riluzole attenuated morphine-induced tolerance to analgesia and apoptosis in addition to preventing the morphine-induced increase of glutamate in the lumbar spinal cord of rats.

Background and Purpose: Current strategies to ameliorate cardiac ischaemic and reperfusion damage, including block of the sodium-hydrogen exchanger, are therapeutically ineffective. Here we propose a different approach, block of the persistent sodium current (INaP)., Experimental Approach: Left ventricular pressure was measured as an index of functional deficit in isolated, Langendorff perfused, hearts from adult rats, subjected to 30 min global ischaemia and reperfusion with vehicle only (control) or riluzole (1-10 microM) in the perfusate. Cell shortening and intracellular Ca2+ concentrations [Ca2+](i) were measured in adult rat isolated myocytes subjected to hypoxia and re-oxygenation. The block of transient and persistent sodium currents by concentrations of riluzole between 0.01 and 100 microM were assessed in rat isolated myocytes using patch clamp techniques., Key Results: In perfused hearts, riluzole produced a concentration-dependent cardioprotective action, with minor protection from 1 microM and produced rapid and almost complete recovery upon reperfusion from 3 and 10 microM. In isolated myocytes, riluzole at 3 and 10 microM greatly attenuated or prevented the hypoxia- and reperfusion-induced rise in [Ca2+](i) and the contractile deficit. In patch clamp experiments, riluzole blocked the persistent sodium current with an IC(50) of 2.7 microM, whereas the block of the transient sodium current was only apparent at concentrations above 30 microM., Conclusions and Implications: Riluzole preferentially blocked INaP and was protective in cardiac ischaemia and reperfusion. Thus block of the persistent sodium current would be a viable method of ameliorating cardiac ischaemic and reperfusion damage.

N-acetylaspartate (NAA) is present at very high concentrations in the brain and is used as a non-invasive marker of neuronal viability in magnetic resonance spectroscopy. N-acetylaspartylglutamate (NAAG) is an acetylated dipeptide formed from NAA, and may be an agonist of the mGluR3 receptor. Both NAA and NAAG are synthesized primarily in neurons. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder resulting in motor neuron death, and progressive paralysis. Levels of both NAA and NAAG are reported to be decreased in ALS. Riluzole is a glutamatergic modulating agent used to treat ALS, but there are conflicting results in the literature concerning the recovery of NAA after riluzole treatment. We studied the effects of riluzole on the biosynthesis of both NAA and NAAG in SH-SY5Y human neuroblastoma cells. We used two methodologies to examine the effect; one involving radiolabel incorporation from corresponding substrates into NAA and NAAG, and the other involving the measurement of endogenous NAA and NAAG levels using HPLC. We show that riluzole treatment, which decreases glutamatergic neuronal excitation, decreases the synthesis and levels of both NAA and NAAG in SH-SY5Y cells in a dose and time dependant manner. These results suggest that the synthesis of NAA and NAAG may be coupled to glutamatergic neurotransmission, and further investigations along these lines are warranted., (Published by Elsevier B.V.)

More than 30 phase II or III clinical trials have been carried out in amyotrophic lateral sclerosis (ALS). Only riluzole, however, has been shown to extend survival and/or time to tracheostomy. Many early ALS trials lacked solid pharmacodynamic and pharmacokinetic data for the treatment being tested, challenging the interpretation of the efficacy and pathway relevance. Understanding of the genetics and pathophysiology of ALS has improved considerably in the past decade, but biomarkers of disease activity remain lacking. A more efficient approach to early phase clinical trials is needed to accelerate the identification of useful agents for ALS. Here we summarize our current thinking about phase II design options and the potential benefits of a clinical trial network for phase II trials in ALS.

We examined the role of riluzole (RIL)- and flufenamic acid (FFA)-sensitive mechanisms in respiratory rhythmogenesis in rats and hamsters using the in situ arterially perfused preparation. Based on the hypothesis that respiratory networks in animals capable of autoresuscitation would have a greater prevalence of membrane mechanisms that promote endogenous bursting, we predicted that older (weaned) hamsters (a hibernating species) would be more sensitive to the blockade of RIL- and FFA-sensitive mechanisms than age-matched rats and that younger (preweaned) rats would behave more like hamsters. Consistent with this, we found that respiratory motor output in weaned hamsters [>21 days postnatal (P21)] was highly sensitive to RIL (0.2-20 muM), while in young rats (P12-14) it was less so (only affected at higher concentrations of RIL), and weaned rats were not affected at all. On the other hand, respiratory motor output was equally reduced by FFA (0.25-25 muM) in both young and weaned rats but was unaffected in weaned hamsters. Coapplication of RIL and FFA (RIL + FFA) produced greater inhibition of respiration in both young and weaned rats compared with either drug alone. In contrast, in weaned hamsters, FFA coapplication offset the inhibitory effect of RIL alone. Increasing respiratory drive with hypercapnia/acidosis ameliorated the respiratory inhibition produced by RIL + FFA in weaned rats but had no effect in young rats. Data from the present study indicate that respiratory rhythmogenesis in young rats is more dependent on excitatory RIL-sensitive and FFA-sensitive mechanisms than older rats and that fundamental differences exist in the respiratory rhythmogenic mechanisms between rats and hamsters.

