Elias Zilkha

Excitotoxicity in neurological disorders--the glutamate paradox.

Beneficial effects of glutamate‐receptor antagonists in models of neurological disorders are often used to support the notion that endogenous excitotoxicity (i.e. resulting from extracellular accumulation of endogenous glutamate) is a major contributor to neuronal death associated with these conditions. However, this interpretation conflicts with a number of robust and important experimental evidence. Here, emphasis is placed on two key elements: (i) very high extracellular levels of glutamate must be reached to initiate neuronal death, far above those measured in models of neurological disorders; and (ii) changes in extracellular glutamate as measured by microdialysis are not related to changes in the synaptic cleft, i.e. the compartment where neurotransmitter glutamate interacts with its receptors.

Evidence disputing the link between seizure activity and high extracellular glutamate.

Abstract: As seizures in experimental models can be induced by the activation and suppressed by the inhibition of glutamate receptors, it is often proposed that a high extracellular glutamate level subsequent to excessive presynaptic release and/or altered glutamate uptake is epileptogenic. The purpose of this study was to ascertain the link between seizure activity and high extracellular glutamate. To assist the detection of any putative rise in extracellular glutamate during seizures, microdialysis was coupled to enzyme‐amperometric detection of glutamate, which provides maximal sensitivity and time resolution. Electrical activity and field potential were also recorded through the dialysis membrane to confirm that epileptic activity was present at the sampling site. No increase in dialysate glutamate content was detected during picrotoxin‐induced seizures, even when the K+ concentration in the perfusion medium was raised to 50% above that measured previously during paroxysmal activity. In addition, sustained inhibition of glutamate uptake by l‐trans‐pyrrolidine‐2,4‐dicarboxylate increased the extracellular glutamate level >20‐fold but did not produce electrophysiological changes indicative of excessive excitation. These findings indicate that seizures are not necessarily accompanied by an increased extracellular glutamate level and that increased glutamatergic excitation in epilepsy may result from other abnormalities such as increased density of glutamate receptors, enhanced activation subsequent to reduced modulation, or sprouting of glutamatergic synapses.

The Relationship between the Apparent Diffusion Coefficient Measured by Magnetic Resonance Imaging, Anoxic Depolarization, and Glutamate Efflux during Experimental Cerebral Ischemia

A reduction in the apparent diffusion coefficient (ADC) of water measured by magnetic resonance imaging (MRI) has been shown to occur early after cerebrovascular occlusion. This change may be a useful indicator of brain tissue adversely affected by inadequate blood supply. The objective of this study was to test the hypothesis that loss of membrane ion homeostasis and depolarization can occur simultaneously with the drop in ADC. Also investigated was whether elevation of extracellular glutamate ([GLU]e) would occur before ADC changes. High-speed MRI of the trace of the diffusion tensor (15-second time resolution) was combined with simultaneous recording of the extracellular direct current (DC) potential and on-line [GLU]e from the striatum of the anesthetized rat. After a control period, data were acquired during remote middle cerebral artery occlusion for 60 minutes, followed by 30 minutes of reperfusion, and cardiac arrest-induced global ischemia. After either focal or global ischemia, the ADC was reduced by 10 to 25% before anoxic depolarization occurred. After either insult, the time for half the maximum change in ADC was significantly shorter than the corresponding DC potential parameter (P < 0.05). The [GLU]e remained at low levels during the entire period of varying ADC and DC potential and did not peak until much later after either ischemic insult. This study demonstrates that ADC changes can occur before membrane depolarization and that high [GLU]e has no involvement in the early rapid ADC decrease.

Intracerebral microdialysis : electrophysiological evidence of a critical pitfall

Abstract: Using microdialysis probes incorporating an electrode for the recording of extracellular field potentials, we have found that microdialysis markedly inhibited the propagation of spreading depression. This effect was independent of the microdialysis flow rate and did not result from tissue injury following probe implantation. Increasing the K+ concentration in the perfused artificial CSF dose‐dependently restored the propagation of spreading depression and revealed a large, synchronous transient increase in extracellular glutamate. These findings clearly illustrate that microdialysis can influence the experimental or pathological conditions under study, by buffering transient changes in the extracellular fluid composition. Epileptic seizures and ischaemia are two important conditions that may be prone to such a detrimental interaction.

