RS-100642-198, a novel sodium channel blocker, provides differential neuroprotection against hypoxia/hypoglycemia, veratridine or glutamate-mediated neurotoxicity in primary cultures of rat cerebellar neurons

The present study investigated the effects of RS-100642-198 (a novel sodium channel blocker), and two related compounds (mexiletine and QX-314), inin vitro models of neurotoxicity. Neurotoxicity was produced in primary cerebellar cultures using hypoxia/hypoglycemia (H/H), veratridine or glutamate where, in vehicle-treated neurons, 65%, 60% and 75% neuronal injury was measured, respectively. Dose-response neuroprotection experiments were carried out using concentrations ranging from 0.1-500 μM. All the sodium channel blockers were neuroprotective against H/H-induced injury, with each exhibiting similar potency and efficacy. However, against veratridine-induced neuronal injury only RS-100642-198 and mexiletine were 100% protective, whereas QX-314 neuroprotection was limited (i.e. only 54%). In contrast, RS 100642-198 and mexiletine had no effect against glutamate-induced injury, whereas QX-314 produced a consistent, but very limited (i.e. 25%), neuroprotection. Measurements of intraneuronal calcium ([Ca²⁺];) mobilization revealed that glutamate caused immediate and sustained increases in [Ca²⁺]_i which were not affected by RS-100642-198 or mexiletine. However, both drugs decreased the initial amplitude and attenuated the sustained rise in [Ca²⁺]_i mobilization produced by veratridine or KC1 depolarization. QX-314 produced similar effects on glutamate-, veratridineor KC1-induced [Ca²⁺]_i dynamics, effectively decreasing the amplitude and delaying the initial spike in [Ca²⁺]_i, and attenuating the sustained increase in [Ca²⁺]_i mobilization. By using differentin vitro models of excitotoxicity, a heterogeneous profile of neuroprotective effects resulting from sodium channel blockade has been described for RS-100642-198 and related drugs, suggesting that selective blockade of neuronal sodium channels in pathological conditions may provide therapeutic neuroprotection against depolarization/excitotoxicity via inhibition of voltage-dependent Na⁺ channels.     

Calpain activation and Na+/Ca2+ exchanger degradation occur downstream of calcium deregulation in hippocampal neurons exposed to excitotoxic glutamate

Delayed calcium deregulation (DCD) plays an essential role in glutamate excitotoxicity, a major detrimental factor in stroke, traumatic brain injury, and various neurodegenerations. In the present study, we examined the role of calpain activation and Na⁺/Ca²⁺ exchanger (NCX) degradation in DCD and excitotoxic cell death in cultured hippocampal neurons. Exposure of neurons to glutamate caused DCD accompanied by secondary mitochondrial depolarization. Activation of calpain was evidenced by detecting NCX isoform 3 (NCX3) degradation products. Degradation of NCX isoform 1 (NCX1) was below the detection limit of Western blotting. Degradation of NCX3 was detected only after 1 hr of incubation with glutamate, whereas DCD occurred on average within 15 min after glutamate application. Calpeptin, an inhibitor of calpain, significantly attenuated NCX3 degradation but failed to inhibit DCD and excitotoxic neuronal death. Calpain inhibitors I, III, and VI also failed to influence DCD and glutamate‐induced neuronal death. On the other hand, MK801, an inhibitor of the NMDA subtype of glutamate receptors, added shortly after the initial glutamate‐induced jump in cytosolic Ca²⁺, completely prevented DCD and activation of calpain and strongly protected neurons against excitotoxicity. Taken together, our results suggest that, in glutamate‐treated hippocampal neurons, the initial increase in cytosolic Ca²⁺ that precedes DCD is insufficient for sustained calpain activation, which most likely occurs downstream of DCD. © 2009 Wiley‐Liss, Inc.     

