Tihomir P. Obrenovitch

Altered glutamatergic transmission in neurological disorders: from high extracellular glutamate to excessive synaptic efficacy.

This review is a critical appraisal of the widespread assumption that high extracellular glutamate, resulting from enhanced pre-synaptic release superimposed on deficient uptake and/or cytosolic efflux, is the key to excessive glutamate-mediated excitation in neurological disorders. Indeed, high extracellular glutamate levels do not consistently correlate with, nor necessarily produce, neuronal dysfunction and death in vivo. Furthermore, we exemplify with spreading depression that the sensitivity of an experimental or pathological event to glutamate receptor antagonists does not imply involvement of high extracellular glutamate levels in the genesis of this event. We propose an extension to the current, oversimplified concept of excitotoxicity associated with neurological disorders, to include alternative abnormalities of glutamatergic transmission which may contribute to the pathology, and lead to excitotoxic injury. These may include the following: (i) increased density of glutamate receptors; (ii) altered ionic selectivity of ionotropic glutamate receptors; (iii) abnormalities in their sensitivity and modulation; (iv) enhancement of glutamate-mediated synaptic efficacy (i.e. a pathological form of long-term potentiation); (v) phenomena such as spreading depression which require activation of glutamate receptors and can be detrimental to the survival of neurons. Such an extension would take into account the diversity of glutamate-receptor-mediated processes, match the complexity of neurological disorders pathogenesis and pathophysiology, and ultimately provide a more elaborate scientific basis for the development of innovative treatments.

Extracellular Glutamate During Focal Cerebral Ischaemia in Rats: Time Course and Calcium Dependency

Abstract: The time course of changes in extracellular glutamic acid levels and their Ca2+ dependency were studied in the rat striatum during focal cerebral ischaemia, using microdialysis. Ischaemia‐induced changes were compared with those produced by high K+‐evoked local depolarization. To optimize time resolution, glutamate was analysed continuously as the dialysate emerged from the microdialysis probe by either enzyme fluorimetry or biosensor. The Ca2+ dependency of glutamate changes was examined by perfusing the probe with Ca2+‐free medium. With normal artificial CSF, ischaemia produced a biphasic increase in extracellular glutamate, which started from the onset of ischaemia. During the first phase lasting ∼10 min, dialysate glutamate level increased from 5.8 ± 0.9 µM· min−1 to 35.8 ± 6.2 µM where it stabilized for ∼3 min. During the second phase dialysate glutamate increased progressively to its maximum (82 ± 8 µM), reached after 55 min of ischaemia, where it remained for as long as it was recorded (3 h). The overall changes in extracellular glutamate were similar when Ca2+ was omitted from the perfusion medium, except that the first phase was no longer detectable and, early in ischaemia, extracellular glutamate increased at a significantly slower rate than in the control group (2.2 ± 1 µM· min−1; p < 0.05). On the basis of these data, we propose that most of the glutamate released in the extracellular space in severe ischaemia is of metabolic origin, probably originating from both neurons and glia, and caused by altered glutamate uptake mechanisms. Comparison with high K+‐induced glutamate release did not suggest that glutamate “exocytosis,” early after middle cerebral artery occlusion, was markedly limited by deficient ATP levels.

Investigation into the role of N-acetylaspartate in cerebral osmoregulation.

Abstract: Marked abnormalities of the magnetic resonance intensity of N‐acetylaspartate (NAA) have been reported in patients with various neurological disorders, but the neurochemical consequences of these alterations are difficult to assess because the function of NAA remains speculative. The purpose of this study was to examine whether NAA plays a role in protecting neurons against osmotic stress. Intracerebral microdialysis was used to expose a small region of the rat dorsolateral striatum to an increasingly hyposmotic environment and to measure resulting changes in NAA extracellular concentrations. NAA changes in the extracellular fluid (ECF) were compared with those of the amino acids, in particular, taurine, known to be involved in brain osmoregulation. Stepped increases in cellular hydration produced by hyposmotic perfusion media induced a marked increase in ECF NAA, reflecting a redistribution of NAA from intra‐to extracellular space. Parallel experiments showed that, of all the extracellular amino acids measured, only taurine markedly increased with hyposmolar perfusion medium, indicating that the ECF NAA increase associated with hyposmotic stress was a specific response and not passive leakage out of the cells. As NAA is predominantly neuronal, it may contribute to the protection of neurons against swelling (i.e., regulatory volume decrease). In conditions with impaired blood‐brain barrier and cytotoxic oedema, efflux of intracellular NAA subsequent to sustained cellular swelling might lead to a reduction in total brain NAA detectable by magnetic resonance spectroscopy. Alternatively, redistribution of NAA from intra‐to extracellular space implies changes in its chemical environment that may alter its magnetic resonance visibility.

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.

Combined intracerebral microdialysis and electrophysiological recording: methodology and applications.