Riluzole is clinically approved for the treatment of motoneuron disease. We have previously shown that this drug is neuroprotective for both sensory neurons and motoneurons and promotes neurite outgrowth [Bergerot A, Shortland PJ, Anand P, Hunt SP, Carlstedt T (2004) Exp Neurol 187(2):359-366; Shortland PJ, Leinster VH, White W, Robson LG (2006) Eur J Neurosci 24:3343-3353]. This study explored the effects of exogenous administration of 0.1 muM riluzole on the neurite growth of specific subpopulations of adult rat dorsal root ganglion (DRG) neurons in vitro. Neuronal branching and neurite length were measured in calcitonin gene related peptide (CGRP), Griffonia simplicifolia Isolectin B4 (IB4), N52 and parvalbumin positive neuronal subpopulations. Riluzole was found to enhance neurite branching in both CGRP and IB4 positive neurons compared to vehicle treated cultures. However, neurite length was only significantly increased in CGRP positive neurons in riluzole treated cultures. The results suggest that riluzole affects specific subpopulations of sensory neurons in vitro and that its effects may be mediated through activation of neurotrophic factor receptors, since neurite outgrowth could be blocked by the administration of K252a (at 10 nM). Riluzole may offer a new pharmacological approach to promote sensory regeneration following small fibre neuropathies., (Copyright 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

Descending noradrenergic inhibition is an important endogenous pain-relief mechanism which can be activated by local glutamate signaling. In the present study, we examined the effect of glutamate transporter activation by riluzole in the regulation of activity of locus coeruleus (LC) neurons, which provide the major inhibitory descending noradrenergic projection to the spinal cord. Local injection of riluzole into the LC dose-dependently reduced hypersensitivity in rats after L5-L6 spinal nerve ligation (SNL). This anti-hypersensitivity effect of LC-injected riluzole was blocked by intrathecal administration of the alpha2-adrenoceptor antagonist idazoxan and intra-LC co-injection of the AMPA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and the gap-junction blockers, carbenoxolone (CBX) and meclofenamic acid (MEC). In brainstem slices from normal rats, riluzole increased phosphorylated cAMP response element binding protein (pCREB) expressing nuclei in dopamine-beta-hydroxylase (DbetaH) containing cells in the LC. This riluzole-induced pCREB activation in LC neurons was also blocked by CNQX and CBX. In the primary astrocyte culture, riluzole enhanced glutamate-induced glutamate release. Contrary to expectations, these results suggest that activation of glutamate transporters in the LC results in increase of extracellular glutamate signaling, possibly via facilitation of glutamate release from astrocytes, and activation of LC neurons to induce descending inhibition, and that this paradoxical action of glutamate transporters in the LC requires gap-junction connections., (2009 Elsevier B.V. All rights reserved.)

Several studies have reported the neuroprotective effects of lithium (Li) suggesting its potential in the treatment of neurological disorders, among of them amyotrophic lateral sclerosis (ALS). Although the cause of motoneuron (MN) death in ALS remains unknown, there is evidence that glutamate-mediated excitotoxicity plays an important role. In the present study we used an organotypic culture system of chick embryo spinal cord to explore the presumptive neuroprotective effects of Li against kainate-induced excitotoxic MN death. We found that chronic treatment with Li prevented excitotoxic MN loss in a dose dependent manner and that this effect was mediated by the inhibition of glycogen synthase kinase-3beta (GSK-3beta) signaling pathway. This neuroprotective effect of Li was potentiated by a combined treatment with riluzole. Nevertheless, MNs rescued by Li displayed structural changes including accumulation of neurofilaments, disruption of the rough endoplasmic reticulum and free ribosome loss, and accumulation of large dense core vesicles and autophagic vacuoles. Accompanying these changes there was an increase in immunostaining for (a) phosphorylated neurofilaments, (b) calcitonin gene-related peptide (CGRP) and (c) the autophagic marker LC3. Chronic Li treatment also resulted in a reduction in the excitotoxin-induced rise in intracellular Ca(2+) in MNs. In contrast to the neuroprotection against excitotoxicity, Li was not able to prevent normal programmed (apoptotic) MN death in the chick embryo when chronically administered in ovo. In conclusion, these results show that although Li is able to prevent excitotoxic MN death by targeting GSK-3beta, this neuroprotective effect is associated with conspicuous cytopathological changes.