Intracellular acidification induced by membrane depolarization in rat hippocampal slices: roles of intracellular Ca and glycolysis

To elucidate the mechanism of pH_i changes induced by membrane depolarization, the variations in pH_i and [Ca²⁺]_i induced by a number of depolarizing agents, including high K⁺, veratridine, N-methyl-

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-aspartate (NMDA) and ouabain, were investigated in rat hippocampal slices by the fluorophotometrical technique using BCECF or fura-2. All of these depolarizing agents elicited a decrease in pH_i and an elevation of intracellular calcium ([Ca²⁺]_i) in the CA1 pyramidal cell layer. The increases in [Ca²⁺]_i caused by the depolarizing agents almost completely disappeared in the absence of Ca²⁺ (0 mM Ca²⁺ with 1 mM EGTA). In Ca²⁺ free media, pH_i acid shifts produced by high K⁺, veratridine or NMDA were attenuated by 10–25%, and those produced by ouabain decreased by 50%. Glucose-substitution with equimolar amounts of pyruvate suppressed by two-thirds the pH_i acid shifts induced by both high K⁺ and NMDA. Furthermore, lactate contents were significantly increased in hippocampal slices by exposure to high K⁺, veratridine or NMDA but not by ouabain. These results suggest that the intracellular acidification produced by these depolarizing agents, with the exception of ouabain, is mainly due to lactate accumulation which may occur as a result of accelerated glycolysis mediated by increased Na⁺–K⁺ ATPase activity. A Ca²⁺-dependent process may also contribute to the intracellular acidification induced by membrane depolarization. Since an increase in H⁺ concentration can attenuate neuronal activity, glycolytic acid production induced by membrane depolarization may contribute to the mechanism that prevents excessive neuronal excitation.     

Involvement of the glutamate transporter and the sodium–calcium exchanger in the hypoxia-induced increase in intracellular Ca in rat hippocampal slices

Hippocampal slices prepared from adult rats were loaded with fura-2 and the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in the CA1 pyramidal cell layer was measured. Hypoxia (oxygen–glucose deprivation) elicited a gradual increase in [Ca²⁺]_i in normal Krebs solution. At high extracellular sodium concentrations ([Na⁺]_o), the hypoxia-induced response was attenuated. In contrast, hypoxia in low [Na⁺]_o elicited a significantly enhanced response. This exaggerated response to hypoxia at a low [Na⁺]_o was reversed by pre-incubation of the slice at a low [Na⁺]_o prior to the hypoxic insult. The attenuation of the response to hypoxia by high [Na⁺]_o was no longer observed in the presence of antagonist to glutamate transporter. However, antagonist to Na⁺–Ca²⁺ exchanger only slightly influenced the effects of high [Na⁺]_o. These observations suggest that disturbance of the transmembrane gradient of Na⁺ concentrations is an important factor in hypoxia-induced neuronal damage and corroborates the participation of the glutamate transporter in hypoxia-induced neuronal injury. In addition, the excess release of glutamate during hypoxia is due to a reversal of Na⁺-dependent glutamate transporter rather than an exocytotic process.     

Regenerative glutamate release by presynaptic NMDA receptors contributes to spreading depression

Spreading depression (SD) is a slowly propagating neuronal depolarization that underlies certain neurologic conditions. The wave-like pattern of its propagation suggests that SD arises from an unusual form of neuronal communication. We used enzyme-based glutamate electrodes to show that during SD induced by transiently raising extracellular K⁺ concentrations ([K⁺]_o) in rat brain slices, there was a rapid increase in the extracellular glutamate concentration that required vesicular exocytosis but unlike fast synaptic transmission, still occurred when voltage-gated sodium and calcium channels (VGSC and VGCC) were blocked. Instead, presynaptic N-methyl-D-aspartate (NMDA) receptors (NMDARs) were activated during SD and could generate substantial glutamate release to support regenerative glutamate release and propagating waves when VGSCs and VGCCs were blocked. In calcium-free solutions, high [K⁺]_o still triggered SD-like waves and glutamate efflux. Under such a condition, glutamate release was blocked by mitochondrial Na⁺/Ca²⁺ exchanger inhibitors that likely blocked calcium release from mitochondria secondary to NMDA-induced Na⁺ influx. Therefore presynaptic NMDA receptor activation is sufficient for triggering vesicular glutamate release during SD via both calcium entry and release from mitochondria by mitochondrial Na⁺/Ca²⁺ exchanger. Our observations suggest that presynaptic NMDARs contribute to a cycle of glutamate-induced glutamate release that mediate high [K⁺]_o-triggered SD.     

