Giti Garthwaite

NMDA RECEPTOR ACTIVATION INDUCES NITRIC-OXIDE SYNTHESIS FROM ARGININE IN RAT-BRAIN SLICES

Activation of N-methyl-D-aspartate (NMDA) receptors in rat cerebellum leads to the release of endothelium-derived relaxing factor, now identified as nitric oxide (NO), a stimulator of soluble guanylate cyclase. L-NG-monomethylarginine (L-NMMA), which blocks NO synthesis from L-arginine in several tissues, including a crude synaptosomal preparation from brain, inhibited the elevation of cyclic GMP induced by NMDA in rat cerebellar slices. D-NMMA was ineffective. L-Arginine, but not its D enantiomer, augmented the response to NMDA and reversed the inhibition by L-NMMA. The results indicate that stimulation of NMDA receptors results in the activation of the enzyme which catalyzes the formation of NO from L-arginine.

NEUROTOXICITY OF EXCITATORY AMINO-ACID RECEPTOR AGONISTS IN RAT CEREBELLAR SLICES - DEPENDENCE ON CALCIUM-CONCENTRATION

In slices of developing rat cerebellum, a 30-min application of the excitatory amino acid receptor agonist, N-methyl-D-aspartate (NMDA), led to the necrosis of differentiating granule cells and deep nuclear neurones. The corresponding effect of another agonist, kainate, was the death of Golgi cells. The toxic effects of both agonists were prevented if the concentration of calcium in the exposing solution was reduced to 0.3 mM from the control level of 2.5 mM. A lesser reduction (to 1 mM) was enough to prevent 90% of the NMDA-induced necrosis of granule cells. The results indicate that an important component of the acute neurotoxic effects of excitatory amino acids is calcium-dependent and suggest reasons why this may not have been revealed in some previous studies.

Glutamate Toxicity: An Experimental and Theoretical Analysis

In slices of 8‐day‐old rat cerebellum, the lowest concentration of glutamate that induced toxicity (30 min exposure; 90 min recovery) was 100 μM, but the damage only occurred in the outermost regions. As the concentration was raised, the band of necrosis became progressively deeper until, at 3 mM, it was uniform across the slice thickness. At a test concentration of 300 μM, the width of the necrotic band did not change when either the exposure time or the recovery period was varied between 30 min and 3 h. These results are predicted by a theoretical model in which the diffusion of glutamate into brain tissue is countered by cellular uptake of the amino acid, and they argue against the idea that glutamate toxicity is inherently self‐propagating. When slices were examined immediately after exposure (300 μM), a prominent swelling of glial cells was present at the slice surface. Swelling per se did not appear to compromise their uptake function, and the model predicts that cellular swelling, by reducing the rate of diffusion of glutamate, protects against glutamate toxicity. The damage produced by 3 mM glutamate, which was primarily exerted against granule cells, was prevented by W‐methyl‐D‐aspartate (NMDA) receptor blockade, whereas antagonists acting at α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA) receptors were ineffective. Under conditions of energy deprivation, the neurotoxic potency of glutamate was markedly enhanced and a normally non‐toxic concentration (30 μM) became maximally toxic towards granule cells. Dark vacuolar degeneration of Purkinje cells was also present, and this could be inhibited by blocking AMPA receptors. The results and theoretical analysis suggest that intact brain tissue is remarkably resistant to glutamate toxicity, chiefly because of the formidable properties of the uptake system. However, under special circumstances, glutamate can become a potent neurotoxin and its toxicity can then involve both NMDA and AMPA receptors.