Glutamatergic abnormalities may underlie bipolar disorder (BD). The glutamate-modulating drug riluzole may be efficacious in bipolar depression, but few in vivo studies have examined its effect on glutamatergic neurotransmission. We conducted an exploratory study of the effect of riluzole on brain glutamine/glutamate (Gln/Glu) ratios and levels of N-acetylaspartate (NAA). We administered open-label riluzole 100-200 mg daily for 6 weeks to 14 patients with bipolar depression and obtained imaging data from 8-cm(3) voxels in the anterior cingulate cortex (ACC) and parieto-occipital cortex (POC) at baseline, day 2, and week 6 of treatment, using two-dimensional J-resolved proton magnetic resonance spectroscopy at 4 T. Imaging data were analyzed using the spectral-fitting package, LCModel; statistical analysis used random effects mixed models. Riluzole significantly reduced Hamilton Depression Rating Scale (HAM-D) scores (d=3.4; p<0.001). Gln/Glu ratios increased significantly by day 2 of riluzole treatment (Cohen's d=1.2; p=0.023). NAA levels increased significantly from baseline to week 6 (d=1.2; p=0.035). Reduction in HAM-D scores was positively associated with increases in NAA from baseline to week 6 in the ACC (d=1.4; p=0.053), but was negatively associated in the POC (d=9.6; p<0.001). Riluzole seems to rapidly increase Gln/Glu ratios-suggesting increased glutamate-glutamine cycling, which may subsequently enhance neuronal plasticity and reduce depressive symptoms. Further investigation of the Gln/Glu ratio as a possible early biomarker of response to glutamate-modulating therapies is warranted.

Background: Blockade of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamate receptors is a promising pharmacological strategy in the treatment of neurodegenerative diseases. The aim of the study is to elucidate if there are direct interactions of riluzole and phenobarbital with AMPA-type receptor channels and to determinethe molecular pharmacological mechanisms., Methods: The patch-clamp technique was used combining an ultrafast solution exchange system to investigate the interaction of riluzole and phenobarbital with recombinant AMPA-type glutamate receptor channels (homomeric GluR2flipGQ or nondesensitizing GluR2L504Y)., Results: A dose-dependent decrease in the relative peak current amplitude (rAmp) and the relative area-under-the-current curve (rAUC) were found after preincubation with 0.1 mmol/l or higher concentrations of riluzole. Furthermore, in coapplication experiments with GluR2L504Y, the application of 1 or 3 mmol/l riluzole showed a decrease in the current decay time constant, and a reopening current was observed at 3 mmol/l riluzole. Phenobarbital blocks AMPA receptor channels dose-dependently in the coapplication experiments, and reopening currents after removing glutamate and blocker were observed. A slight block effect after preincubation should indicate an additional competitive block effect., Conclusion: Riluzole and phenobarbital modulate AMPA-type receptor channels separately, which could be both characterized as a combination of open-channel block and competitive-block mechanism., (Copyright 2009 S. Karger AG, Basel.)