Altered Na-channel function as an in vitro model of the ischemic penumbra: action of lubeluzole and other neuroprotective drugs

Veratridine blocks Na⁺-channel inactivation and causes a persistant Na⁺-influx. Exposure of hippocampal slices to 10 μM veratridine led to a failure of synaptic transmission, repetitive spreading depression (SD)-like depolarizations of increasing duration, loss of Ca⁺-homeostasis, a large reduction of membrane potential, spongious edema and metabolic failure. Normalization of the amplitude of the negative DC shift evoked by high K⁺ ACSF 80 min after veratridine exposure was taken as the primary endpoint for neuroprotection. Compounds whose mechanism of action includes Na⁺-channel modulation were neuroprotective (IC₅₀-values in μM): tetrodotoxin 0.017, verapamil 1.18, riluzole 1.95, lamotrigine ≥10, and diphenylhydantoin 16.1. Both NMDA (MK-801 and APH) and non-NMDA (NBQX) excitatory amino acid antagonists were inactive, as were NOS-synthesis inhibitors (nitro-

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-arginine and

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-NAME), Ca²⁺-channel blockers (cadmium, nimodipine), and a K⁺-channel blocker (TEA). Lubeluzole significantly delayed the time before the slices became epileptic, postponed the first SD-like depolarization, allowed the slices to better recover their membrane potential after a larger number of SD-like DC depolarizations, preserved Ca²⁺ and energy homeostasis, and prevented the neurotoxic effects of veratridine (IC₅₀-value 0.54 μM). A concentration of lubeluzole, which was 40× higher than its IC₅₀-value for neuroprotection against veratridine, had no effect on repetitive Na⁺-dependent action potentials induced by depolarizing current in normal ACSF. The ability of lubeluzole to prevent the pathological consequences of excessive Na⁺-influx, without altering normal Na⁺-channel function may be of benefit in stroke.     

Monitoring of glutamate‐induced excitotoxicity by mitochondrial oxygen consumption

Dysfunction of mitochondrial activity is often associated with the onset and progress of neurodegenerative diseases. Membrane depolarization induced by Na⁺ influx increases intracellular Ca²⁺ levels in neurons, which upregulates mitochondrial activity. However, overlimit of Na⁺ influx and its prolonged retention ultimately cause excitotoxicity leading to neuronal cell death. To return the membrane potential to the normal level, Na⁺/K⁺‐ATPase exchanges intracellular Na⁺ with extracellular K⁺ by consuming a large amount of ATP. This is a reason why mitochondria are important for maintaining neurons. In addition, astrocytes are thought to be important for supporting neighboring neurons by acting as energy providers and eliminators of excessive neurotransmitters. In this study, we examined the meaning of changes in the mitochondrial oxygen consumption rate (OCR) in primary mouse neuronal populations. By varying the medium constituents and using channel modulators, we found that pyruvate rather than lactate supported OCR levels and conferred on neurons resistance to glutamate‐mediated excitotoxicity. Under a pyruvate‐restricted condition, our OCR monitoring could detect excitotoxicity induced by glutamate at only 10 μM. The OCR monitoring also revealed the contribution of the N‐methyl‐D‐aspartate receptor and Na⁺/K⁺‐ATPase to the toxicity, which allowed evaluating spontaneous excitation. In addition, the OCR monitoring showed that astrocytes preferentially used glutamate, not glutamine, for a substrate of the tricarboxylic acid cycle. This mechanism may be coupled with astrocyte‐dependent protection of neurons from glutamate‐mediated excitotoxicity. These results suggest that OCR monitoring would provide a new powerful tool to analyze the mechanisms underlying neurotoxicity and protection against it.     