Evidence Against High Extracellular Glutamate Promoting the Elicitation of Spreading Depression by Potassium

This study ascertains whether high extracellular glutamate contributes to the initiation of spreading depression (SD) by K+. Two microdialysis probes, each incorporating an electrode to record the extracellular direct current (DC) potential at the elicitation site, were implanted symmetrically in the cortex of anesthetized rats. Recurrent SD was triggered by perfusion of 130 mM K+ through the microdialysis probe for 20 min. On one side, this medium was supplemented with increasing concentrations of glutamate (0.1–1 mM) or of the selective glutamate uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylate (L-trans-PDC; 1–10 mM). The effects of L-trans-PDC on extracellular glutamate and basal DC potential were studied in separate experiments. Application of K+ for 20 min consistently elicited five to seven waves of SD. Increasing the concentration of glutamate in the perfusion medium did not alter SD elicitation. Application of L-trans-PDC concentration dependently increased the dialysate levels of glutamate (by ∼ 19-fold with 10 mM L-trans-PDC) but, unexpectedly, reduced SD elicitation. These data do not support the hypothesis that SD is elicited because high extracellular glutamate resulting from exocytosis and/or reversal of glutamate uptake depolarizes adjacent neurons. As SD elicitation requires activation of N-methyl-D-aspartate (NMDA) receptors, these results also illustrate that sensitivity of a pathological or experimental event to NMDA receptor antagonists does not necessarily imply involvement of increased extracellular glutamate. This does not rule out a selective action of glutamate, transiently released from presynaptic vesicles, on immediately juxtaposed postsynaptic receptors.

EFFECTS OF PHARMACOLOGICAL INHIBITION OF GLUTAMATE-UPTAKE ON ISCHAEMIA-INDUCED GLUTAMATE EFFLUX AND ANOXIC DEPOLARIZATION LATENCY

Abstract It has been proposed that deficient glutamate uptake, by increasing the extracellular concentration of this excitatory neurotransmitter, may contribute to the pathophysiology of cerebral ischaemia. This study aimed to examine whether pharmacological inhibition of glutamate uptake altered the kinetics of ischaemia-induced glutamate efflux, and precipitated anoxic depolarisation. Microdialysis was used for application of the glutamate-uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylate (L-trans-PDC), recording of the EEG and extracellular direct current (DC) potential with an electrode within the probe, and continuous monitoring of changes in extracellular glutamate. L-trans-PDC was applied locally from 8 min prior to cardiac arrest to the end of the recording period. L-trans-PDC (2.5 mM) barely altered the time course of postmortem glutamate efflux in the cortex. Only the maximum rate of efflux during the first exocytotic phase, and the concentration reached at the end of this phase, appeared slightly increased. L-trans-PDC (5 mM) reduced significantly the delay between EEG silence and anoxic depolarization in the cerebral cortex (59.2 ± 9.2 s vs. 79.7 ± 11.5 s; n = 6), but not in the striatum and hippocampus. These effects contrast with the marked increase in dialysate glutamate that L-trans-PDC produces in all these three brain regions. Together, these data do not support the hypothesis that inhibition of glutamate uptake plays a critical role, early in cerebral ischaemia. However, a contribution of reversed glutamate uptake to the secondary Ca2+-independent phase of ischaemia-induced glutamate efflux cannot be ruled out.

Effect of probenecid on depolarizations evoked by N-methyl-D-aspartate (NMDA) in the rat striatum

Abstract Kynurenic acid is an endogenous, competitive antagonist of the N-methyl-D-aspartate (NMDA) receptor glycine site. Accordingly, increasing the brain extracellular concentration of this metabolite may be a suitable alternative to administration of exogenous NMDA antagonists for the treatment of neurological disorders involving excessive NMDA-receptor activation. As competitive inhibition of organic anion transport by probenecid increased brain extracellular levels of kynurenic acid, the purpose of this study was to examine whether intracerebral application of probenecid reduced depolarizations evoked at the same tissue site by NMDA. Microdialysis probes incorporating an electrode were implanted into the striatum of rats and perfused with artificial cerebrospinal fluid. Local depolarizations were produced by perfusing 200 μM NMDA for 2 min, either alone, or co-applied with 1, 5 or 20 mM probenecid. The lowest concentration of probenecid had no effect. At 5 mM, probenecid abolished the hyperpolarization which consistently followed NMDA-responses, but the slight decrease in depolarization amplitude did not reach significance. Inhibition of post-depolarization hyperpolarization suggests that sustained, high extracellular concentrations of probenecid reduce the capacity of the tissue to recover from a depolarizing stimulus, presumably because intensive transport of probenecid imposes a heavy load on Na+, K+-ATPase. At 20 mM, probenecid inhibited NMDA-evoked depolarization by approximately 60% (from 4.7 ± 0.7 mV to 2.1 ± 0.2 mV; n = 6, P <>; 0.005). This effect was more marked 30 min after returning to perfusion with normal artificial cerebrospinal fluid, suggesting that high concentrations of probenecid may be toxic to nerve cells, or initiate long-lasting effects linked to inhibition of the transport of important organic anions. These data suggest that inhibition of organic anion transport is not, by itself, sufficient to protect against neurological disorders involving excessive NMDA-receptor activation. However, results from other studies suggest that it may be a valid strategy for enhancing the neuroprotective actions of treatments which stimulate kynurenic acid synthesis, or those of exogenous glutamate receptor antagonists.