Breathing, chewing, and walking are critical life-sustaining behaviors in mammals that consist essentially of simple rhythmic movements. Breathing movements in particular involve the diaphragm, thorax, and airways but emanate from a network in the lower brain stem. This network can be studied in reduced preparations in vitro and using simplified mathematical models that make testable predictions. An iterative approach that employs both in vitro and in silico models argues against canonical mechanisms for respiratory rhythm in neonatal rodents that involve reciprocal inhibition and pacemaker properties. We present an alternative model in which emergent network properties play a rhythmogenic role. Specifically, we show evidence that synaptically activated burst-generating conductances-which are only available in the context of network activity-engender robust periodic bursts in respiratory neurons. Because the cellular burst-generating mechanism is linked to network synaptic drive we dub this type of system a group pacemaker., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Amyotrophic lateral sclerosis (ALS) is a universally fatal neurodegenerative disease of the human motor system. Aetiological mechanisms implicated in the development of ALS have been linked to the glutamatergic neurotransmitter system, with destruction of motor neurons triggered through excessive activation of glutamate receptors at the synaptic cleft. This 'excitotoxicity' theory of ALS gave rise to the development of therapeutic approaches and ultimately clinical trials involving riluzole, initially thought to act solely as an inhibitor of glutamate release. Subsequent effects of riluzole have been postulated to include indirect antagonism of glutamate receptors, in addition to inactivation of neuronal voltage-gated Na+ channels. Riluzole remains the only disease-modifying therapy available to patients with ALS. Despite having been clinically available since the mid-1990 s, the in vivo pharmacological targets of riluzole have been poorly defined. An improved understanding concerning the potential neuroprotective mechanisms of riluzole may unearth pathophysiological processes that mediate neurodegeneration in ALS. The present review summarises the known chemical and pharmacological properties of riluzole. The failure of other putative neuroprotective therapies to demonstrate positive treatment outcomes in this intractable disease will be reviewed. Finally, the hypothesis that Na+ conductances may be involved in the processes of neuronal and axonal degeneration in ALS will be explored.

This work characterizes the properties of wild-type (WT) mouse motoneurons in the second postnatal week and compares these at the same age and in the same conditions to those of two different SOD1 mutant lines used as models of human amyotrophic lateral sclerosis (ALS), the SOD1(G93A) low expressor line and SOD1(G85R) line, to describe any changes in the functional properties of mutant motoneurons (Mns) that may be related to the pathogenesis of human ALS. We show that very early changes in excitability occur in SOD1 mutant Mns that have different properties from those of WT animals. The SOD1(G93A-Low) low expressor line displays specific differences that are not found in other mutant lines including a more depolarized membrane potential, larger spike width, and slower spike rise slope. With current pulses SOD1(G93A-Low) were hyperexcitable, but both mutants had a lower gain with current ramps stimulation. Changes in the threshold and intensities of Na(+) and Ca(2+) persistent inward currents were also observed. Low expressor mutants show reduced total persistant inward currents compared with WT motoneurons in the same recording conditions and give arguments toward modifications of the balance between Na(+) and Ca(2+) persistent inward currents. During the second week postnatal, SOD1(G93A-Low) lumbar motoneurons appear more immature than those of SOD1(G85R) compared with WT and we propose that different time course of the disease, possibly linked with different toxic properties of the mutated protein in each model, may explain the discrepancies between excitability changes described in the different models.

Glutamate receptor-mediated excitotoxicity and mitochondrial dysfunction appear to play an important role in motoneuron (MN) degeneration in amyotrophic lateral sclerosis (ALS). In the present study we used an organotypic slice culture of chick embryo spinal cord to explore the responsiveness of mature MNs to different excitotoxic stimuli and mitrochondrial inhibition. We found that, in this system, MNs are highly vulnerable to excitotoxins such as glutamate, N-methyl-D-aspartate (NMDA), and kainate (KA), and that the neuroprotective drug riluzole rescues MNs from KA-mediated excitotoxic death. MNs are also sensitive to chronic mitochondrial inhibition induced by malonate and 3-nitropropionic acid (3-NP) in a dose-dependent manner. MN degeneration induced by treatment with mitochondrial toxins displays structural changes similar to those seen following excitotoxicity and can be prevented by applying either the antiexcitotoxic drug 6-cyano-7-nitroquinoxaline-2,3-dione disodium (CNQX) or riluzole. Excitotoxicity results in an increased frequency of normal spontaneous Ca2+ oscillations in MNs, which is followed by a sustained deregulation of intracellular Ca2+. Tolerance to excitotoxic MN death resulting from chronic exposure to excitotoxins correlates with a reduced excitotoxin-induced increase in intracellular Ca2+ and increased thapsigargin-sensitive Ca2+ stores.

Bipolar disorder and major depressive disorder are common, chronic, and recurrent mood disorders that affect the lives of millions of individuals worldwide. Growing evidence suggests that glutamatergic system dysfunction is directly involved in mood disorders. This article describes the role of the "tripartite glutamatergic synapse," comprising presynaptic and postsynaptic neurons and glial cells, in the pathophysiology and therapeutics of mood disorders. Glutamatergic neurons and glia directly control synaptic and extrasynaptic glutamate levels/ release through integrative effects that target glutamate excitatory amino acid transporters, postsynaptic density proteins, ionotropic receptors (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid [AMPA], N-methyl-D-aspartate [NMDA], and kainate), and metabotropic receptors. This article also explores the glutamatergic modulators riluzole and ketamine, which are considered valuable proof-of-concept agents for developing the next generation of antidepressants and mood stabilizers. In therapeutically relevant paradigms, ketamine preferentially targets postsynaptic AMPA/NMDA receptors, and riluzole preferentially targets presynaptic voltage-operated channels and glia.