Two sides of the same coin: Sodium homeostasis and signaling in astrocytes under physiological and pathophysiological conditions

The intracellular sodium concentration of astrocytes is classically viewed as being kept under tight homeostatic control and at a relatively stable level under physiological conditions. Indeed, the steep inwardly directed electrochemical gradient for sodium, generated by the Na⁺/K⁺‐ATPase, contributes to maintain the electrochemical gradient of K⁺ and the highly K⁺‐based negative membrane potential, and is a central element in energizing membrane transport. As such it is tightly coupled to the homeostasis of extra‐ and intracellular potassium, calcium or pH and to the reuptake of transmitters such as glutamate. Recent studies, however, have demonstrated that this picture is far too simplistic. It is now firmly established that transmitters, most notably glutamate, and excitatory neuronal activity evoke long‐lasting sodium transients in astrocytes, the properties of which are distinctly different from those of activity‐related glial calcium signals. From these studies, it emerges that sodium homeostasis and signaling are two sides of the same coin: sodium‐dependent transporters, primarily known for their role in ion regulation and homeostasis, also generate relevant ion signals during neuronal activity. The functional consequences of activity‐related sodium transients are manifold and are just coming into view, enabling surprising and important new insights into astrocyte function and neuron‐glia interaction in the brain. The present review will highlight current knowledge about the mechanisms that contribute to sodium homeostasis in astrocytes, present recent data on the spatial and temporal properties of activity‐related glial sodium signals and discuss their functional consequences with a special emphasis on pathophysiological conditions. GLIA 2013;61:1191–1205     

Glutamate receptor-mediated calcium entry in neurons derived from P19 embryonal carcinoma cells

We have examined the control of calcium elevation by glutamate in neurons derived from the mouse P19 embryonal carcinoma cell line. Following transient exposure to retinoic acid, P19 cells differentiate into neurons that express both NMDA and non-NMDA glutamate receptor subtypes. Fluorescence videomicroscopy using the indicator fura-2 revealed concentration-dependent elevation in cytosolic calcium levels with exposure to NMDA or kainate. Replacement of extracellular sodium with N-methylglucamine significantly reduced the action of kainate. Exposure to high K⁺ medium also elicited an elevation of cytosolic calcium in P19 cells, which was partially inhibited by the calcium channel antagonist nimodipine. These experiments suggest that the elevation in calcium produced by kainate involves the activation of voltage-gated calcium channels as a consequence of membrane depolarization, in contrast to direct calcium entry through NMDA receptor channels. Whole-cell recordings revealed that P19 NMDA receptors were highly permeable to calcium (P_Ca/P_Na = 5.6 ± 0.2). In most cells, channels gated by kainate displayed low permeability to calcium; the median permeability ratio, P_Ca/P_Na, was 0.053 (range 0.045 to 0.132). Activation of peak currents by NMDA, glycine, and kainate was half-maximal at 24 μM, 240 nM, and 81 μM, respectively. In addition, cadmium-sensitive currents through voltage-gated calcium channels were recorded in P19 cells bathed in barium/TEA chloride. Staining with antibodies directed against AMPA receptor subunits revealed widespread immunoreactivity for anti-GluR-B/C and anti-GluR-B/D. About half of the P19 cells were stained with antibodies selective for GluR-D but there was little or no immunoreactivity for the GluR-A subunit. © 1996 Wiley-Liss, Inc.     

Altered transporter‐mediated neocortical GABA release in Rasmussen encephalitis

To learn whether epileptic seizures in Rasmussen encephalitis (RE) may be promoted by insufficient γ‐aminobutyric acid (GABA) release. ³H‐GABA was released from neocortical synaptosomes through transporter reversal following intrasynaptosomal Na⁺ accumulation by veratridine that prevents inactivation of Na⁺ channels. Tissues of three RE patients were compared with those of nine non‐RE. In RE, the release was markedly reduced. In non‐RE, the extracellular Ca²⁺ concentration ([Ca²⁺]_e) was inversely related to the amount of release. In RE, the percental decline of additional release upon withdrawal was linked with the presurgical duration of epilepsy. Permanent opening of Na⁺ channels by veratridine resembles maximal frequency of action potentials corresponding to epileptic seizures. These are preceded by a fall in [Ca²⁺]_e. Zero [Ca²⁺]_e increased release through the Na⁺/Ca²⁺ exchanger additionally elevating intrasynaptosomal Na⁺. This enhanced GABA release probably reflects an antiseizure mechanism. In RE, the additional release gets lost over epilepsy duration.     