Effects of probenecid on the elicitation of spreading depression in the rat striatum

Spreading depression (SD) is a wave of cellular depolarization which contributes to neuronal damage in experimental focal ischaemia, and may also underlie the migraine aura. The purpose of this study was to examine the effects of probenecid, an inhibitor of organic anion transport, on K+-evoked SD in vivo. Microdialysis electrodes were implanted in the rat striatum, and recurrent SD elicited by perfusion of artificial cerebrospinal fluid containing 160 mM K+ for 20 min. Probenecid was administered either directly through the microdialysis probe, starting 50 min before application of high K+, or intravenously. SD was markedly reduced by perfusion of 5 mM probenecid through the microdialysis probe. In contrast, a high intravenous dose of probenecid (250 mg/kg) only slightly inhibited SD elicitation 90 min after treatment, despite clear changes in the amplitude and spectrum of the electroencephalogram, as early as 10 min after drug administration, confirming that probenecid readily penetrated the central nervous system. As SD is inhibited by hypercapnia, we have examined the possibility that probenecid may inhibit SD through extracellular acidification subsequent to blockade of lactate transport. Perfusion of 1-20 mM probenecid increased dose-dependently the dialysate levels of lactate, but without extracellular acidosis since the dialysate pH was not significantly reduced. How probenecid inhibits SD deserves further investigation because it may help identify novel strategies to suppress this phenomenon, now recognized deleterious to neuronal function and survival.

Effects of L-701,324, a high-affinity antagonist at the N-methyl-D-aspartate (NMDA) receptor glycine site, on the rat electroencephalogram

L-701,324 (7-chloro-4-hydroxy-3-(3-phenoxy) phenyl-2-(1H)-quinolone) is a novel, orally active antagonist at the N-methyl-D-aspartate (NMDA) receptor glycine site. As NMDA receptor antagonism is generally associated with anaesthetic effects, we have examined the electroencephalographic alterations produced by doses of L-701,324 that effectively reduce NMDA-evoked responses in vivo. Microdialysis probes incorporating an electrode were implanted in the striatum of rats and perfused with artificial cerebrospinal fluid (ACSF). Under light halothane anaesthesia, 12 consecutive depolarizations were elicited by switching to ACSF containing 200 µM NMDA for 2 or 3 min, every 20 min. NMDA-evoked depolarizations and EEG were recorded with the microdialysis electrode. L-701,324 (5 or 10 mg kg-1 i.v.) or vehicle were administered 5 min after the 3rd NMDA stimulus. L-701,324 dose-dependently inhibited NMDA-evoked depolarizations, with 10 mg kg-1 reducing these responses by 50 % for at least 3 h. The average amplitude of the EEG in the window 0.25-6 Hz (low frequencies) and 6-21 Hz (high frequencies) did not change in the control group. At the higher dose of 10 mg kg-1 L-701,324 transiently increased the amplitude of low frequencies by around 20 %. In contrast, both 5 and 10 mg kg-1 significantly reduced the high frequencies to around 70 % of control, and this action was sustained with the higher dose. Analysis of the relative EEG power spectra confirmed a small, but persistent shift from high to low EEG frequencies. Our results suggest that L-701,324 slightly strengthened halothane anaesthesia at doses inhibiting effectively NMDA receptor function. Accordingly, the resulting anticonvulsant and neuroprotective actions of L-701,324 may not be associated with marked anaesthesia-like side-effects.

Intracerebral Microdialysis Markedly Inhibits the Propagation of Cortical Spreading Depression

It is accepted that the ionic composition of the medium perfused through a microdialysis probe should match that of the extracellular fluid (ECF) under physiological conditions. In contrast, the possibility that control artificial cerebrospinal fluid may influence the experimental or pathological conditions under study, by buffering changes in the ECF composition, has been neglected. Spreading depression (SD) is a propagating transient suppression of electrical activity due to cellular depolarization which may contribute to neuronal damage in focal ischaemia, and underlie the migraine aura Here we report that microdialysis markedly inhibits SD propagation, by buffering the sudden increase in extracellular K+ associated with this event. This effect is independent of the microdialysis flow rate and does not result from tissue injury following probe implantation. This finding clearly illustrates that microdialysis can influence the pathological conditions under investigation.