Mechanisms of AMPA Neurotoxicity in Rat Brain Slices

The mechanisms underlying the neurodegenerative effects of the glutamate receptor agonist, AMPA (α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate), were studied using brain slice preparations of young rat (8–9 days old) cerebellum and hippocampus. Rapid AMPA toxicity (exerted on some cerebellar interneurons) was inhibited by including the appropriate receptor blocker, CNQX (6‐cyano‐7‐nitroquinoxaline‐2,3‐dione, 10 μM), in the exposing solution. The degeneration of other neurons, including Purkinje cells and hippocampal pyramidal neurons, persisted. It could, however, be largely prevented if CNQX was included for 1.5 h during the post‐incubation period, suggesting that an enduring ‘rebound’ AMPA receptor activation was responsible for this delayed type of degeneration, not the exposure itself. In cerebellar slices, independent evidence for the occurrence, postexposure, of persisting AMPA receptor stimulation was obtained electrophysiologically. Omission of Ca2+ during the exposure period (and for 10 min beforehand) markedly reduced rapid AMPA toxicity but was ineffective in protecting most of the Purkinje cells. However, if the slices were previously starved of Ca2+ for 1 h, then most of these neurons survived, even if the ion was reinstated during the recovery period. Slow AMPA toxicity, which takes place during long (2 h) exposures, could be inhibited either by CNQX or by omission of Ca2+ (30 min preincubation). The results indicate that the rapid oedematous necrosis induced by AMPA, like that caused by N‐methyl‐d‐aspartate and kainate, is likely to involve excessive influx of Ca2+. In contrast, the induction of the delayed mechanism, as well as its ‘expression’ during the postincubation period, probably depends on intracellular Ca2+, rather than Ca2+ influx.

AMPA Neurotoxicity in Rat Cerebellar and Hippocampal Slices: Histological Evidence for Three Mechanisms.

Excitatory amino acid‐induced death of central neurons may be mediated by at least two receptor types, the so‐called NMDA (N‐methyl‐d‐aspartate) and AMPA (α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazoleproprionate) receptors. We have studied the neurodegenerative mechanisms set in motion by AMPA receptor activation using incubated slices of 8‐day‐old rat cerebellum and hippocampus. In both preparations, AMPA induced a pattern of degeneration that differed markedly from the one previously shown to be elicited by NMDA. In cerebellar slices, AMPA induced the degeneration of most Purkinje cells together with a population of Golgi cells; in hippocampal slices the neurons were affected in the order CA3 > CA1 > dentate granule cells. Three mechanisms could be discerned: an acute one in which neurons (e.g. cerebellar Golgi cells) underwent a rapid degeneration; a delayed one in which the neurons (Purkinje cells and hippocampal neurons) appeared to be only mildly affected immediately after a 30 min exposure but then underwent a protracted degeneration during the postincubation period (1.5–3 h); and finally a slow toxicity, which took place during long (2 h) exposures to AMPA (3–30 μM). Although Purkinje cells were vulnerable in both cases, the efficacy of AMPA was higher for the delayed mechanism than for the slow one. The pathology displayed by the acutely destroyed Golgi neurons was a classical oedematous necrosis, whereas most neurons vulnerable to the delayed and slow mechanisms displayed a ‘dark cell degeneration’, whose cytological features bore a close resemblance to those of neurons irreversibly damaged by ischaemia, hypoglycaemia or status epilepticus in vivo.

Amino acid neurotoxicity: Intracellular sites of calcium accumulation associated with the onset of irreversible damage to rat cerebellar neurones in vitro

Electron microscopy and the combined oxalate-pyroantimonate technique were used to locate calcium in intracerebellar nucleus neurones of rat cerebellar slices subjected to a neurotoxic concentration of N-methyl-D-aspartate. After a sub-lethal exposure period (5 min) calcium pyroantimonate deposits were found in swollen cisterns of the Golgi apparatus and, in lesser amounts, in the nuclei. Deposits were more prominent in the nuclei after a just-lethal exposure (10 min) when they were additionally observed within a population of swollen mitochondria and also apparently free in the dendritic and somatic cytoplasm. The results support the proposal that amino acid neurotoxicity is a consequence of an intracellular Ca2+ overload brought about by excessive Ca2+ influx.