Glutamate increases the [Ca]i but stimulates Ca-independent release of [H]GABA in cultured chick retina cells

The effect of glutamate of [Ca²⁺]_i and on [³H]γ-aminobutyric acid (GABA) release was studied on cultured chick embryonic retina cells. It was observed that glutamate (100 μM) increases the [Ca²⁺]_i by Ca²⁺ influx through Ca²⁺ channels sensitive to nitrendipine, but not to ω-conotoxin GVIA (ω-Cg Tx) (50%), and by other channels insensitive to either Ca²⁺ channel blocker. Mobilization of Ca²⁺ by glutamate required the presence of external Na⁺, suggesting that Na⁺ mobilization through the ionotropic glutamate receptors is necessary for the Ca²⁺ channels to open. The increase in [Ca²⁺]_i was not related to the release of [³H]GABA induced by glutamate, suggesting that the pathway for the entry of Ca²⁺ triggered by glutamate does not lead to exocytosis. In fact, the glutamate-induced release of [³H]GABA was significantly depressed by Ca_o²⁺, but it was dependent on Na_o⁺, just as was observed for the [³H]GABA release induced by veratridine (50 μM). The veratridine-induced release could be fully inhibited by TTX, but this toxin had no effect on the glutamate-induced [³H]GABA release. Both veratridine- and glutamate-induced [³H]GABA release were inhibited by 1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,5,6-tetrahydro-3-pyridine-carboxylic acid (NNC-711), a blocker of the GABA carrier. Blockade of the NMDA and non-NMDA glutamate receptors with MK-801 and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), respectively, almost completely blocked the release of [³H]GABA evoked by glutamate. Continuous depolarization with 50 mM K⁺ induced maximal release of [³H]GABA of about 1.5%, which is much smaller than the release evoked by glutamate under the same conditions (6.0–6.5%). Glycine (3 μM) stimulated [³H]GABA release induced by 50 mM K⁺, and this effect was blocked by MK-801, suggesting that the effect of K⁺ on [³H]GABA release was partially mediated through the NMDA receptor which probably was stimulated by glutamate released by K⁺ depolarization. We conclude that glutamate induces Ca²⁺-independent release of [³H]GABA through reversal of the GABA carrier due to Na⁺ entry through the NMDA and non-NMDA, TTX-insensitive, channels. Furthermore the GABA carrier seems to be inhibited by Ca²⁺ entering by the pathways open by glutamate. This Ca²⁺ does not lead to exocytosis, probably because the Ca²⁺ channels used are located at sites far from the active zones.     

Veratridine delays apoptotic neuronal death induced by NGF deprivation through a Na+-dependent mechanism in cultured rat sympathetic neurons

Superior-cervical ganglion (SCG) cells dissociated from newborn rats depend on nerve growth factor (NGF) for survival. Membrane depolarization with elevated K+ is known to prevent neuronal death following NGF deprivation and/or to promote survival via a Ca2+-dependent mechanism. Here we have exploited the possibility of whether or not a Na+-dependent pathway for neuronal survival is present in these cells. Veratridine (ec50=40 nM), a voltage-dependent Na+ channel activator, significantly delayed the onset of apoptotic cell death in NGF-deprived SCG neurons that had been cultured for 7 days in the presence of NGF. This effect was blocked completely by Na+ channel blockers including tetrodotoxin (TTX, 1 μM), benzamil (25 μM) and flunarizine (1 μM), but was not attenuated by nimodipine (1 μM), an L-type Ca²⁺ channel blocker. The saving effect of veratridine on cultured neurons was observed even in low Ca²⁺ media (0–1.0 mM), but was completely abolished in a low Na⁺ medium (38 mM). Sodium-binding benzofuran isophthalate was employed as a fluorescent probe for monitoring the level of cytoplasmic free Na⁺, which revealed a sustained increase in its level (12.9 mM, 307% of that of control) in response to veratridine (0.75 μM). The TTX or flunarizine completely blocked veratridine-induced Na⁺ influx in these cultured neurons. Moreover, no appreciable increase in intracellular Ca²⁺ was detected under these conditions. Though Na⁺ channels were effectual in SCG neurons which were freshly isolated from newborn rats, the Na⁺-dependent saving effect of veratridine was not observed in these young neurons. These lines of evidence suggest that the death-suppressing effect of veratridine on cultured SCG neurons depends on the Na⁺ influx via voltage-dependent Na⁺ channels, and suggests the presence of Na⁺-dependent regulatory mechanism(s) in neuronal survival.     