Several lines of evidence strongly suggest that brain-derived neurotrophic factor (BDNF) is associated with the formation, storage and recall of memory in the hippocampus and that it is important to maintain a considerable level of hippocampal BDNF in order to keep normal functions. BDNF can be synthesized in an activity-dependent manner. In fact, kainic acid or AMPA enhances BDNF levels in hippocampal granule neurons. However, the mechanisms of BDNF production are largely unclear. Recently, we have found that riluzole, which blocks voltage-gated sodium channels and thereby reduces glutamate release, actually strengthens immunoreactivity of BDNF in hippocampal granule neurons of rats. Therefore, we examined the riluzole-activated signaling pathways for BDNF production. Riluzole increased levels of phospho-p38 mitogen-activated protein kinase (p38 MAPK), as well as BDNF levels. Inhibition of p38 MAPK by SB203580 reduced riluzole effects, while activation of p38 MAPK by anisomycin increased levels of BDNF, suggesting that p38 MAPK can mediate BDNF production. Riluzole-induced elevation of phospho-activating transcription factor-2, a transcription factor downstream of p38 MAPK, was also observed. A blocker of N-type voltage-gated calcium channels reduced the effects of riluzole on BDNF production and p38 MAPK activation. We also examined a possible involvement of the adenosine A1 receptor in BDNF production because riluzole can influence ecto-nucleotide levels. An A1 receptor agonist inhibited riluzole-induced elevation of BDNF levels, whereas an antagonist not only increased levels of BDNF and active p38 MAPK but also augmented riluzole effects. These results indicate that, in the rat hippocampus, there is an in vivo signaling pathway for BDNF synthesis mediated by p38 MAPK, and that N-type voltage-gated calcium channels and/or adenosine A1 receptors contribute to p38 MAPK activation.

The mechanisms underlying the disruption of glutamate-glutamine cycle (Glu-Gln cycle) in manganism are still unknown. To approach the concrete mechanisms, the rats were i.p. injected with different doses of MnCl(2) (0, 8, 40, and 200 micromol/kg), and the levels of Mn, Glu, and Gln, the morphological and ultrastructural changes, activities of Na(+)-K(+)-ATPase, GS, and PAG, mRNA and protein expression of GS, GLAST, and GLT-1 in the striatum were investigated. In addition, the effect of 21.35 micromol/kg riluzole (Na(+) channel blocker) was studied at 200 micromol/kg MnCl(2). It was observed that (1) Mn and Glu levels and PAG activity increased; (2) many pathological changes occurred; (3) Gln levels, Na(+)-K(+)-ATPase and GS activities, and GS, GLAST, and GLT-1 mRNA and protein expression inhibited, does dependently. Furthermore, the research indicated that pretreatment of riluzole reversed toxic effects of MnCl(2) significantly. These results suggested that Glu-Gln cycle was disrupted by Mn exposure dose dependently; riluzole might antagonize Mn neurotoxicity.

The brainstem nucleus hypoglossus contains motoneurons that provide the exclusive motor nerve supply to the tongue. In addition to voluntary tongue movements, tongue muscles rhythmically contract during a wide range of physiological activities, such as respiration, swallowing, chewing and sucking. Hypoglossal motoneurons are destroyed early in amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease often associated with a deficit in the transport system of the neurotransmitter glutamate. The present study shows how periodic electrical discharges of motoneurons are mainly produced by a neuronal network that drives them into bursting mode via glutamatergic excitatory synapses. Burst activity is, however, modulated by the intrinsic properties of motoneurons that collectively synchronize their discharges via gap junctions to create 'group bursters'. When glial uptake of glutamate is blocked, a distinct form of pathological bursting spontaneously emerges and leads to motoneuron death. Conversely, H(2)O(2)-induced oxidative stress strongly increases motoneuron excitability without eliciting bursting. Riluzole (the only drug currently licensed for the treatment of ALS) suppresses bursting of hypoglossal motoneurons caused by blockage of glutamate uptake and limits motoneuron death. These findings highlight how different patterns of electrical oscillations of brainstem motoneurons underpin not only certain physiological activities, but also motoneuron death induced by glutamate transporter impairment.