A microdialysis probe is described that can simultaneously monitor indices of electrical activity, ionic homeostasis and changes in the composition of the extracellular fluid at the same brain site in anaesthetised laboratory animals. The probe is no larger than its conventional counterpart and avoids tissue injury problems due to implantation of separate recording electrodes. Examples are given of its application to the study of changes following probe implantation, cerebral ischaemia and local high K(+)-induced depolarisation.

Extracellular neuroactive amino acids in the rat striatum during ischaemia : comparison between penumbral conditions and ischaemia with sustained anoxic depolarisation

Abstract: Changes in the extracellular levels of excitatory and inhibitory amino acid transmitters were studied in the rat striatum during penumbral ischaemia using intracerebral microdialysis. Effects of penumbral forebrain ischaemia were compared with those of ischaemia with sustained anoxic depolarisation and K+ (100 mM). Comparisons were also made between different groups of animals at 2 and 24 h after dialysis probe implantation. The K+ stimulus did not provoke any release of excitatory amino acids in the 24‐h group, probably reflecting a decrease of functional synapses adjacent to the probe. During 30 min of penumbral ischaemia, excitatory amino acids did not reach critical concentrations in the extracellular fluid, and increases in levels of inhibitory/modulatory amino acids were similar. On the other hand, severe transient ischaemia resulted in massive synchronous release of many neuroactive excitatory and inhibitory compounds, in both the 2‐ and 24‐h groups. These and other data suggest that changes during severe ischaemia may arise from both neurotransmitter and metabolic pools. It is concluded that is‐ chaemic damage in the penumbra may not be related to extracellular neuroactive amino acid changes generated within this region.

High Extracellular Glutamate and Neuronal Death in Neurological Disorders: Cause, Contribution or Consequence?

ABSTRACT: In models of neurological disorders, increased extracellular glutamate and beneficial effects produced by glutamate‐receptor antagonists are consistently taken as supporting evidence of excitotoxicity. This systematic interpretation is over‐simplified and potentially misleading.

Intracerebral microdialysis combined with recording of extracellular field potential: a novel method for investigation of depolarizing drugs in vivo

1 The purpose of this study was to examine whether depolarizations evoked by excitatory amino acids can be recorded quantitatively, in vivo, with a microelectrode incorporated within a microdialysis probe. 2 Microdialysis probes incorporating a chlorided silver wire were implanted in the striatum of anaesthetized rats and perfused with artificial cerebrospinal fluid (ACSF). Increasing concentrations of excitatory amino acids were applied for 2 min via the microdialysis probe, and the extracellular direct current (d.c.) potential was recorded between the microdialysis electrode and a reference electrode placed under the scalp. 3 N‐methyl‐D‐aspartate (NMDA, 25–500 μm), α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid (AMPA, 5–1000 μm), kainate (5–500 μm), and glutamate (0.25–100 μm) evoked concentration‐dependent depolarizations with maxima ranging from 7 to 10 mV, i.e. 3 to 10 times larger than those recorded from brain slices in vitro. Depolarizations evoked by glutamate receptor agonists applied by microdialysis shared several features with those recorded from brain slices. The most characteristic were: steep onset and recovery of NMDA and glutamate responses; marked post‐depolarization hyper‐polarization with NMDA; and very slow recovery after kainate application. At high concentrations (500 μm), NMDA occasionally initiated spreading depression. The relative potency of glutamate and NMDA was of the same order of magnitude to that obtained with the cortical wedge and hippocampal slices, glutamate being 100 to 400 times less potent than NMDA. 4 Two consecutive series of NMDA‐stimuli within the same procedure evoked comparable depolarizations, indicating that reliable quantitative analysis of drug action can be performed, with each animal serving as its own control. This is relevant to the study of drugs acting on glutamate receptors especially antagonists. The remarkable inter‐animal reproducibility is also a valuable feature. 5 Pretreatment with dizocilpine maleate (MK‐801, 2 mg kg−1, i.p.) reduced by 65% the responses evoked by NMDA (500 μm). The non‐NMDA antagonist 6,7‐dinitroquinoxaline‐2,3‐dione (DNQX, 100 μm) applied via the microdialysis probe reduced by around 78% the responses to AMPA and kainate (250 μm). The fact that drugs, especially antagonists, can be administered either systemically, or directly through the dialysis probe to by‐pass the blood‐brain barrier or avoid peripheral effects, is especially relevant for neuropharmacological studies. 6 Intracerebral microdialysis combined with in vivo recording of extracellular field potential is a novel and valuable method for the quantitative analysis of the action of drugs acting on glutamate receptors. This method should prove especially useful for comparing the sensitivitiy of specific brain structures to selective glutamate receptor agonists under normal conditions and when the neuronal microenvironment is altered. It should also be useful for investigating the action of other depolarizing agents, such as veratridine, and their antagonists.

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.