Astrocytes restrict discharge duration and neuronal sodium loads during recurrent network activity

Influx of sodium ions into active neurons is a highly energy‐expensive process which must be strictly limited. Astrocytes could play an important role herein because they take up glutamate and potassium from the extracellular space, thereby dampening neuronal excitation. Here, we performed sodium imaging in mouse hippocampal slices combined with field potential and whole‐cell patch‐clamp recordings and measurement of extracellular potassium ([K⁺]_o). Network activity was induced by Mg²⁺‐free, bicuculline‐containing saline, during which neurons showed recurring epileptiform bursting, accompanied by transient increases in [K⁺]_o and astrocyte depolarizations. During bursts, neurons displayed sodium increases by up to 22 mM. Astrocyte sodium concentration increased by up to 8.5 mM, which could be followed by an undershoot below baseline. Network sodium oscillations were dependent on action potentials and activation of ionotropic glutamate receptors. Inhibition of glutamate uptake caused acceleration, followed by cessation of electrical activity, irreversible sodium increases, and swelling of neurons. The gliotoxin NaFAc (sodium‐fluoroacetate) resulted in elevation of astrocyte sodium concentration and reduced glial uptake of glutamate and potassium uptake through Na⁺/K⁺‐ATPase. Moreover, NaFAc extended epileptiform bursts, caused elevation of neuronal sodium, and dramatically prolonged accompanying sodium signals, most likely because of the decreased clearance of glutamate and potassium by astrocytes. Our experiments establish that recurrent neuronal bursting evokes sodium transients in neurons and astrocytes and confirm the essential role of glutamate transporters for network activity. They suggest that astrocytes restrict discharge duration and show that an intact astrocyte metabolism is critical for the neurons' capacity to recover from sodium loads during synchronized activity. GLIA 2015;63:936–957     

Multiple episodes of sodium depletion in the rat: a remodeling of the electrical properties of median preoptic nucleus neurons

In rat brain, the detection and integration of chemosensory and neural signals are achieved, inter alia, by the median preoptic nucleus (MnPO) during a disturbance of the hydromineral balance. This is allowed through the presence of the sodium (Na⁺) sensor neurons. Interestingly, enkephalins and mu‐opioid receptors (μ‐ORs) are known for their role in ingestive behaviors and have previously been shown to regulate the excitability of MnPO neurons following a single Na⁺ depletion. However, little is known about the role of these μ‐ORs in the response enhancement following repeated Na⁺ challenge. Therefore, we used whole‐cell recordings in acute brain slices to determine neuronal plasticity in the electrical properties of the MnPO Na⁺ sensor‐specific neuronal population following multiple Na⁺ depletions. Our results show that the population of Na⁺ sensor neurons was represented by 80% of MnPO neurons after a single Na⁺ depletion and was reduced after three Na⁺ depletions. Interestingly, the subpopulation of Na⁺ sensors responding to D‐Ala²,N‐MePhe⁴,Gly‐ol‐enkephalin (DAMGO), a specific μ‐OR agonist, represented 11% of MnPO neurons after a single Na⁺ depletion and the population doubled after three Na⁺ depletions. Moreover, Na⁺ sensor neurons displayed modifications in the discharge pattern distribution and shape of calcium action potentials after three Na⁺ depletions but these changes did not occur in Na⁺ sensors responding to DAMGO. Thus, the reinforced μ‐OR functionality in Na⁺ sensors might take place to control the neuronal hyperexcitability and this plasticity in opioid‐sensitive and Na⁺ detection MnPO networks might sustain the enhanced salt ingestion induced by repeated exposure to Na⁺ depletion.     