Receptor-linked ionic channels mediate N-methyl-d-aspartate neurotoxicity in rat cerebellar slices

In young rat cerebellar slices, histological methods showed that the neurotoxic potency of N-methyl-D-aspartate (NMDA) towards granule cells and intracerebellar nucleus neurons was increased 2- to 3-fold on removal of Mg ions, which have a blocking effect on NMDA-activated ion channels. The depolarizing potency of NMDA on granule cells, recorded using a gap method, was similarly enhanced whereas that of kainate, a non-NMDA receptor agonist, was unchanged. The neurotoxic potency of kainate (towards Golgi cells) was also unaltered by removal of Mg2+. In Mg2+-containing medium, neuronal depolarization induced either by kainate or by high K+ potentiated NMDA toxicity, apparently by reducing the channel block by Mg2+. The results strongly support the hypothesis that excessive Ca2+ influx through NMDA/Mg2+-gated ion channels mediates NMDA toxicity. They also have clear implications regarding the likely mechanism of toxicity of agonists, such as glutamate, able to activate both NMDA and non-NMDA receptors.

Differential sensitivity of rat cerebellar cells in vitro to the neurotoxic effects of excitatory amino acid analogues

The neurotoxic effects of N-methyl-D-aspartate, quisqualate and kainate were studied in slice preparations of adult rat cerebellum. Only the inhibitory interneurones (basket, stellate and Golgi cells) were affected by N-methyl-D-aspartate in concentrations up to 300 microM. Quisqualate (10-100 microM) affected both Purkinje cells and inhibitory interneurones but spared granule cells. Kainate affected Purkinje cells and inhibitory interneurones at a concentration of 10 microM but at 30 microM also damaged granule cells. The relative potencies of these compounds and the vulnerability of the different cell types to their neurotoxic effects are in accordance with the predictions of the excitotoxic hypothesis and therefore do not support a special mechanism for kainate neurotoxicity in the cerebellum.

Quinolinate mimics neurotoxic actions of N-methyl-d-aspartate in rat cerebellar slices

Incubation of slices of immature rat cerebellum for 30 min with quinolinate (QUIN), an endogenous neurotoxin, resulted in the selective necrosis of granule cells and intracerebellar nucleus neurones. Concentration of QUIN in the millimolar range were needed for these effects. The same neuronal populations were also selectively killed by N-methyl-D-aspartate (NMDA) but the toxic potency of NMDA was 40-fold higher than that of QUIN. Depolarizing responses of granule cells to brief applications of QUIN and NMDA were recorded using a gap method. Dose-response curves to the two compounds appeared parallel but NMDA was 30-fold more potent than QUIN. The depolarizing and toxic actions of QUIN and NMDA were inhibited by the NMDA antagonist, 2-amino-5-phosphonopentanoate. We conclude that the selective toxicity of QUIN in this tissue arises from its activity on NMDA receptors.

Mechanisms of Excitatory Amino Acid Neurotoxicity in Rat Brain Slices

Excitatory amino acids (EAA), in addition to their roles in neurotransmission and synaptic plasticity, have also been implicated in a variety of neurodegenerative disorders of the central nervous system. The work of Olney and colleagues who showed that glutamate and related excitants are toxic to central neurones in vivo through acting on EAA receptors (see Olney, 1984) coupled with the realization that EAAs (glutamate in particular) are likely to be the principal excitatory transmitters in the brain, provided the key theoretical link between brain physiology and brain pathology. The most convincing experimental evidence to date that the neurotoxic action of endogenous EAAs contributes to neuronal loss in vivo is in the acute conditions of ischaemia (Simon et al., 1984a; Gill et al., 1988), hypoglycaemia (Weiloch, 1985) and trauma (Faden et al., 1989) where EAA antagonists have been shown to provide significant protection. Their involvement in more chronic conditions such as Alzheimer’s disease, Huntington’s chorea and Parkinsonism, which are more difficult to model experimentally, is more speculative (Choi, 1988).