Local anesthetics inhibit glutamate release from rat cerebral cortex synaptosomes

Local anesthetics have been widely used for regional anesthesia and the treatment of cardiac arrhythmias. Recent studies have also demonstrated that low‐dose systemic local anesthetic infusion has neuroprotective properties. Considering the fact that excessive glutamate release can cause neuronal excitotoxicity, we investigated whether local anesthetics might influence glutamate release from rat cerebral cortex nerve terminals (synaptosomes). Results showed that two commonly used local anesthetics, lidocaine and bupivacaine, exhibited a dose‐dependent inhibition of 4‐AP‐evoked release of glutamate. The effects of lidocaine or bupivacaine on the evoked glutamate release were prevented by the chelation of extracellular Ca²⁺ ions and the vesicular transporter inhibitor bafilomycin A1. However, the glutamate transporter inhibitor dl ‐threo‐beta‐benzyl‐oxyaspartate did not have any effect on the action of lidocaine or bupivacaine. Both lidocaine and bupivacaine reduced the depolarization‐induced increase in [Ca²⁺]_C but did not alter 4‐AP‐mediated depolarization. Furthermore, the inhibitory effect of lidocaine or bupivacaine on evoked glutamate release was prevented by blocking the Ca_v2.2 (N‐type) and Ca_v2.1 (P/Q‐type) channels, but it was not affected by blocking of the ryanodine receptors or the mitochondrial Na⁺/Ca²⁺ exchange. Inhibition of protein kinase C (PKC) and protein kinase A (PKA) also prevented the action of lidocaine or bupivacaine. These results show that local anesthetics inhibit glutamate release from rat cortical nerve terminals. This effect is linked to a decrease in [Ca²⁺]_C caused by Ca²⁺ entry through presynaptic voltage‐dependent Ca²⁺ channels and the suppression of the PKA and PKC signaling cascades. Synapse 67:568–579, 2013 . © 2013 Wiley Periodicals, Inc.     

Kainate‐induced toxicity in the hippocampus: potential role of lithium

Crespo‐Biel N, Camins A, Canudas AM, Pallàs M. Kainate‐induced toxicity in the hippocampus: potential role of lithium.
Bipolar Disord 2010: 12: 425–436. © 2010 The Authors. Journal compilation © 2010 John Wiley & Sons A/S. Objectives: We investigated the neuroprotective effects of lithium in an experimental neurodegeneration model gated to kainate (KA) receptor activation. Methods: The hippocampus from KA‐treated mice and hippocampal cell cultures were used to evaluate the pathways regulated by chronic lithium pretreatment in both in vivo and in vitro models. Results: Treatment with KA, as measured by fragmentation of α‐spectrin and biochemically, induced the activation of calpain resulting in p35 cleavage to p25, indicating activation of cyclin‐dependent kinase 5 (cdk5) and glycogen synthase kinase‐3ß (GSK‐3ß) and an increase in tau protein phosphorylation. Treatment with lithium reduced calpain activation and reduced the effects of cdk5 and GSK‐3ß on tau. KA treatment of cultures resulted in neuronal demise. According to nuclear condensed cell counts, the addition of lithium to neuronal cell cultures (0.5–1 mM) a few days before KA treatment had neuroprotective and also antiapoptotic effects. The action of lithium on calpain/cdk5 and GSK‐3ß pathways produced similar results in vivo. As calpain is activated by an increase in intracellular calcium, we showed that lithium reduced calcium concentrations in basal and KA‐treated hippocampal cells, which was accompanied by an increase in NCX3, a Na⁺/Ca²⁺ exchanger pump. Conclusion: A robust neuroprotective effect of lithium in the excitotoxic process induced by KA in mouse hippocampus was demonstrated via modulation of calcium entry and the subsequent inhibition of the calpain pathway. These mechanisms may act in an additive way with other mechanisms previously described for lithium, suggesting that it may be useful as a possible therapeutic strategy for Alzheimer’s disease.     

Glutamate receptors communicate with Na+/K+-ATPase in rat cerebellum granule cells

Two glutamate receptor agonists, NMDA (N-methyl-d-aspartic acid) and ACPD (cis-(1S/3R)-1-aminocyclopentane-1,3-dicarboxylic acid), induce the reactive oxygen species (ROS) production in rat cerebellum granule cells, whereas the third one, 3-HPG (3-hydroxyphenylglycine), decreases this parameter. The simultaneous presence of 3-HPG, together with NMDA or ACPD, prevents the generation of ROS by neuronal cells. A similar effect of these ligands on Na⁺/K⁺-ATPase can be demonstrated: NMDA and ACPD inhibited the enzyme activity, but 3-HPG activated Na⁺/K⁺-ATPase and prevented its inhibition by NMDA or ACPD. In terms of current classification, NMDA is an agonist of ionotropic glutamate receptors of the so-called NMDA class, whereas ACPD and 3-HPG belong to metabotropic agonists, the former primarily being an activator of metabotropic glutamate receptors (mGluRs) of groups 2 and 3, and the latter, that of mGluRs of groups 1 and 5. Thus, the data presented illustrate the existence of diverse mechanisms of the cross talk between Na⁺/K⁺-ATPase and different glutamate receptors, as well as that between glutamate receptors of different classes.     

Abnormal activity of the Na/Ca exchanger enhances glutamate transmission in experimental autoimmune encephalomyelitis

It is increasingly accepted that excessive glutamate release plays a key role in the pathophysiology of grey matter damage in multiple sclerosis (MS). The mechanisms causing abnormal glutamate transmission in this disorder are however largely unexplored. By means of electrophysiological recordings from single striatal neurons in slices, we found that the presymptomatic and acute phases of experimental autoimmune encephalomyelitis (EAE), a preclinical model of MS, are associated with enhanced synaptic release of glutamate. The reverse mode of action of axonal Na⁺/Ca⁺⁺ exchanger, secondary to abnormal functioning of voltage-dependent Na⁺ channels, was identified as a major cause of this alteration. In fact, inhibition of the Na⁺/Ca⁺⁺ exchanger with bepridil or with KB-R7943, which selectively blocks the reverse mode of the exchanger, reduced the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) recorded from striatal neurons in EAE mice but not in control animals. In the presence of tetrodotoxin (TTX), a blocker of voltage-dependent Na⁺ channels, the effect of bepridil was normalized in acute (25 days post-immunization) EAE mice, indicating that axonal accumulation of Na⁺ ions flowing through voltage-dependent Na⁺ channels plays a role in the abnormal activity of the Na⁺/Ca⁺⁺ exchanger in EAE.Our data reveal an important role of Na⁺/Ca⁺⁺ exchanger and of voltage-dependent Na⁺ channels in the pathological process of EAE, and provide a rationale for the use of neuroprotective strategies since the very early stages of MS.     

N-methyl-d-aspartate and hypoxia induced Ca-changes in the CA1 region of the hippocampal slice

Hypoxic neuronal depolarization was accompanied by a large decrease in extracellular [Ca²⁺]. After reoxygenation, the time at which [Ca²⁺] normalized was correlated with the extent of recovery of N-methyl-d-aspartate (NMDA) and synaptic responses. There was no evidence that the NMDA receptor system was more disrupted following hypoxia than the receptors involved in synaptic transmission. The Na⁺/K⁺ pump appeared to be better able to recover from hypoxia than the NMDA responses or synaptic transmission.     

Anoxia-Induced Neuronal Injury: Role of NaEntry and Na-Dependent Transport

An important cause of anoxia-induced nerve injury involves the disruption of the ionic balance that exists across the neuronal membrane. This loss of ionic homeostasis results in an increase in intracellular calcium, sodium, and hydrogen and is correlated with cell injury and death. Using time-lapse confocal microscopy, we have previously reported that nerve cell injury is mediated largely by sodium and that removing extracellular sodium prevents the anoxia-induced morphological changes. In this study, we hypothesized that sodium enters neurons via specific mechanisms and that the pharmacologic blockade of sodium entry would prevent nerve damage. In cultured neocortical neurons we demonstrate that replacing extracellular sodium with NMDG⁺prevents anoxia-induced morphological changes. With sodium in the extracellular fluid, various routes of sodium entry were examined, including voltage-sensitive sodium channels, glutamate receptor channels, and sodium-dependent chloride–bicarbonate exchange. Blockade of these routes had no effect. Amiloride, however, prevented the morphological changes induced by anoxia lasting 10, 15, or 20 min. At doses of 10 μM–1 mM, amiloride protected neurons in a dose-dependent fashion. We argue that amiloride acts on a Na⁺-dependent exchanger (e.g., Na⁺–Ca²⁺) and present a model to explain these findings in the context of the neuronal response to anoxia.